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Synthesis and Evaluation of Aryl Quinolines as HIV-1 Integrase Multimerization Inhibitors
Abstract
HIV-1 integrase multimerization inhibitors have recently been established as an effective class of antiretroviral agents due to their potent ability to inhibit viral replication. Specifically, quinoline-based inhibitors have been shown to effectively impair HIV-1 replication, highlighting the importance of these heterocyclic scaffolds. Pursuant of our endeavors to further develop a library of quinoline-based candidates, we have implemented a structure activity relationship study of trisubstituted 4-arylquinoline scaffolds that examined the integrase multimerization properties of substitution patterns at the 4-position of the quinoline. Substrates consisting of substituted phenyl rings, heteroaromatics, or polycyclic moieties were examined utilizing an integrase abberant multimerization in vitro assay. Para-chloro-4-phenylquinoline 11b and 2,3-benzo[b][1,4]dioxine 15f showed noteworthy EC50 values of 0.10 μM and 0.08 µM, respectively.
... Allosteric HIV-1 Integrase Inhibitors (ALLINIs) [18][19][20][21][22], which are also known as LEDGINs (LEDGF/p75 Inhibitors) [23], NCINIs (Noncatalytic Site Integrase Inhibitors) [24], or INLAIs (IN-LEDGF Allosteric Inhibitors) [25], selectively bind at this LEDGF/p75 IBD binding pocket, away from the IN catalytic site and potently inhibit HIV-1 replication. Importantly, these compounds retain full potency against clinical strains resistant to the FDA-approved IN catalytic inhibitors [23]. ...
... At Boehringer Ingelheim, a similar series of quinolines ( Figure 3) with antiviral activities were identified through a high throughput screening (HTS) campaign targeting the 3P enzymatic activity of IN [26]. Testing of the initial hit compound 4 ( Figure 3) showed an in vitro inhibition of the 3P catalytic function of IN at 9.0 µM using a FRET-based assay Allosteric HIV-1 Integrase Inhibitors (ALLINIs) [18][19][20][21][22], which are also known as LEDGINs (LEDGF/p75 Inhibitors) [23], NCINIs (Noncatalytic Site Integrase Inhibitors) [24], or INLAIs (IN-LEDGF Allosteric Inhibitors) [25], selectively bind at this LEDGF/p75 IBD binding pocket, away from the IN catalytic site and potently inhibit HIV-1 replication. Importantly, these compounds retain full potency against clinical strains resistant to the FDA-approved IN catalytic inhibitors [23]. ...
... Substitutions added to position 4 of the quinoline scaffold aim to maximize interaction between the compound and the hydrophobic pocket defined by the residue pair W132/L102 of subunit 2 (yellow residues in Figure 5A). Thus, compounds with aromatic groups on position 4 are highly favored, with a preference for substituted phenyl groups [22]. Extensive exploration of the chemical space demonstrates that several single para substitutions of the phenyl group (Table 1) improve the compound's multimerization EC 50 s compared with the unsubstituted derivative 8. Substitutions with fluorine (compound 10) or fluorinecontaining groups such as trifluoromethyl and trifluoromethoxy (compounds 12 and 14) slightly decrease EC 50 s by 2-4 folds while substitution for either a methyl or a methoxy group (compounds 11 and 13) resulted in a six-fold improvement in the EC 50 values. ...
Allosteric HIV-1 Integrase (IN) Inhibitors or ALLINIs bind at the dimer interface of the IN, away from the enzymatic catalytic site, and disable viral replication by inducing over-multimerization of IN. Interestingly, these inhibitors are capable of impacting both the early and late stages of viral replication. To better understand the important binding features of multi-substituted quinoline-based ALLINIs, we have surveyed published studies on IN multimerization and antiviral properties of various substituted quinolines at the 4, 6, 7, and 8 positions. Here we show how the efficacy of these inhibitors can be modulated by the nature of the substitutions at those positions. These features not only improve the overall antiviral potencies of these compounds but also significantly shift the selectivity toward the viral maturation stage. Thus, to fully maximize the potency of ALLINIs, the interactions between the inhibitor and multiple IN subunits need to be simultaneously optimized.
... Another example of 3-hydroxypyridin-4-one synthesis was reported by Sirous et al., Scheme 10 [26]. 3-Hydroxy group of the commercially available maltol (42) was protected by the reaction with benzyl bromide and further reactions with benzyl-or phenetylamines gave 1-substituted pyridine-4-ones 43. Cleavage of the benzyl group was carried out using boron tribromide or a mixture of hydrochloric and acetic acids. ...
... This protocol was patented in 2014 [41]. [42]. The synthetic scheme is quite similar to the one mentioned above (Scheme 18), and it starts from commercially available 4-hydroxy-2-methyl-quinoline 80 that has undergone direct bromination and reaction with POCl3 to give intermediate 91 in 65% yield. ...
... Acylation with benzoyl chloride provided amides 101. [42]. The synthetic scheme is quite similar to the one mentioned above (Scheme 18), and it starts from commercially available 4-hydroxy-2-methyl-quinoline 80 that has undergone direct bromination and reaction with POCl3 to give intermediate 91 in 65% yield. ...
