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An overview of ProTide technology and its implications to drug discovery
Abstract
Introduction
The ProTide technology is a phosphate (or phosphonate) prodrug method devised to deliver nucleoside monophosphate (or monophosphonate) intracellularly bypassing the key challenges of antiviral and anticancer nucleoside analogues. Three new antiviral drugs, exploiting this technology, have been approved by the FDA while others are in clinical studies as anticancer agents.
Areas covered
The authors describe the origin and development of this technology and its incredible success in transforming the drug discovery of antiviral and anticancer nucleoside analogues. As evidence, discussion on the antiviral ProTides on the market, and those currently in clinical development is included. The authors focus on how the proven capacity of this technology to generate new drug candidates has stimulated its application to non-nucleoside-based molecules.
Expert opinion
The ProTide approach has been extremely successful in delivering blockbuster antiviral medicines and it seems promising in oncology. Its application to non-nucleoside-based small molecules is recently emerging and proving effective in other therapeutic areas. However, investigations to explain the lack of activity of certain ProTide series and comprehensive structure activity relationship studies to identify the appropriate phosphoramidate motifs depending on the parent molecule are in our opinion mandatory for the future development of these compounds.
... ProTide derivatives of antiviral and anticancer nucleoside analogues used in the clinics or ongoing clinical trials (*Acelarin, the SP isomer of NUC-1031, has received a fast track designation from the US FDA for the treatment of biliary tract cancer (BTC) in September 2021, however the clinical phase III for the combination of NUC-1031 and cisplatin [41] was discontinued for advanced BTC in March 2022). nucleoTIDE) developed by McGuigan's group in the 90s [15][16][17][18][19][20], which led to several marketed drugs. As an example, Sofosbuvir [21,22] was the first FDA (US Food and Drug Administration) approved livertargeting drug for the cure of hepatitis C in 2013 and Remdesivir [14,[23][24][25]] is a C-nucleoside based Pro-Tide exhibiting broad spectrum antiviral activity against RNA viruses, also licensed by Gilead Sciences (Fig. 3). ...
... Several ProTides are also under clinical investigation for antitumoral applications [39][40][41]. A number of recent reviews have focused on ProTides [14,16,18,19,42,43]. ...
... Their hydrolysis is mediated by carboxylicester hydrolases or carboxypeptidase-type enzymes (CPY), leading to the release of the carboxylic acid Fig. (4). General mechanism of the bioactivation of ProTides (Adapted from [19]). ...
Nucleoside analogues are widely used as anti-infectious and antitumoral agents. However, their clinical use may face limitations associated with their physico-chemical properties, pharmacokinetic parameters, and/or their peculiar mechanisms of action. Indeed, once inside the cells, nucleoside analogues require to be metabolized into their corresponding (poly-)phosphorylated derivatives, mediated by cellular and/or viral kinases, in order to interfere with nucleic acids biosynthesis. Within this activation process, the first-phosphorylation step is often the limiting one and to overcome this limitation numerous prodrug approaches have been proposed. Herein, we will focus on recent literature data (from 2015 and onwards) related to new prodrug strategies, the development of original synthetic approaches and novel applications of nucleotide prodrugs (namely pronucleotides) leading to the intracellular delivery of 5’-monophosphate nucleoside analogues.
... ProTide technology is a phosphate prodrug method designed to deliver nucleoside monophosphates intracellularly. It has been successful in transforming the drug discovery of antiviral and anticancer nucleoside analogs by improving therapeutic efficiency and reducing side effects [7]. ...
... Patent protection may be available for novel prodrug compositions that significantly outperform already available medications, giving them market exclusivity and financial benefits. [7] 3. Structure-Activity Relationships (SAR) and Prodrug Optimization: Structure-activity relationships (SAR) study the connection between a molecule's three-dimensional structure and its biological activity. SAR is critical in determining the structural characteristics of the lead chemical that are necessary for its biological activity in the context of prodrug optimization. ...
Prodrugs, which are chemically altered substances that go through biotransformation to release the active ingredient, offer better medication stability and specialised delivery. Prodrug creation has had a major influence on contemporary drug discovery throughout history, from the isolation of active molecules during the Scientific Revolution through the use of traditional herbal treatments. Structure-activity correlations, optimization approaches, and design principles all take strategic changes into account. Enzymatic, pharmacological, and pH-dependent activation mechanisms allow for controlled drug release and therapeutic activity at a specific spot. Prodrug carriers and nanoparticle-based approaches have the potential to transport drugs to particular areas of the body, opening the door to personalised medicine and disease-specific therapies. Successful case studies highlight transformational medicines for a range of disorders despite obstacles in toxicity, bioavailability, and stability. Through clinical trials, regulatory inspection, and post-marketing monitoring, the clinical development process is rigorously evaluated. Looking ahead, prodrug uses in gene therapy and biologics, tailored therapeutics, and developing trends provide interesting opportunities for pharmacological interventions. Prodrugs' full potential will be unlocked through embracing innovation and solving problems, transforming medication delivery and patient care.
... Another approach providing phosphorylated NAs is McGuigan's ProTide technology based on phosphoroamidites [114]. The first work was published in 1993, and since then, this approach has been successfully applied to improve the pharmacokinetics of a number of NAs [115]. ...
... Notably, the number of possible modifications of NAs to improve the pharmacokinetic properties is immense. The issue is discussed in more detail in the corresponding literature [21,22,111,114,135,136]. Here, the most significant approaches that allow for the enhancement of the effectiveness of NAs are listed. ...
The search for new drugs has been greatly accelerated by the emergence of new viruses and drug-resistant strains of known pathogens. Nucleoside analogues (NAs) are a prospective class of antivirals due to known safety profiles, which are important for rapid repurposing in the fight against emerging pathogens. Recent improvements in research methods have revealed new unexpected details in the mechanisms of action of NAs that can pave the way for new approaches for the further development of effective drugs. This review accounts advanced techniques in viral polymerase targeting, new viral and host enzyme targeting approaches, and prodrug-based strategies for the development of antiviral NAs.
... This compound is then hydrolyzed by an amidase, Although nucleoside derivatives generally act in di-or triphosphate form, these species are not employed as such because of their high polarity, which hampers their absorption across cell membranes. Phosphoramidates and related prodrugs, developed by McGuigan, are generally described as ProTide (PROdrug + nucleoTIDE), and have found widespread application in recent years [40]. The ionizable groups of these compounds are blocked by lipophilic moieties, allowing a good membrane permeability. ...
... Although nucleoside derivatives generally act in di-or triphosphate form, these species are not employed as such because of their high polarity, which hampers their absorption across cell membranes. Phosphoramidates and related prodrugs, developed by McGuigan, are generally described as ProTide (PROdrug + nucleoTIDE), and have found widespread application in recent years [40]. The ionizable groups of these compounds are blocked by lipophilic moieties, allowing a good membrane permeability. ...
In spite of advances in vaccination, control of the COVID-19 pandemic will require the use of pharmacological treatments against SARS-CoV2. Their development needs to consider the existence of two phases in the disease, namely the viral infection and the inflammatory stages. The main targets for antiviral therapeutic intervention are: (a) viral proteins, including the spike (S) protein characteristic of the viral cover and the viral proteases in charge of processing the polyprotein arising from viral genome translation; (b) host proteins, such as those involved in the processes related to viral entry into the host cell and the release of the viral genome inside the cell, the elongation factor eEF1A and importins. The use of antivirals targeted at host proteins is less developed but it has the potential advantage of not being affected by mutations in the genome of the virus and therefore being active against all its variants. Regarding drugs that address the hyperinflammatory phase of the disease triggered by the so-called cytokine storm, the following strategies are particularly relevant: (a) drugs targeting JAK kinases; (b) sphingosine kinase 2 inhibitors; (c) antibodies against interleukin 6 or its receptor; (d) use of the traditional anti-inflammatory corticosteroids.
... 15 This technique presents an intriguing approach for generating nucleotide prodrugs with demonstrated antiviral and anticancer activities. 16,17 The structure comprises three essential components: (i) an aryl moiety, (ii) an amino acid, and (iii) esters ( Figure 1). Therefore, using this method, we modified ACV analogs, to avoid the initial stage of bioactivation in cells that require the TK enzyme. ...
Acyclovir (ACV) is a vital treatment for herpes simplex (HSV) and varicella-zoster virus (VZV) infections that inhibit viral DNA polymerase. Phosphoramidate ProTides-ACV, a promising technology, circumvents the reliance on thymidine kinase (TK) for activation. Twelve novel single isomers of phosphoramidate ProTide-ACV were synthesized. Successful isomer separation was achieved, emphasizing the importance of single isomers in medical advancements. The enzymatic hydrolysis kinetics of the synthesized compounds were investigated by using carboxypeptidase Y (CPY). The results revealed a faster conversion for the isomer Rp- than for the Sp-diastereomer. Hydrolysis experiments confirmed steric hindrance effects, particularly with the tert-butyl and isopropyl groups. Molecular modeling elucidated the mechanisms of hydrolysis, supporting the results of the experiments. This research sheds light on the potential of phosphoramidate ProTides-ACV, bridging the gap in understanding their biological and metabolic properties, while supporting future investigations into anti-HSV activity. Preliminary screening revealed that three of the four single isomers demonstrated superior antiviral efficacy against wild-type HSV-1 compared to acyclovir, with isomer 24a ultimately reducing the viral yield at 200 μM. These findings emphasize the importance of isolating racemic ACV-ProTides as pure single isomers for future drug development.