Human immunodeficiency virus (HIV) causes one of the most dangerous diseases—acquired immunodeficiency syndrome (AIDS). An estimated about 40 million people are currently living with HIV worldwide, most of whom are already on antiretroviral therapy. This makes the development of effective drugs to combat this virus very relevant. Currently, one of the dynamically developing areas of organic and medicinal chemistry is the synthesis and identification of new compounds capable of inhibiting HIV-1 integrase—one of the HIV enzymes. A significant number of studies on this topic are published annually. Many compounds inhibiting integrase incorporate pyridine core. Therefore, this review is an analysis of the literature on the methods for the synthesis of pyridine-containing HIV-1 integrase inhibitors since 2003 to the present.
... This binding pocket ( Figure 1) has become of significant interest as an IN alternative target option. Allosteric IN inhibitors (ALLINIs) [16][17][18][19][20], which are also known as LED-GINs (LEDGF/p75 Inhibitors) [21], NCINIs (Noncatalytic Site Integrase Inhibitors) [22], or While originally designed to block IN-LEDGF/p75 interactions, these ALLINIs have been shown to also impact IN functions by triggering an aberrant multimerization of the viral enzyme [16,22,24]. Additionally, several studies on the antiviral mode of action of ALLINIs have revealed that these compounds not only inhibit integration but also target the late stages of HIV-1 replication by yielding non-infectious virions. ...
... We and others have found that large aromatic substitutions at this site greatly enhance multimerization properties of the compounds by maximizing interaction with the hydrophobic pocket [20,33], including residues W132 and L102 ( Figure 1). We found that a para-chloro-4-phenylquinoline substitution (Figures 1 and 2) showed effective inhibition with an in vitro IN multimerization EC 50 of 100 nM due to the stabilizing Cl-π interaction between the aromatic side chain of W132 and the chlorobenzene group [20]. Our most potent compound from this series was the 2,3-benzo [1,4] dioxanyl group at the 4-position of the quinoline (Figure 2), which showed an in vitro IN multimerization EC 50 of 80 nM [20]. ...
... We found that a para-chloro-4-phenylquinoline substitution (Figures 1 and 2) showed effective inhibition with an in vitro IN multimerization EC 50 of 100 nM due to the stabilizing Cl-π interaction between the aromatic side chain of W132 and the chlorobenzene group [20]. Our most potent compound from this series was the 2,3-benzo [1,4] dioxanyl group at the 4-position of the quinoline (Figure 2), which showed an in vitro IN multimerization EC 50 of 80 nM [20]. This observation led to the hypothesis that larger, multicyclic structures have the capability to rotate within the pocket, allowing for optimal interactions. ...
Allosteric HIV-1 integrase (IN) inhibitors, or ALLINIs, are a new class of antiviral agents that bind at the dimer interface of the IN, away from the enzymatic catalytic site and block viral replication by triggering an aberrant multimerization of the viral enzyme. To further our understanding of the important binding features of multi-substituted quinoline-based ALLINIs, we have examined the IN multimerization and antiviral properties of substitution patterns at the 6 or 8 position. We found that the binding properties of these ALLINIs are negatively impacted by the presence of bulky substitutions at these positions. In addition, we have observed that the addition of bromine at either the 6 (6-bromo) or 8 (8-bromo) position conferred better antiviral properties. Finally, we found a significant loss of potency with the 6-bromo when tested with the ALLINI-resistant IN A128T mutant virus, while the 8-bromo analog retained full effectiveness.
... LEDGF/p75 also binds the IN tetramer through its C-terminal integrase binding domain by inserting a small loop into a vshaped cavity located at the IN CCD dimer interface (10,14). Allosteric IN inhibitors (ALLINIs) (15)(16)(17)(18)(19), which are also known as LEDGINs (LEDGF/p75 inhibitors) (20), noncatalytic site integrase inhibitors (21), or IN-LEDGF allosteric inhibitors (22), selectively bind at the IN CCD dimer interface at the LEDGF/p75 binding pocket, away from the IN catalytic site and potently inhibit HIV-1 replication in cell culture. While originally designed to block IN-LEDGF/p75 interactions, these compounds have been shown to also impact IN functions by inducing aberrant IN multimerization (15,21,23). ...
... For example, early works have shown that the 3-α-tert-butoxy acetic acid side-chain (quinoline scaffold position numbers in Fig. 2) is optimal for IN anchoring (15,16,20) because it provides several hydrogen bond interactions with T174 and H171 residues that mimic the LEDGF/p75 binding pattern. More recent derivatization efforts have focused on the position 4 (19,29). We and others have found that large aromatic substitutions at this site greatly enhance multimerization properties of the compounds by maximizing interaction with the hydrophobic pocket (19,29) including residues W132 and L102 (Fig. 2). ...
... More recent derivatization efforts have focused on the position 4 (19,29). We and others have found that large aromatic substitutions at this site greatly enhance multimerization properties of the compounds by maximizing interaction with the hydrophobic pocket (19,29) including residues W132 and L102 (Fig. 2). In the present work, to further understand and quantify the specific contribution of each feature of these inhibitors toward multimerization, we have kept constant the 3α-tert-butoxy acetic acid side-chain and our previously optimized position 4 with a 1,4-benzo dioxane group (19). ...
During the integration step, HIV-1 integrase (IN) interacts with viral DNA and the cellular cofactor LEDGF/p75 to effectively integrate the reverse transcript into the host chromatin. Allosteric HIV-1 integrase inhibitors (ALLINIs) are a new class of antiviral agents that bind at the dimer interface of the IN catalytic core domain and occupy the binding site of LEDGF/p75. While originally designed to block IN-LEDGF/p75 interactions during viral integration, several of these compounds have been shown to also severely impact viral maturation through an IN multimerization mechanism. In this study, we tested the hypothesis that these dual properties of ALLINIs could be decoupled toward late stage viral replication effects by generating additional contact points between the bound ALLINI and a third subunit of IN. By sequential derivatization at position 7 of a quinoline-based ALLINI scaffold, we show that IN multimerization properties are enhanced by optimizing hydrophobic interactions between the compound and the C-terminal domain of the third IN subunit. These features not only improve the overall anti-viral potencies of these compounds but also significantly shift the ALLINIs selectivity toward the viral maturation stage. Thus, we demonstrate that in order to fully maximize the potency of ALLINIs, the interactions between the inhibitor and all three IN subunits need to be simultaneously optimized.
... These factors contributed to inconsistent yields and reproducibility in our hands. Our group's interest in TBS-MAC (1) is from our research in the development of new organocatalyzed reactions and the synthesis of small molecule inhibitors of the HIV integrase enzyme [20,31,32]. Due to our need for TBS-MAC on several projects, a reliable and scalable synthesis of both acetylmalononitrile (7) and TBS-MAC (1) was required to access these valuable synthons. ...
This paper describes the three-step synthesis of TBS-MAC, a masked acyl cyanide (MAC) and a versatile one-carbon oxidation state three synthon. We have developed a scalable and detailed synthesis that involves: (1) acetylation of malononitrile to form the sodium enolate, (2) protonation of the enolate to form acetylmalononitrile, and (3) epoxidation of the enol, rearrangement to an unstable alcohol, and TBS-protection to form the title compound. Both the sodium enolate and acetylmalononitrile are bench-stable precursors to the intermediate hydroxymalononitrile, which can be converted to other MAC reagents beyond TBS by varying the protecting group (Ac, MOM, EE, etc.).
... Quinolines and isoquinolines are the important heterocyclic scaffolds and are known to possess several pharmacological activities such as anti-viral (De la Guardia et al., 2018), antileishmanial (Tempone et al., 2005), anti-tuberculosis (Nayyar et al., 2007), anti-inflammatory (Wen et al., 2015), anti-cancer (Pragathi et al., 2020) agents as well as inhibitors for HIV-1 integrase (Jentsch et al., 2018). These biological properties attracted the researchers to investigate these structural classes for their antimicrobial activity. ...
The current study reports synthesis of 2-aminoquinolines and 1-aminoisoquinolines derivatives and their characterization. Further, in vitro studies were conducted to determine antimicrobial activities. Compound 3 h showed maximum activity against B. subtilis (IC50: 0.10±0.02 µM) and E. coli (IC50: 0.13±0.01 µM) whereas compound 3i showed higher antimicrobial activity against E. coli (IC50: 0.11±0.01) and C. viswanathii (IC50: 0.10±0.05 µM). Safety profiles of the most potent derivatives were evaluated utilizing cell viability assay using RAW 264.7 and HeLa cell lines and in vitro hemolytic assay was carried out freshly isolated RBC from healthy rat. Furthermore, in silico studies, like molecular docking, binding free energy calculations and ADME predictions were done to get the best lead candidates. Additionally, molecular dynamic simulation for 100 ns was performed to know stability of protein and ligand complex. The active compounds were found to be non-toxic and non-hemolytic and hold great promise to become newer antimicrobial agents.
... Quinolines represent an important class of heterocyclic compounds, which widely occur as a core structural motif in natural products (McCormick et al., 1996;Subbaraju et al., 2004;McCauley et al., 2020), pharmaceuticals (Gorka et al., 2013;Kokatla et al., 2013;Jentsch et al., 2018), functional materials (Tong et al., 2003;Kim et al., 2005;Zhang et al., 2014), organocatalysis or ligands (Biddle et al., 2007;Zhang and Sigman., 2007;Esteruelas et al., 2016), and valuable building blocks (Wan et al., 2016;Duan et al., 2018;Wang et al., 2019;Ankade et al., 2021). Due to their great importance, considerable efforts have been focused on the development of efficient synthetic methods to their structures and modifications over the past years. ...
A transition-metal-free method for the construction of 3-substituted or 3,4-disubstituted quinolines from readily available N,N-dimethyl enaminones and o-aminobenzyl alcohols is reported. The direct oxidative cyclocondensation reaction tolerates broad functional groups, allowing the efficient synthesis of various quinolines in moderate to excellent yields. The reaction involves a C (sp³)-O bond cleavage and a C=N bind and a C=C bond formation during the oxidative cyclization process, and the mechanism was proposed.
... Several other quinoline analogues have been synthetized, which exhibit low micromolar inhibitory potency against HIV-1 IN as recently reviewed by Wadhwa et al. (Wadhwa et al. 2018). A series of multisubstituted quinolines, with the focus on the substitution pattern of the 4-phenyl moiety and incorporation of heteroaromatic or polycyclic substituents at the C(4)-position, were prepared and examined for their ability to trigger HIV-1 IN multimerisation via binding to an allosteric site (Jentsch et al. 2018). 4-(4-Chlorophenyl)quinoline and 2,3-benzo[b][1,4]dioxine showed highest potency with EC 50 values (half maximal eradication concentration) of 0.1 and 0.08 μM, respectively. ...