... Once inside, these small molecules decompose, allowing the preactivated compounds to carry out their intended function. Tenofovir sulfonamide (TAF) is another variant of the tenofovir prodrug, which was developed using proTide technology [98]. TAF exhibits greater antiviral activity than TDF and possesses superior distribution within lymphatic tissues. ...
The treatment of HIV presents significant challenges due to the low solubility and bioavailability of antiretroviral drugs. These limitations impact the absorption of the drugs in the gastrointestinal tract, leading to reduced effectiveness. Patients often exhibit varying responses due to inconsistent levels of the drug in their bloodstream. Achieving desired plasma concentrations is challenging and often requires higher doses that increase the risk of side effects. Developing drugs with low solubility is a complex and costly process, slowing down overall drug development. Traditional formulation strategies have proven ineffective in addressing these issues, necessitating exploration of innovative techniques. Nanocrystallization has emerged as a transformative approach that enhances solubility and dissolution rates by reducing drug particles to the nanometer scale. This increased surface area improves drug solubility and bioavailability. Nanocrystallized drugs demonstrate a faster onset of action, higher drug load, and enhanced safety and efficiency, offering a potential solution to the challenges posed by the poor water solubility of many antiretroviral drugs. This article delves into nanocrystal formulations of HIV drugs and examines the effectiveness of commonly used stabilizers and surfactants in nanocrystal production. The aim is to determine whether the popularization of nanocrystallization could potentially overcome the prevalent issues of low solubility and bioavailability in existing antiretroviral therapies.
... In the last step, a phosphoramide-type enzyme once again hydrolyzes the phosphorus-nitrogen (P-N) bond, producing the triphosphorylated nucleoside. This nucleoside is then released by cellular kinases, enabling its effectiveness ediagainst virus-encoded DNA or RNA polymerases (Figure 3) [37,38]. ...
The effective treatment of human immunodeficiency virus (HIV), also known as acquired immunodeficiency syndrome (AIDS), has transformed a highly lethal disease into a chronic and manageable condition. However, better methods need to be developed for eliminating the virus from reservoirs, enhancing patient access and adherence to therapy, and improving treatment in the long term to reduce adverse effects. From the perspective of drug discovery, one promising strategy is the development of anti-HIV prodrugs. This approach aims to enhance the efficacy and safety of treatment, promoting the development of more appropriate and convenient systems for patients. In this review, we discussed the use of the prodrug approach for HIV antiviral agents and emphasized nucleoside reverse transcriptase inhibitors. We comprehensively described various strategies that are used to enhance factors such as water solubility, bioavailability, pharmacokinetic parameters, permeability across biological membranes, chemical stability, drug delivery to specific sites/organs, and tolerability. These strategies might help researchers conduct better studies in this field. We also reported successful examples from the primary therapeutic classes while discussing the advantages and limitations. In this review, we highlighted the key trends in the application of the prodrug approach for treating HIV/AIDS..
... Magnesium ions and pyrophosphate create two ionic bond contacts close to RDV-MP, which are absent in all other configurations of the SARS-CoV-2 RdRp complex with RNA. The triphosphate occupying the nucleotide input site may prevent nucleotide triphosphates (NTP) from entering the active site, providing a possible mechanism for remdesivir's inhibitory effect [203,204]. Other nucleotides, including favipiravir, ribavirin, IDX-184, galidesivir, EIDD-2801 and molnupiravir, effectively hinder SARS-CoV-2 replication by targeting RdRp [205] (Fig. 26). ...
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic since 2019, spreading rapidly and posing a significant threat to human health and life. With over 6 billion confirmed cases of the virus, the need for effective therapeutic drugs has become more urgent than ever before. RNA-dependent RNA polymerase (RdRp) is crucial in viral replication and transcription, catalysing viral RNA synthesis and serving as a promising therapeutic target for developing antiviral drugs. In this article, we explore the inhibition of RdRp as a potential treatment for viral diseases, analysing the structural information of RdRp in virus proliferation and summarizing the reported inhibitors’ pharmacophore features and structure–activity relationship profiles. We hope that the information provided by this review will aid in structure-based drug design and aid in the global fight against SARS-CoV-2 infection.
Graphical Abstract
... Another interesting possibility for use of the P-S substitution is the creation of non-hydrolyzable antiviral ribonucleotide analogs. Most clinically used monophosphorylated ribonucleotide analogs are readily delivered to cells as prodrugs that require intracellular activation by histidine triad nucleotide-binding protein 1 (HINT-1) (78,79), the so-called ProTide strategy (7,(80)(81)(82). Similar strategies can be used for P-S-substituted ribonucleoside monophosphates. ...
Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3′-to-5′ proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3′-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3′-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.
... Another interesting possibility for use of the P-S substitution is the creation of nonhydrolyzable antiviral ribonucleotide analogs. Most clinically used monophosphorylated ribonucleotide analogs are readily delivered to cells as prodrugs that require intracellular activation by histidine triad nucleotide-binding protein 1 (HINT-1) (77,78), the so-called ProTide strategy (7,(79)(80)(81). Similar strategies can be used for P-S-substituted ribonucleoside monophosphates. ...
Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3'-to-5' proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3'-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3'-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.
... To improve cell permeability and pharmacokinetic performance, nucleoside/nucleotide analogs are often structurally modified to be ester prodrugs [2]. The ProTide technology has been a powerful tool to improve the cell permeability and the intracellular activation of nucleoside/nucleotide antivirals and anticancer drugs [3,4]. The most recent ProTide technology is to structurally modify nucleoside/nucleotide analogs to aryloxy phosphoramidite prodrugs by masking two of the oxygens of the monophosphate and monophosphonate The putative intracellular activation pathway of TAF. ...
ProTide technology is a powerful tool for the design of nucleoside/nucleotide analog prodrugs. ProTide prodrug design improves cell permeability and enhances intracellular activation. The hydrolysis of the ester bond of a ProTide is a determinant of the intracellular activation efficiency and final antiviral efficacy of the prodrug. The hydrolysis is dictated by the catalytic activity and abundance of activating enzymes. The antiviral agents tenofovir alafenamide (TAF) and sofosbuvir (SBV) are typical ProTides. Both TAF and SBV have also been proposed to treat patients with COVID-19. However, the mechanisms underlying the activation of the two prodrugs in the lung remain inconclusive. In the present study, we profiled the catalytic activity of serine hydrolases in human lung S9 fractions using an activity-based protein profiling assay. We evaluated the hydrolysis of TAF and SBV using human lung and liver S9 fractions and purified enzymes. The results showed that CatA and CES1 were involved in the hydrolysis of the two prodrugs in the human lung. More specifically, CatA exhibited a nearly 4-fold higher hydrolytic activity towards TAF than SBV, whereas the CES1 activity on hydrolyzing TAF was slightly lower than that for SBV. Overall, TAF had a nearly 4-fold higher hydrolysis rate in human lung S9 than SBV. We further analyzed protein expression levels of CatA and CES1 in the human lung, liver, and primary cells of the two tissues using proteomics data extracted from the literature. The relative protein abundance of CatA to CES1 was considerably higher in the human lung and primary human airway epithelial cells than in the human liver and primary human hepatocytes. The findings demonstrated that the high susceptivity of TAF to CatA-mediated hydrolysis resulted in efficient TAF hydrolysis in the human lung, suggesting that CatA could be utilized as a target activating enzyme when designing antiviral ester prodrugs for the treatment of respiratory virus infection.
In this study, we report for the first time a microwave-accelerated synthesis of purine and pyrimidine nucleoside triphosphate prodrugs, whose γ phosphate is masked with an aryloxy moiety and an amino acid ester (γ-ProTriP). The synthetic utility of this method is illustrated by the synthesis of triphosphate prodrugs of clofarabine and gemcitabine, two FDA-approved anticancer drugs. These new prodrugs showed good chemical and rat serum stability. Remarkably the clofarabine prodrug showed significant cytotoxicity against a panel of cancer cell lines.
The global prevalence of RNA virus infections has presented significant challenges to public health in recent years, necessitating the expansion of its alternative therapeutic library. Due to its evolutional conservation, RNA‐dependent RNA polymerase (RdRp) has emerged as a potential target for broad‐spectrum antiviral nucleoside analogues. However, after over half a century of structural modification, exploring unclaimed chemical space using frequently‐used structural substitution methods to design new nucleoside analogues is challenging. In this study, we explore the use of the “ring‐opening” strategy to design new base mimics, thereby using these base mimics to design new nucleoside analogues with broad‐spectrum antiviral activities. A total of 29 compounds were synthesized. Their activity against viral RdRp was initially screened using an influenza A virus RdRp high‐throughput screening model. Then, the antiviral activity of 38a was verified against influenza virus strain A/PR/8/34 (H1N1), demonstrating a 50% inhibitory concentration (IC 50 ) value of 9.95 μM, which was superior to that of ribavirin (the positive control, IC 50 = 11.43 μM). Moreover, 38a also has inhibitory activity against coronavirus 229E with an IC 50 of 30.82 μM. In addition, compounds 42 and 46f exhibit an 82% inhibition rate against vesicular stomatitis virus at a concentration of 20 μM and hardly induce cytotoxicity in host cells. This work demonstrates the feasibility of designing nucleoside analogues with “ring‐opening” bases and suggests the “ring‐opening” nucleosides may have greater polarity, and designing prodrugs is an important aspect of optimizing their antiviral activity. Future research should focus on enhancing the conformational restriction of open‐loop bases to mimic Watson‐Crick base pairing better and improve antiviral activity.