Infective diseases have become health threat of a global proportion due to appearance and spread of microorganisms resistant to majority of therapeutics currently used for their treatment. Therefore, there is a constant need for development of new antimicrobial agents, as well as novel therapeutic strategies. Quinolines and quinolones, isolated from
plants, animals, and microorganisms, have demonstrated numerous biological activities
such as antimicrobial, insecticidal, antiinflammatory, antiplatelet, and antitumor. For
more than two centuries quinoline/quinolone moiety has been used as a scaffold for drug
development and even today it represents an inexhaustible inspiration for design and development of novel semi-synthetic or synthetic agents exhibiting broad spectrum of
bioactivities. The structural diversity of synthetized compounds provides high and
selective activity attained through different mechanisms of action, as well as low toxicity
on human cells. This review describes quinoline and quinolone derivatives with antibacterial, antifungal, anti-virulent, antiviral, and anti-parasitic activities with the focus on the last 10 years literature.
Anlotinib is a tyrosine kinase inhibitor. It inhibits tumour growth by inhibiting the phosphorylation of angiogenesis-related receptors and attenuating the expression of related signals downstream of this pathway. Anlotinib has shown good antitumour activity and tolerability in patients with tumours, and multitargeted inhibition of angiogenesis does not lead to drug resistance due to excessive bypass activation. Moreover, its antitumour activity is superior to that of sunitinib, a conventional angiogenesis inhibitor. Results from several clinical studies have indicated that anlotinib improves progression-free survival and overall survival. Most adverse effects of anlotinib treatment were found to be alleviated by dose adjustment and symptomatic supportive therapy in several clinical trials. Therefore, anlotinib is a promising drug for oncology patients that is safe, effective, and tolerable, allowing patients with advanced cancer to benefit from drug therapy. This article reviews the basic information, antitumour mechanisms, clinical applications, clinical trial findings, and adverse effects of anlotinib and describes the problems in anlotinib research. It concludes with an outlook on future work.
Human immunodeficiency virus type 1 (HIV-1), a lentivirus that causes acquired immunodeficiency syndrome (AIDS), poses a serious threat to global public health. Since the advent of the first drug zidovudine, a number of anti-HIV agents acting on different targets have been approved to combat HIV/AIDS. Among the abundant heterocyclic families, quinoline and isoquinoline moieties are recognized as promising scaffolds for HIV inhibition. This review intends to highlight the advances in diverse chemical structures and abundant biological activity of quinolines and isoquinolines as anti-HIV agents acting on different targets, which aims to provide useful references and inspirations to design and develop novel HIV inhibitors for medicinal chemists.
Continued emerging resistance of pathogens against the clinically approved candidates and their associated limitations continuously demand newer agents having better potency with a more suited safety profile. Quinoline nuclei containing scaffolds of natural and synthetic origin have been documented for diverse types of pharmacological activities, and a number of drugs are clinically approved. In the present review, we unprecedentedly covered the biological potential of 4‐substituted quinoline and elaborated a rationale for its special privilege to afford the significant number of approved clinical drugs, particularly against infectious pathogens. Compounds with 4‐substituted quinoline are well documented for antimalarial activity, but in the last two decades, they have been extensively explored for activity against cancer, tuberculosis, and several other pathogens including viruses, bacteria, fungi, and other infectious pathogens. In the present study, the anti‐infective spectrum of this scaffold is discussed against viruses, mycobacteria, malarial parasites, and fungal and bacterial strains, along with recent updates in this area, with special emphasis on the structure–activity relationship. This review covers the biological potential of 4‐substituted quinoline and gives a rationale for its special privilege to afford many approved clinical drugs, particularly against infectious pathogens. The anti‐infection spectrum of these compounds against viruses, mycobacteria, malarial parasites, and fungal and bacterial strains is discussed, along with recent updates in this area, with special emphasis on the structure–activity relationship.
A facile transition metal-free decarboxylative C4 selective C-H difluoroarylmethylation of 8-aminoquinolines has been developed. This strategy proceeds under simple aqueous conditions and displays a broad substrate scope and excellent functional...
Based on the structural skeleton of natural products boeravinones, two types of 6H-chromeno[3,4-b]quinoline derivatives were designed and synthesized by nitrogen atom substitution strategy. Then, their cytotoxic activities were evaluated against six human tumor cell lines including HepG2 (hepatocellular carcinoma), A2780 (ovarian cancer), Hela (cervical cancer), HCT116 (colorectal cancer), SW1990 (pancreatic cancer), and MCF7 (breast cancer). The results showed that compounds ZML-8 and ZML-14 exhibited robust inhibitory activities against HepG2 cells with IC50 values of 0.58 and 1.94 μM, respectively. In addition, ZML-8 and ZML-14 showed higher selectivity against HepG2 and L-02 cells than Topotecan. Mechanistically, ZML-8 and ZML-14 not only induced cell cycle arrest in the G2/M phase and cell apoptosis, but also dose-dependently inhibited topoisomerase I activity and induced DNA damage in HepG2 cells. Molecular docking showed that ZML-8 and ZML-14 could interact with topoisomerase I-DNA complex with a similar binding mode to Topotecan. Inhibitory activities of these two compounds on topoisomerase I were then confirmed in both cell-free systems and in whole-cell lysates. Taken together, compounds ZML-8 and ZML-14 merit further development as a new generation of non-camptothecin topoisomerase I inhibitors for the treatment of cancer.
As the continuation of our studies on novel and effective anti-cancer agents, a series of novel quinoline-indole derivatives were firstly designed and synthesized by molecular hybridization strategy and Lewis acid-catalyzed coupling reactions. Their antiproliferative potency on gastric cancer cell line MGC-803, colon cancer cell line HCT-116, and esophageal cancer cell line Kyse450 of all the targeted compounds was explored using methyl thiazolyl tetrazolium (MTT) assay. 2-Chloro-4-(5-methoxy-1H-indol-3-yl)quinoline (9b) exhibited potently inhibitory activity against MGC-803, HCT-116, and Kyse450 cells with IC50 values of 0.58, 0.68 and 0.59 μmol•L⁻¹. Further mechanism studies suggested that compound 9b inhibited the cell colony formation of MGC-803 and HGC-27 cells. Compound 9b induced an intrinsic apoptosis and downregulated the levels of apoptosis related proteins in MGC-803 and HGC-27 cells. Meanwhile, compound 9b arrested MGC-803 and HGC-27 cells at the G2/M phase. Taken together, these results indicated that compound 9b might be a valuable lead compound for anticancer agents.
In search of new environmentally friendly and effective antifungal agents, a series of 4‐aminoquinolines bearing a 1,3‐benzodioxole moiety were prepared and their structures were fully elucidated by spectroscopic analyses. The antifungal activities of all the target compounds against five phytopathogenic fungi were evaluated in vitro . The results revealed that most of the newly synthesized compounds exhibited obvious inhibitory activities at the concentration of 50 μg/mL. Among them, 2‐(furan‐2‐yl)‐6,7‐methylenedioxy‐ N ‐( p ‐tolyl)quinolin‐4‐amine hydrochloride ( 7m ) displayed more promising antifungal potency with EC 50 values of 10.3 and 14.0 μg/mL against C. lunata and A. alternate , respectively. Particularly, the EC 50 value of 7m against C. lunata was 7.3‐fold as potent as the standard azoxystrobin. There were some significant morphological alterations in the mycelia of C. lunata when treated with 7m at 50 μg/mL. Additionally, the preliminary structure–activity relationships (SARs) were also discussed. Thus, this study suggests that 4‐aminoquinolines bearing a 1,3‐benzodioxole moiety are interesting scaffolds for the development of novel antifungal agents.
This study presents an efficient strategy for constructing 1,2-difunctionalized quinoline derivatives via the multicomponent cascade coupling of N-heteroaromatics with alkyl halides and different terminal alkynes. This reaction was achieved through sequential functionalization at the one- and two-positions of quinolines, which displayed a broad substrate scope, environmental friendliness, excellent functional group tolerance, high atom efficiency, and chemoselectivity. The multicomponent coupling involved the abnormal construction of new C-N, C═C, and C═O bonds in one pot. The applicability of this method was further demonstrated by the late-stage functionalization of complex drug molecules under the established conditions.
Reaction of 6-methyl-4,5-dioxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carboxaldehyde (1) with hydroxylamine hydrochloride under different reaction conditions produced a novel pyrano[3,2-c]quinoline derivatvies 2-5. Ring opening ring closure reactions of pyrano[3,2-c]quinoline-3-carbonitrile 2 with some nitrogen nucleophiles afforded the novel 3-heteroaryl-4-hydroxyquinolines. While, treatment of carbonitrile 3 with some nitrogen nucleohilic reagents produced a diversity of annulated pyrano[3,2-c]quinolines. The chemical reactivity of the prepared pyrano[3,2-c]quinoline-3-carbonitriles 2 and 3 is widely different depending on the different reactivity of pyran-2-one and pyran-4-one moieties toward the nucleophilic reagents as well as the functional groups present in pyrone rings.
An effective annulation of ynones and (iso)quinoline N-oxides was developed to deliver various functionalized 3-((iso)quinolin-1-yl)-4 H -chromen-4-ones and 13 H -isoquinolino[2,1- a ]quinolin-13-ones in moderate to excellent yields, respectively. This protocol exhibits high regioselectivity and broad...
A useful and convenient method for C-H bond arylation of 8-aminoquinoline motifs on the remote C4 position was developed. This method shows good functional group tolerance toward various Grignard reagents and aminoquinoline via a nickel catalysis, giving the desired arylated products in good yields. The present method affords an efficient access to construct multisubstituted aminoquinolines.
Allosteric HIV-1 integrase inhibitors (ALLINIs) are a new class of potential antiretroviral therapies with a unique mechanism of action and drug resistance profile. To further extend this class of inhibitors via a scaffold hopping approach, we have synthesized a series of analogues possessing an isoquinoline ring system. Lead compound 6l binds in the v-shaped pocket at the IN dimer interface and is highly selective for promoting higher-order multimerization of inactive IN over inhibiting IN-LEDGF/p75 binding. Importantly, 6l potently inhibited HIV-1NL4-3 (A128T IN) which confers marked resistance to arche-typal quinoline-based ALLINIs. Thermal degradation studies indicated that at elevated temperatures the acetic acid side chain of specific isoquinoline derivatives undergo decarboxylation reactions. This reactivity has implications for the synthesis of various ALLINI analogues.
Retroviral Integrase Structure and DNA Recombination Mechanism, Page 1 of 2
Abstract
Retroviruses are the only animal viruses that require the stable integration of genetic information into the genome of the host cell as an obligate step in replication. All members of the virus family Retroviridae accordingly carry with them integrase, which is a specialized DNA recombination enzyme. Integration is required for efficient expression of retroviral genes by the host transcriptional machinery and hence productive virus replication. The integrase encoded by human immunodeficiency virus type 1 (HIV-1) is thus an important antiviral target in the fight against HIV/AIDS ( 1 ). Integration additionally ensures replication and segregation of viral genes to daughter cells during cell division. Stable association of HIV-1 with cellular DNA underlies the notoriously incurable nature of AIDS despite highly active antiretroviral therapy (HAART) ( 2 ).