Protein O‐GlcNAcylation is a ubiquitous posttranslational modification of cytosolic and nuclear proteins involved in numerous fundamental regulation processes. Investigation of O‐GlcNAcylation by metabolic glycoengineering (MGE) has been carried out for two decades with peracetylated N‐acetylglucosamine (GlcNAc) and N‐acetylgalactosamine derivatives modified with varying reporter groups. Recently, it has been shown that these derivatives can result in non‐specific protein labeling termed S‐glyco modification. Here, we report norbornene‐modified GlcNAc derivatives with a protected phosphate at the anomeric position and their application in MGE. These derivatives overcome two limitations of previously used O‐GlcNAc reporters. They do not lead to detectable S‐glyco modification, and they efficiently react in the inverse‐electron‐demand Diels‐Alder (IEDDA) reaction, which can be carried out even within living cells. Using a derivative with an S‐acetyl‐2‐thioethyl‐protected phosphate, we demonstrate the protein‐specific detection of O‐GlcNAcylation of several proteins and the protein‐specific imaging of O‐GlcNAcylation inside living cells by Förster resonance energy transfer (FRET) visualized by confocal fluorescence lifetime imaging microscopy (FLIM).
Protein O−GlcNAcylation is a ubiquitous posttranslational modification of cytosolic and nuclear proteins involved in numerous fundamental regulation processes. Investigation of O−GlcNAcylation by metabolic glycoengineering (MGE) has been carried out for two decades with peracetylated N‐acetylglucosamine (GlcNAc) and N‐acetylgalactosamine derivatives modified with varying reporter groups. Recently, it has been shown that these derivatives can result in non‐specific protein labeling termed S‐glyco modification. Here, we report norbornene‐modified GlcNAc derivatives with a protected phosphate at the anomeric position and their application in MGE. These derivatives overcome two limitations of previously used O−GlcNAc reporters. They do not lead to detectable S‐glyco modification, and they efficiently react in the inverse‐electron‐demand Diels–Alder (IEDDA) reaction, which can be carried out even within living cells. Using a derivative with an S‐acetyl‐2‐thioethyl‐protected phosphate, we demonstrate the protein‐specific detection of O−GlcNAcylation of several proteins and the protein‐specific imaging of O−GlcNAcylation inside living cells by Förster resonance energy transfer (FRET) visualized by confocal fluorescence lifetime imaging microscopy (FLIM).
Antiretroviral Therapy (ART) is an effective treatment for human immunodeficiency virus (HIV) which has transformed the highly lethal disease, acquired immunodeficiency syndrome (AIDS), into a chronic and manageable condition. However, better methods need to be developed for enhancing patient access and adherence to therapy and for improving treatment in the long term to reduce adverse effects. From the perspective of drug discovery, one promising strategy is the development of anti-HIV prodrugs. This approach aims to enhance the efficacy and safety of treatment, promoting the development of more appropriate and convenient systems for patients. In this review, we discussed the use of the prodrug approach for HIV antiviral agents and emphasized nucleoside reverse transcriptase inhibitors. We comprehensively described various strategies that are used to enhance factors such as water solubility, bioavailability, pharmacokinetic parameters, permeability across biological membranes, chemical stability, drug delivery to specific sites/organs, and tolerability. These strategies might help researchers conduct better studies in this field. We also reported successful examples from the primary therapeutic classes while discussing the advantages and limitations. In this review, we highlighted the key trends in the application of the prodrug approach for treating HIV/AIDS.
ProTide prodrug technology has emerged as a promising way for the development of anti-viral and anti-tumor drugs, whereas, there are fewer applications for the treatment of liver cancer. Herein, a series of distinct 3'-ester ProTide prodrugs of 5-fluoro-2'-deoxyuridine (FdUR) were synthesized and evaluated for their anti-liver cancer activity. The most efficient prodrug 11b reached a sub-micromolar activity (IC50 = 0.42 ± 0.13 μM) against HepG2 and over 100-fold and 200-fold improvements compared to 5-FU, respectively. 11b also demonstrated favorable selectivity towards normal liver cells L-02 (IC50 > 100 μM). In vitro metabolic stability studies revealed that 11b is stable in the plasma and could be activated rapidly in the liver, which supported that 11b is liver-targeted. Importantly, to more accurately evaluate the anti-HCC activity of 11b, the liver orthotopic model was built and 11b significantly suppressed tumor growth (TGI = 75.5%) at a dose of 60 mg/kg/2d in vivo without obvious toxicity. Overall, these promising results indicated that 11b could serve as a safe and effective prodrug of 5-FU nucleoside for liver cancer therapy.
The metabolism in eukaryotic cells is a highly ordered system involving various cellular compartments, which fluctuates based on physiological rhythms. Organelles, as the smallest independent sub-cell unit, are important contributors to cell metabolism and drug metabolism, collectively designated intracellular metabolism. However, disruption of intracellular spatiotemporal metabolism can lead to disease development and progression, as well as drug treatment interference. In this review, we systematically discuss spatiotemporal metabolism in cells and cell subpopulations. In particular, we focused on metabolism compartmentalization and physiological rhythms, including the variation and regulation of metabolic enzymes, metabolic pathways, and metabolites. Additionally, the intricate relationship among intracellular spatiotemporal metabolism, metabolism-related diseases, and drug therapy/toxicity has been discussed. Finally, approaches and strategies for intracellular spatiotemporal metabolism analysis and potential target identification are introduced, along with examples of potential new drug design based on this.
3'-Deoxyadenosine (3'-dA, Cordycepin, 1) is a nucleoside analogue with anticancer properties, but its clinical development has been hampered due to its deactivation by adenosine deaminase (ADA) and poor cellular uptake due to low expression of the human equilibrative transporter (hENT1). Here, we describe the synthesis and characterization of NUC-7738 (7a), a 5'-aryloxy phosphoramidate prodrug of 3'-dA. We show in vitro evidence that 7a is an effective anticancer drug in a panel of solid and hematological cancer cell lines, showing its preferential cytotoxic effects on leukemic stem cells. We found that unlike 3'-dA, the activity of 7a was independent of hENT1 and kinase activity. Furthermore, it was resistant to ADA metabolic deactivation. Consistent with these findings, 7a showed increased levels of intracellular 3'-deoxyadenosine triphosphate (3'-dATP), the active metabolite. Mechanistically, levels of intracellular 3'-dATP were strongly associated with in vitro potency. NUC-7738 is now in Phase II, dose-escalation study in patients with advanced solid tumors.
A range of lipophilic prodrugs of α-carboxy nucleoside phosphonates, potent inhibitors of HIV-1 reverse transcriptase without requiring prior phosphorylation, were synthesized to evaluate their in vivo potency against HIV in cell culture. A series of prodrug derivatives bearing a free carboxylic acid where the phosphonate was masked with bispivaloyloxymethyl, diisopropyloxycarbonyloxymethyl, bisamidate, aryloxyphosphoramidate, hexadecyloxypropyl, CycloSal, and acycloxybenzyl moieties were synthesized, adapting existing methodologies for phosphonate protection to accommodate the adjacent carboxylic acid moiety. The prodrugs were assayed for anti-HIV activity in CEM cell cultures─the bispivaloyloxymethyl free acid monophosphonate prodrug exhibited some activity (inhibitory concentration-50 (IC50) 59 ± 17 μM), while the other prodrugs were inactive at 100 μM. A racemic bispivaloyloxymethyl methyl ester monophosphonate prodrug was also prepared to assess the suitability of the methyl ester as a carboxylic acid prodrug. This compound exhibited no activity against HIV in cellular assays.
Introduction:
Poor or inconsistent adherence to daily oral pre-exposure prophylaxis (PrEP) has emerged as a key barrier to effective HIV prevention. The advent of potent long-acting (LA) antiretrovirals (ARVs) in conjunction with advances in controlled release technologies have enabled LA ARV drug delivery systems (DDS) capable of providing extended dosing intervals and overcome the challenge of suboptimal drug adherence with daily oral dosing.
Areas covered:
This review discusses the current state of the LA PrEP field, recent advances and emerging technologies, including ARV prodrug modifications and new DDS. Technological challenges, knowledge gaps, preclinical testing considerations, and future directions important in the context of clinical translation and implementation of LA HIV PrEP are discussed.
Expert opinion:
The HIV prevention field is evolving faster than ever and the bar for developing next-generation LA HIV prevention options continues to rise. The requirements for viable LA PrEP products to be implemented in resource-limited settings is challenging, necessitating proactive consideration and product modifications during the design and testing of promising new candidates. If successfully translated, next-generation LA PrEP that are safe, affordable, highly effective, and accepted by both end users and key stakeholders, will offer significant potential to curb the HIV pandemic.
A synthesis of aryloxy phosphoramidate prodrug of alcohols enabled by a transesterification strategy is described here.
Synthetic phosphate‐derived functional groups are important for controlling the function of bioactive molecules in vivo. Herein we describe the development of a new type of biocompatible phosphate analog, a fluorophosphoramidate (FPA) functional group that has characteristic P−F and P−N bonds. We found that FPA with a primary amino group was relatively unstable in aqueous solution and was converted to a monophosphate, while FPA with a secondary amino group was stable. Furthermore, by improving the molecular design of FPA, we developed a reaction in which a secondary amino group is converted to a primary amino group in the intracellular environment and clarified that the FPA group functions as a phosphate prodrug of nucleoside. Various FPA‐gemcitabine derivatives were synthesized and their toxicity to cancer cells were evaluated. One of the FPA‐gemcitabine derivatives showed superior toxicity compared with gemcitabine and its ProTide prodrug, which methodology is widely used in various nucleoside analogs, including anti‐cancer and anti‐virus drugs.