Recently, a new class of HIV-1 integrase (IN) inhibitors with a dual mode of action, called IN-LEDGF/p75 allosteric inhibitors (INLAIs), was described. Designed to interfere with the INLEDGF/p75 interaction during viral integration, unexpectedly, their major impact was on virus maturation. This activity has been linked to induction of aberrant IN multimerization, while inhibition of the IN-LEDGF/p75 interaction accounts for weaker antiretroviral effect at integration. Since these dual activities result from INLAI binding to IN at a single binding site, we expected that these activities co-evolved together, driven by the affinity for IN. Using an original INLAI, MUT-A, and its activity on an Ala-125 (A125) IN variant, we found that these two activities on A125 IN can be fully dissociated: MUT-A-induced IN multimerization and the formation of eccentric condensates in viral particles, that are responsible for inhibition of virus maturation, were lost, while inhibition of the IN-LEDGF/p75 interaction and consequently integration, was fully retained. Hence the mere binding of INLAI to A125 IN is insufficient to promote the conformational changes of IN required for aberrant multimerization. By analyzing the X-ray structures of MUT-A bound to the IN catalytic core domain (CCD) with or without the A125 polymorphism, we discovered that the loss of IN multimerization is due to stabilization of the A125 IN variant CCD dimer, highlighting the importance of the CCD dimerization energy for IN multimerization. Our study reveals that affinity for the LEDGF/p75-binding pocket is not sufficient to induce INLAI-dependent IN multimerization and the associated inhibition of viral maturation.
The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization. Author Summary A promising new class of antivirals called " ALLINIs " (allosteric inhibitors of integrase) potently inhibits HIV replication. Like other drugs, ALLINIs seem to target also the HIV-1 integrase (IN), which is crucial for the replication of this virus, but instead of acting at early phases of HIV replication, they interfere with viral particle assembly and maturation that occur at late stages and induce aggregation of IN. Despite these findings, the structural bases for the effects are still unknown. In this study, we crystallized full-length HIV-1 IN in complex with an ALLINI called GSK1264 and determined its structure to 4.4 Å.
Employing a scaffold hopping approach, a series of allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) have been synthesized based on an indole scaffold. These compounds incorporate the key elements utilized in quinoline-based ALLINIs for binding to the IN dimer interface at the principal LEDGF/p75 binding pocket. The most potent of these compounds displayed good activity in the LEDGF/p75 dependent integration assay (IC50 = 4.5 μM) and, as predicted based on the geometry of the five- versus six-membered ring, retained activity against the A128T IN mutant that confers resistance to many quinoline-based ALLINIs.
The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3'-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications.
Background
Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an important new class of anti-HIV-1 agents. ALLINIs bind at the IN catalytic core domain (CCD) dimer interface occupying the principal binding pocket of its cellular cofactor LEDGF/p75. Consequently, ALLINIs inhibit HIV-1 IN interaction with LEDGF/p75 as well as promote aberrant IN multimerization. Selection of viral strains emerging under the inhibitor pressure has revealed mutations at the IN dimer interface near the inhibitor binding site.ResultsWe have investigated the effects of one of the most prevalent substitutions, H171T IN, selected under increasing pressure of ALLINI BI-D. Virus containing the H171T IN substitution exhibited an ~68-fold resistance to BI-D treatment in infected cells. These results correlated with ~84-fold reduced affinity for BI-D binding to recombinant H171T IN CCD protein compared to its wild type (WT) counterpart. However, the H171T IN substitution only modestly affected IN-LEDGF/p75 binding and allowed HIV-1 containing this substitution to replicate at near WT levels. The x-ray crystal structures of BI-D binding to WT and H171T IN CCD dimers coupled with binding free energy calculations revealed the importance of the N¿- protonated imidazole group of His171 for hydrogen bonding to the BI-D tert-butoxy ether oxygen and establishing electrostatic interactions with the inhibitor carboxylic acid, whereas these interactions were compromised upon substitution to Thr171.Conclusions
Our findings reveal a distinct mechanism of resistance for the H171T IN mutation to ALLINI BI-D and indicate a previously undescribed role of the His171 side chain for binding the inhibitor.
The aggregation of amyloid-β (Aβ) peptide and its deposition in parts of the brain form the central processes in the etiology of Alzheimer disease (AD). The low-molecular weight oligomers of Aβ aggregates (2 to 30 mers) are known to be the primary neurotoxic agents whose mechanisms of cellular toxicity and synaptic dysfunction have received substantial attention in the recent years. However, how these toxic agents proliferate and induce widespread amyloid deposition throughout the brain, and what mechanism is involved in the amplification and propagation of toxic oligomer species, are far from clear. Emerging evidence based on transgenic mice models indicates a transmissible nature of Aβ aggregates and implicates a prion-like mechanism of oligomer propagation, which manifests as the dissemination and proliferation of Aβ toxicity. Despite accumulating evidence in support of a transmissible nature of Aβ aggregates, a clear, molecular-level understanding of this intriguing mechanism is lacking. Recently, we reported the characterization of unique replicating oligomers of Aβ42 (12–24 mers) in
vitro called Large Fatty Acid-derived Oligomers (LFAOs) (Kumar et al., 2012, J. Biol. Chem). In the current report, we establish that LFAOs possess physiological activity by activating NF-κB in human neuroblastoma cells, and determine the experimental parameters that control the efficiency of LFAO replication by self-propagation. These findings constitute the first detailed report on monomer – oligomer lateral propagation reactions that may constitute potential mechanism governing transmissibility among Aβ oligomers. These data support the previous reports on transmissible mechanisms observed in transgenic animal models.
Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a promising class of antiretroviral agents for clinical development.
Although ALLINIs promote aberrant IN multimerization and inhibit IN interaction with its cellular cofactor LEDGF/p75 with
comparable potencies in vitro, their primary mechanism of action in infected cells is through inducing aberrant multimerization of IN. Crystal structures
have shown that ALLINIs bind at the IN catalytic core domain dimer interface and bridge two interacting subunits. However,
how these interactions promote higher-order protein multimerization is not clear. Here, we used mass spectrometry-based protein
footprinting to monitor surface topology changes in full-length WT and the drug-resistant A128T mutant INs in the presence
of ALLINI-2. These experiments have identified protein-protein interactions that extend beyond the direct inhibitor binding
site and which lead to aberrant multimerization of WT but not A128T IN. Specifically, we demonstrate that C-terminal residues
Lys-264 and Lys-266 play an important role in the inhibitor induced aberrant multimerization of the WT protein. Our findings
provide structural clues for exploiting IN multimerization as a new, attractive therapeutic target and are expected to facilitate
development of improved inhibitors.
The quinoline-based allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complicated by the multifunctional nature of these inhibitors because they both inhibit IN binding with its cofactor LEDGF/p75 and promote aberrant IN multimerization with similar potencies in vitro. Here we report design of small molecules that allowed us to probe the role of HIV-1 IN multimerization independently from IN-LEDGF/p75 interactions in infected cells. We altered the rigid quinoline moiety in ALLINIs and designed pyridine-based molecules with a rotatable single bond to allow these compounds to bridge between interacting IN subunits optimally and promote oligomerization. The most potent pyridine-based inhibitor, KF116, potently (EC50 of 0.024 µM) blocked HIV-1 replication by inducing aberrant IN multimerization in virus particles, whereas it was not effective when added to target cells. Furthermore, KF116 inhibited the HIV-1 IN variant with the A128T substitution, which confers resistance to the majority of quinoline-based ALLINIs. A genome-wide HIV-1 integration site analysis demonstrated that addition of KF116 to target or producer cells did not affect LEDGF/p75-dependent HIV-1 integration in host chromosomes, indicating that this compound is not detectably inhibiting IN-LEDGF/p75 binding. These findings delineate the significance of correctly ordered IN structure for HIV-1 particle morphogenesis and demonstrate feasibility of exploiting IN multimerization as a therapeutic target. Furthermore, pyridine-based compounds present a novel class of multimerization selective IN inhibitors as investigational probes for HIV-1 molecular biology.
Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a very promising new class of anti-HIV-1 agents that exhibit a multimodal mechanism of action by allosterically modulating IN multimerization and interfering with IN-lens epithelium-derived growth factor (LEDGF)/p75 binding. Selection of viral strains under ALLINI pressure has revealed an A128T substitution in HIV-1 IN as a primary mechanism of resistance. Here, we elucidated the structural and mechanistic basis for this resistance. The A128T substitution did not affect the hydrogen bonding between ALLINI and IN that mimics the IN-LEDGF/p75 interaction but instead altered the positioning of the inhibitor at the IN dimer interface. Consequently, the A128T substitution had only a minor effect on the ALLINI IC50 values for IN-LEDGF/p75 binding. Instead, ALLINIs markedly altered the multimerization of IN by promoting aberrant higher order WT (but not A128T) IN oligomers. Accordingly, WT IN catalytic activities and HIV-1 replication were potently inhibited by ALLINIs, whereas the A128T substitution in IN resulted in significant resistance to the inhibitors both in vitro and in cell culture assays. The differential multimerization of WT and A128T INs induced by ALLINIs correlated with the differences in infectivity of HIV-1 progeny virions. We conclude that ALLINIs primarily target IN multimerization rather than IN-LEDGF/p75 binding. Our findings provide the structural foundations for developing improved ALLINIs with increased potency and decreased potential to select for drug resistance.
Background: The A128T substitution in HIV-1 integrase (IN) confers resistance to allosteric integrase inhibitors (ALLINIs).
Results: The A128T substitution does not significantly alter ALLINI IC50 values for IN-LEDGF/p75 binding but confers marked resistance to ALLINI-induced aberrant integrase multimerization.
Conclusion: Allosteric perturbation of HIV-1 integrase multimerization underlies ALLINI antiviral activity.
Significance: Our findings underscore the mechanism of ALLINI action and will facilitate development of second-generation compounds.
Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the binding site for the host protein lens epithelium-derived growth factor (LEDGF)/transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose-response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles' heel for exploitation in clinical ALLINI development.