Drug development based on phenolic natural products as drug candidates against various diseases has gained much attention in recent years. However, most of those molecules lack therapeutic efficacy in clinical trials, usually due to poor bioavailability. Therefore, a prodrug approach was adopted to address the bioavailability problem of phenolic drugs. This paper describes a mild and convenient method for late-stage ProTide-type prodrug synthesis of phenolic pharmaceuticals, which gives various phosphoramidate prodrugs from unprotected phenolic natural products and drugs in high yield. More importantly, this reaction is amenable for the selective phosphorylation of the phenolic hydroxyl group in the presence of otherwise problematic nucleophilic functional groups like amines and alcohols. We also observed that the chemical release rate of the phenol can be substantially tuned by changing the amino acid residue on the phosphoramidate moiety.
Phosphoramidates obtained according to the ProTide strategy are known for their ability to increase the biological activity of various nucleosides. A series of such prodrugs of SRO-91, a non-natural ribofuranosyl-1,2,3-triazole C-nucleoside obtained by a synthetic sequence involving an indium mediated alkynylation and a Huisgen cycloaddition, was prepared and the antitumor activity on 3 strains of tumor cells was investigated. Two compounds 9a and 9c exhibited interesting cell proliferative inhibitions (IC50 = 2.5-12.1 µM) on two cell lines (pancreas and lung). Moreover, concerning the antiviral activity, another phosphoramidate 14 bearing a different aryl masking group exhibited an IC50 of 5 µM on Crimean-Congo Hemorrhagic Fever orthonairovirus. In both cases, free SRO-91 presented no activity on these cell lines.
HIV reverse transcriptase (RT) inhibitors are the important components of highly active antiretroviral therapies (HAARTs) for anti-HIV treatment and pre-exposure prophylaxis in clinical practice. Many RT inhibitors and their combination regimens have been approved in the past ten years, but a review on their drug discovery, pharmacology, and clinical efficacy is lacking. Here, we provide a comprehensive review of RT inhibitors (tenofovir alafenamide, rilpivirine, doravirine, dapivirine, azvudine and elsulfavirine) approved in the past decade, regarding their drug discovery, pharmacology, and clinical efficacy in randomized controlled trials. Novel RT inhibitors such as islatravir, MK-8504, MK-8507, MK8583, IQP-0528, and MIV-150 will be also highlighted. Future development may focus on the new generation of novel antiretroviral inhibitors with higher bioavailability, longer elimination half-life, more favorable side-effect profiles, fewer drug‒drug interactions, and higher activities against circulating drug-resistant strains.
The naturally occurring nucleotide 3'-deoxy-3',4'-didehydro-cytidine-5'-triphosphate (ddhCTP) was recently found to exert potent and broad-spectrum antiviral activity. However, nucleoside 5'-triphosphates in general are not cell-permeable, which precludes the direct use of ddhCTP as a therapeutic. To harness the therapeutic potential of this endogenous antiviral nucleotide, we synthesized phosphoramidate prodrug HLB-0532247 (1) and found it to result in dramatically elevated levels of ddhCTP in cells. We compared 1 and 3'-deoxy-3',4'-didehydro-cytidine (ddhC) and found that 1 more effectively reduces titers of Zika and West Nile viruses in cell culture with minimal nonspecific toxicity to host cells. We conclude that 1 is a promising antiviral agent based on a novel strategy of facilitating elevated levels of the endogenous ddhCTP antiviral nucleotide.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the novel viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV) potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 μM). Weaker activity is observed in Vero E6 cells (EC50 = 1.65 μM) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminishes lung viral load and improves pulmonary function compared to vehicle treated animals. These data demonstrate that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19.
Background
Remdesivir is an RNA polymerase inhibitor with potent antiviral activity in vitro and efficacy in animal models of coronavirus disease 2019 (Covid-19).
Methods
We conducted a randomized, open-label, phase 3 trial involving hospitalized patients with confirmed SARS-CoV-2 infection, oxygen saturation of 94% or less while they were breathing ambient air, and radiologic evidence of pneumonia. Patients were randomly assigned in a 1:1 ratio to receive intravenous remdesivir for either 5 days or 10 days. All patients received 200 mg of remdesivir on day 1 and 100 mg once daily on subsequent days. The primary end point was clinical status on day 14, assessed on a 7-point ordinal scale.
Results
In total, 397 patients underwent randomization and began treatment (200 patients for 5 days and 197 for 10 days). The median duration of treatment was 5 days (interquartile range, 5 to 5) in the 5-day group and 9 days (interquartile range, 5 to 10) in the 10-day group. At baseline, patients randomly assigned to the 10-day group had significantly worse clinical status than those assigned to the 5-day group (P=0.02). By day 14, a clinical improvement of 2 points or more on the ordinal scale occurred in 64% of patients in the 5-day group and in 54% in the 10-day group. After adjustment for baseline clinical status, patients in the 10-day group had a distribution in clinical status at day 14 that was similar to that among patients in the 5-day group (P=0.14). The most common adverse events were nausea (9% of patients), worsening respiratory failure (8%), elevated alanine aminotransferase level (7%), and constipation (7%).
Conclusions
In patients with severe Covid-19 not requiring mechanical ventilation, our trial did not show a significant difference between a 5-day course and a 10-day course of remdesivir. With no placebo control, however, the magnitude of benefit cannot be determined. (Funded by Gilead Sciences; GS-US-540-5773 ClinicalTrials.gov number, NCT04292899.)
PurposeNUC1031 is a first-in-class ProTide, that is a gemcitabine pro-drug designed to overcome putative mechanisms of resistance, including decreased expression of hENT/hCNT transporters, absence of activating enzymes such as deoxycytidine kinase (dCK) and presence of degrading enzymes such as cytidine deaminase (CDA). We undertook comprehensive pre-clinical evaluation of NUC1031 in biliary tract cancer (BTC) models, given that gemcitabine/cisplatin is a standard first-line therapy in advanced BTC.Methods
Here, we compared the in vitro activity of NUC1031 in comparison to gemcitabine, validate putative mechanism(s) of action, assessed potential biomarkers of sensitivity or resistance, and performed combination studies with cisplatin. We also evaluated the in vivo efficacy of NUC1031 and gemcitabine using a CDA-high cholangiocarcinoma patient-derived xenograft (PDX) model.ResultsIn a panel of BTC cell lines (N = 10), NUC1031 had less potency than gemcitabine in multiple cellular assays. NUC1031 did not demonstrate evidence of greater synergy over gemcitabine in combination with cisplatin. Surprisingly, efficacy of both gemcitabine and NUC1031 was not found to be correlated with hENT/hCTN, dCK or CDA transcript levels. Gemcitabine and NUC1031 showed equivalent efficacy in a CDA-high PDX model in vivo contradicting the primary rationale of NUC1031 design.ConclusionNUC1031 did not exhibit evidence of superior activity over gemcitabine, as a single-agent, or in combination with cisplatin, in either our in vivo or in vitro BTC models. Given that the largest Phase 3 study (ClinicalTrials.gov: NCT0314666) to date in BTC is underway (N = 828) comparing NUC1031/cisplatin to gemcitabine/cisplatin, our results suggest that a more conservative clinical evaluation path would be more appropriate.
The specific targeting of protein‐protein interactions by phosphoserine‐containing small molecules has been scarce due to the dephosphorylation of phosphoserine and its charged nature at physiological pH, which hinder its uptake into cells. To address these issues, we herein report the synthesis of phosphoserine aryloxy triester phosphoramidates as phosphoserine prodrugs that are enzymatically metabolized to release phosphoserine. This phosphoserine‐masking approach was applied to a phosphoserine‐containing inhibitor of 14‐3‐3 dimerization, and the generated prodrugs exhibited improved pharmacological activity. Collectively, this provided a proof of concept that the masking of phosphoserine with biocleavable aryloxy triester phosphoramidate masking groups is a viable intracellular delivery system for phosphoserine‐containing molecules. Ultimately, this will facilitate the discovery of phosphoserine‐containing small‐molecule therapeutics.
The continued emergence of Middle East Respiratory Syndrome (MERS) cases with a high case fatality rate stresses the need for the availability of effective antiviral treatments. Remdesivir (GS-5734) effectively inhibited MERS coronavirus (MERS-CoV) replication in vitro, and showed efficacy against Severe Acute Respiratory Syndrome (SARS)-CoV in a mouse model. Here, we tested the efficacy of prophylactic and therapeutic remdesivir treatment in a nonhuman primate model of MERS-CoV infection, the rhesus macaque. Prophylactic remdesivir treatment initiated 24 h prior to inoculation completely prevented MERS-CoV−induced clinical disease, strongly inhibited MERS-CoV replication in respiratory tissues, and prevented the formation of lung lesions. Therapeutic remdesivir treatment initiated 12 h postinoculation also provided a clear clinical benefit, with a reduction in clinical signs, reduced virus replication in the lungs, and decreased presence and severity of lung lesions. The data presented here support testing of the efficacy of remdesivir treatment in the context of a MERS clinical trial. It may also be considered for a wider range of coronaviruses, including the currently emerging novel coronavirus 2019-nCoV.
Phosphoramidate pro-nucleotides (ProTides) have revolutionized the field of anti-viral and anti-cancer nucleoside therapy, overcoming the major limitations of nucleoside therapies and achieving clinical and commercial success. Despite the translation of ProTide technology into the clinic, there remain unresolved in vivo pharmacokinetic and pharmacodynamic questions. Positron Emission Tomography (PET) imaging using [18F]-labelled model ProTides could directly address key mechanistic questions and predict response to ProTide therapy. Here we report the first radiochemical synthesis of [18F]ProTides as novel probes for PET imaging. As a proof of concept, two chemically distinct radiolabelled ProTides have been synthesized as models of 3′- and 2′-fluorinated ProTides following different radiosynthetic approaches. The 3′-[18F]FLT ProTide was obtained via a late stage [18F]fluorination in radiochemical yields (RCY) of 15–30% (n = 5, decay-corrected from end of bombardment (EoB)), with high radiochemical purities (97%) and molar activities of 56 GBq/μmol (total synthesis time of 130 min.). The 2′-[18F]FIAU ProTide was obtained via an early stage [18F]fluorination approach with an RCY of 1–5% (n = 7, decay-corrected from EoB), with high radiochemical purities (98%) and molar activities of 53 GBq/μmol (total synthesis time of 240 min).
Background:
Although several experimental therapeutics for Ebola virus disease (EVD) have been developed, the safety and efficacy of the most promising therapies need to be assessed in the context of a randomized, controlled trial.
Methods:
We conducted a trial of four investigational therapies for EVD in the Democratic Republic of Congo, where an outbreak began in August 2018. Patients of any age who had a positive result for Ebola virus RNA on reverse-transcriptase-polymerase-chain-reaction assay were enrolled. All patients received standard care and were randomly assigned in a 1:1:1:1 ratio to intravenous administration of the triple monoclonal antibody ZMapp (the control group), the antiviral agent remdesivir, the single monoclonal antibody MAb114, or the triple monoclonal antibody REGN-EB3. The REGN-EB3 group was added in a later version of the protocol, so data from these patients were compared with those of patients in the ZMapp group who were enrolled at or after the time the REGN-EB3 group was added (the ZMapp subgroup). The primary end point was death at 28 days.
Results:
A total of 681 patients were enrolled from November 20, 2018, to August 9, 2019, at which time the data and safety monitoring board recommended that patients be assigned only to the MAb114 and REGN-EB3 groups for the remainder of the trial; the recommendation was based on the results of an interim analysis that showed superiority of these groups to ZMapp and remdesivir with respect to mortality. At 28 days, death had occurred in 61 of 174 patients (35.1%) in the MAb114 group, as compared with 84 of 169 (49.7%) in the ZMapp group (P = 0.007), and in 52 of 155 (33.5%) in the REGN-EB3 group, as compared with 79 of 154 (51.3%) in the ZMapp subgroup (P = 0.002). A shorter duration of symptoms before admission and lower baseline values for viral load and for serum creatinine and aminotransferase levels each correlated with improved survival. Four serious adverse events were judged to be potentially related to the trial drugs.
Conclusions:
Both MAb114 and REGN-EB3 were superior to ZMapp in reducing mortality from EVD. Scientifically and ethically sound clinical research can be conducted during disease outbreaks and can help inform the outbreak response. (Funded by the National Institute of Allergy and Infectious Diseases and others; PALM ClinicalTrials.gov number, NCT03719586.).
A microwave enhanced synthesis of prodrug nucleotide (ProTide) analogues is presented.
The rapid increase in antibiotic-resistant bacteria has emphasized the urgent need to identify new treatments for bacterial infections. One attractive approach, reducing the need for expensive and time-consuming clinical trials, is to repurpose existing clinically approved compounds for use as antibacterial agents. Nucleoside analogues are commonly used for treating viral and fungal infections, as well as for treating cancers, but have received relatively little attention as treatments for bacterial infections. However, a significant number of clinically approved derivatives of both pyrimidines and purines including halogenated, thiolated, and azolated compounds have been shown to have antibacterial activity. In the small number of studies carried out to date, such compounds have shown promise in treating bacterial infections. Here, we review the mechanisms of action and antibacterial activities of nucleoside analogues that can potentially be repurposed for treating infections as well as considering possible limitations in their usage.
Background:
Gemcitabine is used to treat a wide range of tumours, but its efficacy is limited by cancer cell resistance mechanisms. NUC-1031, a phosphoramidate modification of gemcitabine, is the first anti-cancer ProTide to enter the clinic and is designed to overcome these key resistance mechanisms.
Methods:
Sixty-eight patients with advanced solid tumours who had relapsed after treatment with standard therapy were recruited to a dose escalation study to determine the recommended Phase II dose (RP2D) and assess the safety of NUC-1031. Pharmacokinetics and anti-tumour activity was also assessed.
Results:
Sixty-eight patients received treatment, 50% of whom had prior exposure to gemcitabine. NUC-1031 was well tolerated with the most common Grade 3/4 adverse events of neutropaenia, lymphopaenia and fatigue occurring in 13 patients each (19%). In 49 response-evaluable patients, 5 (10%) achieved a partial response and 33 (67%) had stable disease, resulting in a 78% disease control rate. Cmax levels of the active intracellular metabolite, dFdCTP, were 217-times greater than those reported for equimolar doses of gemcitabine, with minimal toxic metabolite accumulation. The RP2D was determined as 825 mg/m2 on days 1, 8 and 15 of a 28-day cycle.
Conclusions:
NUC-1031 was well tolerated and demonstrated clinically significant anti-tumour activity, even in patients with prior gemcitabine exposure and in cancers not traditionally perceived as gemcitabine-responsive.
Small organophosphorus compounds stimulate Vγ9Vδ2 T cells if they serve as ligands of butyrophilin 3A1. Because the most potent natural ligand is (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), which is the last intermediate in bacterial biosynthesis of isoprenoids that is not found in mammalian metabolism, activation of these T cells represents an important component of the immune response to bacterial infections. To identify butyrophilin ligands that may have greater plasma stability, and clinical potential, we have prepared a set of aryl phosphonamidate derivatives (9a-i) of the natural ligand. Testing of these new compounds in assays of T cell response has revealed that this strategy can provide compounds with high potency for expansion of Vγ9Vδ2 T cells (9f, EC50 = 340 pM) and interferon γ production in response to loaded K562 cells (9e, EC50 = 62 nM). Importantly, all compounds of this class display extended plasma stability (t1/2 > 24 h). These findings increase our understanding of metabolism of butyrophilin ligands and the structure-activity relationships of phosphonamidate prodrugs.
The aryloxy triester phosphoramidate prodrug approach has been used with success in drug discovery. Herein, we describe the first application of this prodrug technology to the monophosphate derivative of the phosphoantigen HMBPP and one of its analogues. Some of these prodrugs exhibited specific and potent activation of Vγ9/Vδ2 T-cells, which were then able to lyse bladder cancer cells in vitro. This work highlights the promise of this prodrug technology in the discovery of novel immunotherapeutics.
Emerging viral infections are difficult to control because heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. Severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) successively emerged, causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe. Broad-spectrum therapies capable of inhibiting CoV infections would address an immediate unmet medical need and could be invaluable in the treatment of emerging and endemic CoV infections. We show that a nucleotide prodrug, GS-5734, currently in clinical development for treatment of Ebola virus disease, can inhibit SARS-CoV and MERS-CoV replication in multiple in vitro systems, including primary human airway epithelial cell cultures with submicromolar IC50 values. GS-5734 was also effective against bat CoVs, prepandemic bat CoVs, and circulating contemporary human CoV in primary human lung cells, thus demonstrating broad-spectrum anti-CoV activity. In a mouse model of SARS-CoV pathogenesis, prophylactic and early therapeutic administration of GS-5734 significantly reduced lung viral load and improved clinical signs of disease as well as respiratory function. These data provide substantive evidence that GS-5734 may prove effective against endemic MERS-CoV in the Middle East, circulating human CoV, and, possibly most importantly, emerging CoV of the future.
A synthetic procedure for the preparation of phosphoramidate prodrugs of C-nucleosides is reported. Different phosphorochloridates were reacted with 3′-O-protected N-acetyl-2′-deoxypseudoisocytidine or 3′-O-protected 2′-deoxypseudoisocytidine, followed by acidic hydrolysis of the protecting group. In the presence of the N-acetyl moiety, the enolisable keto group of the nucleobase was able to react (like the 5′-OH) with the phosphorochloridates to give bisphosphorylated derivatives. Epimerisation (β to α) occurred if the amino group of the nucleobase was unprotected. These side reactions demonstrate the peculiar behaviour of C-nucleosides compared to their nucleoside analogues. It was demonstrated that the first enzymatic activation step for this new class of prodrugs can be mediated by carboxypeptidase and that it follows the same pathway and rate reported for ProTides of more conventional nucleoside analogues. These new phosphoramidate derivatives deserve further investigation for their therapeutic potential as anti-cancer agents.
Nucleosides represent a major chemotherapeutic class for treating cancer, however their limitations in terms of cellular uptake, nucleoside kinase-mediated activation and catabolism are well-documented. The monophosphate pro-nucleotides known as ProTides represents a powerful strategy for bypassing the dependence on active transport and nucleoside kinase-mediated activation. Herein, we report the structural tuning of BVdU ProTides. Forty six phosphoramidates were prepared and biologically evaluated against three different cancer cell lines; murine leukemia (L1210), human CD4⁺ T-lymphocyte (CEM) and human cervical carcinoma (HeLa). Twenty-fold potency enhancement compared to BVdU was achieved against L1210 cells. Interestingly, a number of ProTides showed low micromolar activity against CEM and HeLa cells compared to the inactive parent BVdU. The ProTides showed poor, if any measurable toxicity to non-tumourigenic human lung fibroblast cell cultures. Separation of four pairs of the diastereoisomeric mixtures and comparison of their spectral properties, biological activities and enzymatic activation rate is reported.
The present invention is directed to novel compounds that are useful in the treatment of Niemann-Pick diseases, particularly Niemann-Pick type C disease as well other lysosomal storage disorders, particularly sphingolipidoses; endocytic transport abnormalities, defective autophagy, accumulation of free cholesterol, elevated levels of glycosphingolipids, and mycobacterial infection, such as BCG and TB. In particular, the invention relates to phosphoramidates, phosphorodiamidates and other phosphorus derivatives of fingolimod and related S1P receptor modulators. The invention also relates to methods for preparing the compounds and to pharmaceutical compositions containing them.
Loss‐of‐function mutations in the deoxyguanosine kinase (DGUOK) gene result in a mitochondrial DNA (mtDNA) depletion syndrome. DGUOK plays an important role in converting deoxyribonucleosides to deoxyribonucleoside monophosphates via the salvage pathway for mtDNA synthesis. DGUOK deficiency manifests predominantly in the liver; the most common cause of death is liver failure within the first year of life and no therapeutic options are currently available. In vitro supplementation with deoxyguanosine or deoxyguanosine monophosphate (dGMP) were reported to rescue mtDNA depletion in DGUOK‐deficient, patient‐derived fibroblasts and myoblasts. CERC‐913, a novel ProTide prodrug of dGMP, was designed to bypass defective DGUOK while improving permeability and stability relative to nucleoside monophosphates. To evaluate CERC‐913 for its ability to rescue mtDNA depletion, we developed a primary hepatocyte culture model using liver tissue from DGUOK‐deficient rats. DGUOK knockout rat hepatocyte cultures exhibit severely reduced mtDNA copy number (~10%) relative to wild type by qPCR and mtDNA content remains stable for up to eight days in culture. CERC‐913 increased mtDNA content in DGUOK‐deficient hepatocytes up to 2.4‐fold after 4 days of treatment in a dose‐dependent fashion, which was significantly more effective than dGMP at similar concentrations. These early results suggest primary hepatocyte culture is a useful model for the study of mtDNA depletion syndromes and that CERC‐913 treatment can improve mtDNA content in this model.
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The membrane permeability of nucleotide-based drugs, such as sofosbuvir (Sovaldi), requires installation of phosphate-caging groups. One strategy, termed "ProTide", masks the anionic phosphate through an N-linked amino ester and an O-linked aromatic phospho-ester, such that release of the active drug requires consecutive enzymatic liberation by an esterase and then a phosphoramidase, such as Hint1. Because Hint1 is known to be selective for nucleotides, it was not clear if the ProTide approach could be deployed for non-nucleotides. Here, we demonstrate that caging of a phosphate-containing inhibitor of the prolyl isomerase Pin1 increases its permeability. Moreover, this compound was processed by both esterase and phosphoramidase activity, releasing the active molecule to bind and inhibit Pin1 in cells. Thus, Hint1 appears to recognize a broader set of substrates than previously appreciated. It seems possible that other potent, but impermeable, phosphate-containing inhibitors might likewise benefit from this approach.
Background
Although several therapeutic agents have been evaluated for the treatment of coronavirus disease 2019 (Covid-19), none have yet been shown to be efficacious.
Methods
We conducted a double-blind, randomized, placebo-controlled trial of intravenous remdesivir in adults hospitalized with Covid-19 with evidence of lower respiratory tract involvement. Patients were randomly assigned to receive either remdesivir (200 mg loading dose on day 1, followed by 100 mg daily for up to 9 additional days) or placebo for up to 10 days. The primary outcome was the time to recovery, defined by either discharge from the hospital or hospitalization for infection-control purposes only.
Results
A total of 1063 patients underwent randomization. The data and safety monitoring board recommended early unblinding of the results on the basis of findings from an analysis that showed shortened time to recovery in the remdesivir group. Preliminary results from the 1059 patients (538 assigned to remdesivir and 521 to placebo) with data available after randomization indicated that those who received remdesivir had a median recovery time of 11 days (95% confidence interval [CI], 9 to 12), as compared with 15 days (95% CI, 13 to 19) in those who received placebo (rate ratio for recovery, 1.32; 95% CI, 1.12 to 1.55; P<0.001). The Kaplan-Meier estimates of mortality by 14 days were 7.1% with remdesivir and 11.9% with placebo (hazard ratio for death, 0.70; 95% CI, 0.47 to 1.04). Serious adverse events were reported for 114 of the 541 patients in the remdesivir group who underwent randomization (21.1%) and 141 of the 522 patients in the placebo group who underwent randomization (27.0%).
Conclusions
Remdesivir was superior to placebo in shortening the time to recovery in adults hospitalized with Covid-19 and evidence of lower respiratory tract infection. (Funded by the National Institute of Allergy and Infectious Diseases and others; ACCT-1 ClinicalTrials.gov number, NCT04280705.)
Gemcitabine/cisplatin is standard of care for first-line treatment of patients with advanced biliary tract cancer (aBTC); new treatments are needed. NUC-1031 is designed to overcome key cancer resistance mechanisms associated with gemcitabine. The tolerability/efficacy signal of NUC-1031/cisplatin in the Phase Ib ABC-08 study suggested that this combination may represent a more efficacious therapy than gemcitabine/cisplatin for patients with aBTC, leading to initiation of the global NuTide:121 study which will include 828 patients ≥18 years with untreated histologically/cytologically-confirmed aBTC (including cholangiocarcinoma, gallbladder or ampullary cancer); randomized (1:1) to NUC-1031 (725 mg/m ² )/cisplatin (25 mg/m ² ) or gemcitabine (1000 mg/m ² )/cisplatin (25 mg/m ² ), on days 1/8, Q21-days. Primary objectives are overall survival and objective response rate. Secondary objectives: progression-free survival, safety, pharmacokinetics, patient-reported quality of life and correlative studies.
(Investigational new drug (IND) number: 139058, European Clinical Trials database: EudraCT Number 2019-001025-28, ClinicalTrials.gov identifier: NCT04163900).
Background
No specific antiviral drug has been proven effective for treatment of patients with severe coronavirus disease 2019 (COVID-19). Remdesivir (GS-5734), a nucleoside analogue prodrug, has inhibitory effects on pathogenic animal and human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro, and inhibits Middle East respiratory syndrome coronavirus, SARS-CoV-1, and SARS-CoV-2 replication in animal models.
Methods
We did a randomised, double-blind, placebo-controlled, multicentre trial at ten hospitals in Hubei, China. Eligible patients were adults (aged ≥18 years) admitted to hospital with laboratory-confirmed SARS-CoV-2 infection, with an interval from symptom onset to enrolment of 12 days or less, oxygen saturation of 94% or less on room air or a ratio of arterial oxygen partial pressure to fractional inspired oxygen of 300 mm Hg or less, and radiologically confirmed pneumonia. Patients were randomly assigned in a 2:1 ratio to intravenous remdesivir (200 mg on day 1 followed by 100 mg on days 2–10 in single daily infusions) or the same volume of placebo infusions for 10 days. Patients were permitted concomitant use of lopinavir–ritonavir, interferons, and corticosteroids. The primary endpoint was time to clinical improvement up to day 28, defined as the time (in days) from randomisation to the point of a decline of two levels on a six-point ordinal scale of clinical status (from 1=discharged to 6=death) or discharged alive from hospital, whichever came first. Primary analysis was done in the intention-to-treat (ITT) population and safety analysis was done in all patients who started their assigned treatment. This trial is registered with ClinicalTrials.gov, NCT04257656.
Findings
Between Feb 6, 2020, and March 12, 2020, 237 patients were enrolled and randomly assigned to a treatment group (158 to remdesivir and 79 to placebo); one patient in the placebo group who withdrew after randomisation was not included in the ITT population. Remdesivir use was not associated with a difference in time to clinical improvement (hazard ratio 1·23 [95% CI 0·87–1·75]). Although not statistically significant, patients receiving remdesivir had a numerically faster time to clinical improvement than those receiving placebo among patients with symptom duration of 10 days or less (hazard ratio 1·52 [0·95–2·43]). Adverse events were reported in 102 (66%) of 155 remdesivir recipients versus 50 (64%) of 78 placebo recipients. Remdesivir was stopped early because of adverse events in 18 (12%) patients versus four (5%) patients who stopped placebo early.
Interpretation
In this study of adult patients admitted to hospital for severe COVID-19, remdesivir was not associated with statistically significant clinical benefits. However, the numerical reduction in time to clinical improvement in those treated earlier requires confirmation in larger studies.
Funding
Chinese Academy of Medical Sciences Emergency Project of COVID-19, National Key Research and Development Program of China, the Beijing Science and Technology Project.
ProTides comprise an important class of prodrugs currently marketed and developed as antiviral and anticancer therapies. The ProTide technology employs a phosphate masking groups capable of providing more favourable drug-like properties and an intracellular activation mechanism for enzyme-mediated release of a nucleoside monophosphate. Herein we describe the application of phosphoramidate chemistry to 1,3,4-O-acetylated N-acetylmannosamine (Ac3ManNAc) to deliver ManNAc-6-phosphate (ManNAc-6-P), a critical intermediate in sialic acid biosynthesis. Sialic acid deficiency is a hallmark of GNE myopathy, a rare congenital disorder of glycosylation (CDG) caused by mutations in GNE that limit the production of ManNAc-6-P. Synthetic methods were developed to provide a library of Ac3ManNAc-6-phosphoramidates that were evaluated in a series of studies for their potential as a treatment for GNE myopathy. Prodrug 13b showed rapid activation in a carboxylesterase (CPY) enzymatic assay and favourable ADME properties, while also being more effective at increasing sialic acid levels in GNE-deficient cell lines than ManNAc. These results provide a potential platform to address substrate deficiencies in GNE myopathy and other CDGs.
Aryl phosphoramidate prodrugs of fosfoxacin derivatives 15a-b and 8a-b were synthesized and investigated for their ability to target bacteria. No growth inhibition was observed neither for Mycobacterium smegmatis nor for Escherichia coli on solid medium, demonstrating the absence of release of the active compounds in the bacterial cells. Investigation of the stability of the prodrugs and their multienzymatic cleavage in abiotic and biotic conditions showed that the use of aryl phosphoramidate prodrug approach to deliver non-nucleotides compounds is not obvious and might not be appropriate for an antimicrobial drug.
Congenital disorders of glycosylation are a genetically and clinically heterogeneous group of >130 diseases caused by defects in various steps along glycan modification pathways. The vast majority of these monogenic diseases are autosomal recessive and have multi-systemic manifestations, mainly growth failure, developmental delay, facial dysmorphisms, and variable coagulation and endocrine abnormalities. Carbohydrate deficient transferrin (CDT) and protein-linked glycan analysis with mass spectrometry can diagnose some subtypes of congenital disorders of glycosylation (CDG), while many currently rely on massively parallel genomic sequencing for diagnosis. Early detection is important, as a few of these disorders are treatable. Molecular and biochemical techniques continue to further our understanding of this rapidly expanding group of clinically and genetically diverse disorders.
FULL ARTICLE could be read here: https://pubs.acs.org/doi/pdf/10.1021/acsmedchemlett.8b00586
Abstract:
The ProTide prodrug technology has proved very useful in the discovery of nucleotide therapeutics and has successfully led to two FDA-approved drugs. However, with the extensive application of this prodrug approach to nucleotides for nearly three decades, the intellectual property (IP) landscape is becoming congested and, to overcome this, new inventive applications of the ProTide prodrug technology are emerging.
Nucleotide prodrugs (ProTides) based on phosphate or phosphonate compounds are potent and successfully marketed antiviral drugs. Although their biological properties are well explored, experimental evidence on the mechanism of their activation pathway is still missing. In this study, we synthesized two ProTide analogues, which can be activated by UV light. Using 31P and 13C NMR spectroscopy with in situ irradiation, we followed the ProTide activation pathway in various solvents, and we detected the first proposed intermediate and the monoamidate product. Furthermore, we used mass spectrometry (MS) coupled with infrared spectroscopy in the gas phase to detect and to characterize the elusive cyclic pentavalent phosphorane and cyclic acyl phosphoramidate intermediates. Our combined NMR and MS data provided the first experimental evidence of the cyclic intermediates in the activation pathway of ProTide prodrugs.
Unmasked phohate groups of phosphotyrosine-containing molecules carry two negative charges at physiological pH, which compromise their (passive) celular uptake. Also, these phosphate groups are often cleaved off by phosphatases. Together, these ultimately limit the pharmacological efficacy of the phosphotyrosine-containing compounds. To address these drawbacks, we herein present the application of the aryloxy triester phosphoramidate prodrug technology, a monophosphate prodrug technology, to the phosphotyrosine-containing compound ISS-610-Met, an analogue of the anticancer STAT3 dimerization inhibitor ISS-610. Our data shows that the generated ISS-610-Met prodrugs exhibited enhanced pharmacological activity and inhibition of STAT3 downstream signaling compared to the parent compound ISS-610-Met and the known STAT3 dimerization inhibitor ISS-610. These encouraging results provide a compelling proof of concept for the potential of the aryloxy triester phosphoramidate prodrug technology in the discovery of novel therapeutics that contain phosphotyrosine and its phospho mimics.
This synthetic protocol describes two strategies for the preparation of pyrimidine alkenyl acyclic nucleoside phosphonoamidates (ANPs), including linear and trisubstituted alkenyl derivatives. For the first procedure, a bis‐trimethylsilyl ester of the parent alkenyl ANPs is the key intermediate that reacts with the desired amino acid ester and aryl alcohol. For the second procedure, an allyl phosphonoamidate bearing the ProTide promoieties is the key synthon employed as olefin partner for a cross‐metathesis reaction with an alkylated nucleobase.
Prodrugs are molecules with little or no pharmacological activity that are converted to the active parent drug in vivo by enzymatic or chemical reactions or by a combination of the two. Prodrugs have evolved from being serendipitously discovered or used as a salvage effort to being intentionally designed. Such efforts can avoid drug development challenges that limit formulation options or result in unacceptable biopharmaceutical or pharmacokinetic performance, or poor targeting. In the past 10 years, the US Food and Drug Administration has approved at least 30 prodrugs, which accounts for more than 12% of all approved small-molecule new chemical entities. In this Review, we highlight prodrug design strategies for improved formulation and pharmacokinetic and targeting properties, with a focus on the most recently marketed prodrugs. We also discuss preclinical and clinical challenges and considerations in prodrug design and development.
Several hitherto unknown (E)-but-2-enyl nucleoside phosphonoamidate analogs (ANPs) were prepared directed with nitrogen reagents by cross-metathesis in water-under ultrasound irradiation. Two diastereoisomers were formally identified by X-ray diffraction. These compounds were evaluated against a large spectrum of DNA and RNA viruses. Among them, the phosphonoamidate thymine analogue 19 emerged as the best prodrug against varicella-zoster virus (VZV) with EC50 values of 0.33 and 0.39 μM for wild-type and thymidine kinase deficient strains, respectively, and a selectivity index ≥200 μM. This breakthrough approach paves the way for new purine and pyrimidine (E)-but-2-enyl phosphonoamidate analogs.
Prodrugs are substances administered in an inactive form that is then metabolized in the body in vivo into the active compound. The rationale behind administering prodrugs is to optimize absorption, distribution, metabolism, and excretion of these drugs. Since first described in the 1950s, prodrugs continue to be a fertile area of research. There are a number of small pharmaceutical/biotech companies dedicated to using prodrugs for the delivery of older but problematic drugs as well as to developing broad-based prodrug technologies for application to new and future drugs. These volumes represent a comprehensive guide to prodrugs and will guide the reader through the current status of the prodrug concept and its many applications and to highlight its many successes in overcoming formulation and delivery of problematic drugs.
A new family of thirteen phosphoramidate prodrugs (ProTides) of different 6-substituted-5-fluorouridine nucleoside analogues were synthesized and evaluated as potential anticancer agents. In addition, antiviral activity against Chikungunya (CHIKV) virus was evaluated using a cytopathic effect inhibition assay. Although a carboxypeptidase Y assay supported a putative mechanism of activation of ProTides built on 5-fluorouridine with such C6-modifications, the Hint docking studies revealed a compromised substrate-activity for the Hint phosphoramidase-type enzyme that is likely responsible for phosphoramidate bioactivation through P-N bond cleavage and free nucleoside 5'-monophosphate delivery. Our observations may support and explain to some extent the poor in vitro biological activity generally demonstrated by the series of 6-substituted-5-fluorouridine phosphoramidates (ProTides) and will be of guidance for the design of novel phosphoramidate prodrugs.
Background:
New hepatitis C virus (HCV) therapies with pan-genotypic efficacy are needed. The goals of part A of C-CREST-1 and C-CREST-2 were to compare the efficacies of two doses (300 mg or 450 mg once daily) of uprifosbuvir (MK-3682; NS5B inhibitor) in an 8-week regimen combined with grazoprevir (NS3/4A inhibitor; 100 mg once daily) and an NS5A inhibitor, either elbasvir (50 mg once daily) or ruzasvir (MK-8408; 60 mg once daily), and to evaluate the safety and tolerability of these combination regimens in individuals infected with genotypes 1, 2, or 3.
Methods:
Part A of these phase 2, randomised, multicentre, open-label, clinical trials enrolled participants from 11 countries, aged 18 years or older, chronically infected with HCV genotypes 1, 2, or 3, with HCV RNA of at least 10 000 IU/mL, without evidence of cirrhosis, who had not received previous treatment for HCV infection. Within each HCV genotype, participants were randomly assigned (1:1:1:1) with a block size of 4, to open-label treatment to one of four treatment groups: grazoprevir (100 mg/day) plus ruzasvir (60 mg/day) plus uprifosbuvir (300 mg/day); grazoprevir (100 mg/day) plus ruzasvir (60 mg/day) plus uprifosbuvir (450 mg/day); grazoprevir (100 mg/day) plus elbasvir (50 mg/day) plus uprifosbuvir (300 mg/day); or grazoprevir (100 mg/day) plus elbasvir (50 mg/day) plus uprifosbuvir (450 mg/day), according to a computer-generated allocation schedule. Randomisation was centrally implemented using an interactive voice response system and integrated web response system. The primary endpoint was the proportion of participants achieving sustained virological response at 12 weeks (SVR12; HCV RNA less than the lower limit of quantitation at 12 weeks after the end of all study therapy) in the per-protocol analysis set, which included all participants who were randomised and received at least one dose of study drug. The trials are registered with ClinicalTrials.gov, numbers NCT02332707 and NCT02332720.
Findings:
241 participants were randomised between Feb 18, 2015, and March 16, 2015. 240 participants completed 8 weeks of treatment and reached follow-up 12 weeks after the end of treatment. Of the four regimens, grazoprevir plus ruzasvir plus uprifosbuvir 450 mg had the most consistently high SVR12 (>90%) for participants infected with genotype 1 (21 [91%] of 23), genotype 2 (15 [94%] of 16), and genotype 3 (20 [91%] of 22). In particular, among those with genotype 2 infection, the grazoprevir plus ruzasvir plus uprifosbuvir 450 mg regimen had a higher SVR12 (15 [94%] of 16) than the grazoprevir plus ruzasvir plus uprifosbuvir 300 mg regimen (ten [71%] of 14), grazoprevir plus elbasvir plus uprifosbuvir 300 mg regimen (11 [69%] of 16), or grazoprevir plus elbasvir plus uprifosbuvir 450 mg regimen (nine [60%] of 15). Overall, the most common adverse events were headache (55 [23%] of 240), fatigue (47 [20%] of 240), and nausea (32 [13%] of 240). Two (<1%) of 240 participants had serious adverse events (pharyngeal abscess and keratitis), which were not considered drug related by the respective investigators.
Interpretation:
These results support further evaluation of the three-drug direct-acting antiviral agent regimen of grazoprevir 100 mg plus ruzasvir 60 mg plus uprifosbuvir 450 mg among a more diverse HCV-infected population, including those with compensated cirrhosis, previous treatment with an interferon-containing regimen, and HCV-HIV co-infection.
Funding:
Merck & Co, Inc.
The ProTide technology is a prodrug approach developed for the efficient intracellular delivery of nucleoside analogue monophosphates and monophosphonates. In this approach, the hydroxyls of the monophosphate or monophosphonate groups are masked by an aromatic group and an amino acid ester moiety, which are enzymatically cleaved-off inside cells to release the free nucleoside monophosphate and monophosphonate species. Structurally, this represents the current end-point of an extensive medicinal chemistry endeavour that spans almost three decades. It started from the masking of nucleoside monophosphate and monophosphonate groups by simple alkyl groups and evolved into the sophisticated ProTide system as known today. This technology has been extensively employed in drug discovery and it has already led to the discovery of two FDA-approved (antiviral) ProTides. In this work, we will review the development of the ProTide technology, its application in drug discovery and its role in the improvement of drug delivery and efficacy.
There is an urgent need for new antibacterial drugs that are robust against the development of resistance. Drug repurposing is a cost-effective strategy to fast-track the drug development process. Here we examine why the nucleoside and nucleobase analogue drugs in particular present an attractive class for repurposing. Some of these drugs have already been evaluated for their potential as antibacterial agents. In addition to inhibiting bacterial growth and survival, some also act synergistically with antibiotics, and as such can enhance the therapeutic spectrum of currently available antibiotics. Furthermore, nucleoside and nucleobase analogue drugs can inhibit bacterial virulence and biofilm formation. Biofilms are known to impart antibiotic tolerance and are associated with chronic infections. Targeting biofilm formation thus renders pathogens more susceptible to antibiotic treatment and host immune defences. Moreover, specific analogues have properties that make them less susceptible to the development of resistance. Thus, nucleoside and nucleobase analogue drugs ought to be considered as new weapons in our fight against pathogenic bacteria.
Getting phosphorus into healthy shape
ProTide therapeutics play a trick on the body, getting nucleoside analogs where they need to be by decorating them with unnatural phosphoramidates in place of ordinary phosphates. These compounds pose an unusual synthetic challenge because their configuration must be controlled at phosphorus; most methods have been refined to manipulate the geometry of carbon. DiRocco et al. report a metal-free, small-molecule catalyst that attains high selectivity for nucleoside phosphoramidation by activating both reaction partners. Kinetic studies with an early prototype revealed a double role for the catalyst that inspired the rational design of a more active and selective dimeric structure.
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Acyclic nucleoside phosphonates (ANPs) are nowadays one of the key drugs in the treatment of DNA virus and retrovirus infections. In this work, we report the synthesis and antiviral evaluation of phosphonoamidate and diamidates prodrugs of C5-pyrimidine acyclic nucleosides derivatives functionalized with but-2-enyl- chain. In the phosphonoamidate series, the most active compound 15, showed sub-micromolar activity against (TK⁻)-VZV (EC50 = 0.09 μM) and μM activity against HCMV and herpes simplex virus (HSV). Separation of single diastereoisomers for compound 14, showed that 14b had better anti-herpesvirus activity and no cytotoxicity compared to the diastereoisomeric mixture 14. Very interestingly, phosphonodiamidate 21 showed anti-herpesvirus activity with excellent activity against wild-type and thymidine kinase-deficient (TK⁻) varicella-zoster virus (VZV) strains (EC50 = 0.47 and 0.2 μM, respectively) and human cytomegalovirus (HCMV) (EC50 = 3.5–7.2 μM) without any cytotoxicity (CC50 > 100).
A general and efficient method for the synthesis of pronucleotide (ProTide) 5′-phosphoramidate monoesters is reported. This method consists of a highly stereoselective 5′-phosphorylation mediated by dimethylaluminum chloride to afford the desired target ProTides in excellent yields without employing 3′-protection strategies. The application of this methodology to the synthesis of a number of pharmaceutically relevant compounds currently marketed or under investigation in clinical research is demonstrated.
Since loss of function mutations of PINK1 lead to early-onset Parkinson's disease, there has been growing interest in the discovery of small molecules that amplify the kinase activity of PINK1. We herein report the design, synthesis, serum stability and hydrolysis of four kinetin riboside ProTides. These ProTides, along with kinetin riboside, activated PINK1 in cells independent of mitochondrial depolarization. This highlights the potential of modified nucleosides and their phosphate prodrugs as treatments for neurodegenerative diseases.
Previously published S1P receptor modulator benzyl ether derivatives have shown potential as being viable therapeutics for the treatment of neurodegenerative diseases, however, two of the most S1P1-selective compounds are reported as being poorly phosphorylated by kinases in vivo. Phosphoramidates of BED compounds (2a, 2b) were synthesised with the aim of producing kinase-independent S1P receptor modulators. Carboxypeptidase, human serum and cell lysate processing experiments were conducted. ProTide BED analogues were found to have an acceptable level of stability in acidic and basic conditions and in vitro metabolic processing experiments showed that they are processed to the desired pharmacologically active monophosphate. The research describes the development of an entirely novel family of therapeutic agents.
The recent Ebola Virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50=86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1′-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a nonhuman primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3-14 post infection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [Nature 2016, 531, 381-385]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.
Novel antibiotics are urgently needed to combat the rise of infections due to drug-resistant microorganisms. Numerous natural nucleosides and their synthetically modified analogues have been reported to have moderate to good antibiotic activity against different bacterial and fungal strains. Nucleoside-based compounds target several crucial processes of bacterial and fungal cells such as nucleoside metabolism and cell wall, nucleic acid and protein biosynthesis. Nucleoside analogues have also been shown to target many other bacterial and fungal cellular processes though these are not well characterised and may therefore represent opportunities to discover new drugs with unique mechanisms of action. In this perspective we demonstrate that nucleoside analogues, cornerstones of anticancer and antiviral treatments, also have great potential to be repurposed as antibiotics so that an old drug can learn new tricks.
A series of novel aryloxyphosphoramidate nucleoside prodrugs based on l-aspartic acid and l-glutamic acid as amino acid motif has been synthesized and evaluated for antitumoral activity. Depending on the cancer cell line studied and on the nature of the parent nucleoside compound (gemcitabine, 5-iodo-2'-deoxy-uridine, floxuridine or brivudin), the corresponding ProTides are endowed with an improved or decreased cytotoxic activity.
The rationally designed novel anti-HIV drug candidate Stampidine exhibited (a) remarkable subnanomolar to low nanomolar in vitro ARV potency against genotypically and phenotypically NRTI-resistant primary clinical HIV isolates, non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 isolates, clinical non-B subtype HIV- 1 isolates (subtypes A, C, F, and G) originating from South America, Asia, and sub-Saharan Africa with resistance to stavudine, adefovir and tenofovir, as well as recombinant HIV clones containing common patterns of RT mutations responsible for NRTI resistance such as multiple TAMs plus M184V, multiple TAMs plus T69 insertion, and Q151 complex (b) favorable, safety profile in mice, rats, dogs, and cats, and (c) promising prophylactic in vivo anti-retroviral activity in Hu-PBL-SCID mice as well as therapeutic anti-retroviral activity in FIV-infected domestic cats. Notably, in a placebo-controlled Phase I study involving 30 therapy-naïve adult HIV-infected adult patients, formulated GMPgrade oral Stampidine capsules did not cause dose-limiting toxicity at single dose levels ranging from 5 to 25 mg/kg. Taken together, the presented favorable preclinical and early clinical safety/activity profile of Stampidine warrants its further development as a new anti-HIV drug candidate.
We herein report the first copper-catalysed diastereoselective synthesis of P-chiral phosphoramidate prodrugs (ProTides). This procedure allows the synthesis of diastereomeric-enriched mixtures of ProTides. Application of this methodology to the asymmetric phosphorylation of representative examples of biologically relevant purine and pyrimidine nucleoside analogues is presented.