The Feline Immunodeficiency Virus (FIV) capsid protein p24 oligomerizes to form a closed capsid that protects the viral genome. Because of its crucial role in the virion, FIV p24 is an interesting target for the development of therapeutic strategies, although little is known about its structure and assembly. We defined and optimized a protocol to overexpress recombinant FIV capsid protein in a bacterial system. Circular dichroism and isothermal titration calorimetry experiments showed that the structure of the purified FIV p24 protein was comprised mainly of α-helices. Dynamic light scattering (DLS) and cross-linking experiments demonstrated that p24 was monomeric at low concentration and dimeric at high concentration. We developed a protocol for the assembly of the FIV capsid. As with HIV, an increased ionic strength resulted in FIV p24 assembly . Assembly appeared to be dependent on temperature, salt concentration, and protein concentration. The FIV p24 assembly kinetics was monitored by DLS. A limit end-point diameter suggested assembly into objects of definite shapes. This was confirmed by electron microscopy, where FIV p24 assembled into spherical particles. Comparison of FIV p24 with other retroviral capsid proteins showed that FIV assembly is particular and requires further specific study.
Retroviral integrases catalyze two reactions, 3' processing of viral DNA ends, followed by integration of the processed ends into chromosomal DNA. X-ray crystal structures of integrase-DNA complexes from prototype foamy virus, a member of the spumavirus genus of Retroviridae, have revealed the structural basis of integration and how clinicallyrelevant integrase strand transfer inhibitors work. Underscoring the translational potential of targeting viralhost interactions, small molecules that bind at the host factor LEDGF/p75 binding site on HIV-1 integrase promote dimerization and inhibit integrase-viral DNA assembly and catalysis. Here we review recent advances in our knowledge of HIV-1 DNA integration, as well as future research directions.
Targeting the HIV integrase (HIV IN) is a clinically validated approach for designing novel anti-HIV therapies. We have previously described the discovery of a novel class of integration inhibitors, 2-(quinolin-3-yl)acetic acid derivatives, blocking HIV replication at a low micromolar concentration through binding in the LEDGF/p75 binding pocket of HIV integrase, hence referred to as LEDGINs. Here we report the detailed characterization of their mode of action. The design of novel and more potent analogues with nanomolar activity enabled full virological evaluation and a profound mechanistic study. As allosteric inhibitors, LEDGINs bind to the LEDGF/p75 binding pocket in integrase, thereby blocking the interaction with LEDGF/p75 and interfering indirectly with the catalytic activity of integrase. Detailed mechanism-of-action studies reveal that the allosteric mode of inhibition is likely caused by an effect on HIV-1 integrase oligomerization. The multimodal inhibition by LEDGINs results in a block in HIV integration and in a replication deficiency of progeny virus. The allosteric nature of LEDGINs leads to synergy in combination with the clinically approved active site HIV IN strand transfer inhibitor (INSTI) raltegravir, and cross-resistance profiling proves the distinct mode of action of LEDGINs and INSTIs. The allosteric nature of inhibition and compatibility with INSTIs underline an interest in further (clinical) development of LEDGINs.
tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3'-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction.
The multifunctional HIV-1 enzyme integrase interacts with viral DNA and its key cellular cofactor LEDGF to effectively integrate
the reverse transcript into a host cell chromosome. These interactions are crucial for HIV-1 replication and present attractive
targets for antiviral therapy. Recently, 2-(quinolin-3-yl) acetic acid derivatives were reported to selectively inhibit the
integrase-LEDGF interaction in vitro and impair HIV-1 replication in infected cells. Here, we show that this class of compounds impairs both integrase-LEDGF binding
and LEDGF-independent integrase catalytic activities with similar IC50 values, defining them as bona fide allosteric inhibitors of integrase function. Furthermore, we show that 2-(quinolin-3-yl) acetic acid derivatives block the
formation of the stable synaptic complex between integrase and viral DNA by allosterically stabilizing an inactive multimeric
form of integrase. In addition, these compounds inhibit LEDGF binding to the stable synaptic complex. This multimode mechanism
of action concordantly results in cooperative inhibition of the concerted integration of viral DNA ends in vitro and HIV-1 replication in cell culture. Our findings, coupled with the fact that high cooperativity of antiviral inhibitors
correlates with their increased instantaneous inhibitory potential, an important clinical parameter, argue strongly that improved
2-(quinolin-3-yl) acetic acid derivatives could exhibit desirable clinical properties.
Three-dimensional molecular structures can provide detailed information on biological mechanisms and, for cases in which the molecular function affects human health, can significantly aid in the development of therapeutic interventions. For almost 25 years, key components of the lentivirus HIV-1, including the envelope glycoproteins, the capsid and the replication enzymes reverse transcriptase, integrase and protease, have been scrutinized to near atomic-scale resolution. Moreover, structural analyses of the interactions between viral and host cell components have yielded key insights into the mechanisms of viral entry, chromosomal integration, transcription and egress from cells. Here, we review recent advances in HIV-1 structural biology, focusing on the molecular mechanisms of viral replication and on the development of new therapeutics.
Antiretroviral drugs to prevent integration of the HIV viral genome into chromosomes are undergoing clinical trials, yet they have been developed with an imperfect understanding of their mechanism of action. The recent crystal structure of the major viral protein integrase from a related, little‐known retrovirus, has finally provided insight into how the drugs work and, more importantly, how to improve them.
The mandatory integration of the reverse-transcribed HIV-1 genome into host chromatin is catalyzed by the viral protein integrase (IN), and IN activity can be regulated by numerous viral and cellular proteins. Among these, LEDGF has been identified as a cellular cofactor critical for effective HIV-1 integration. The x-ray crystal structure of the catalytic core domain (CCD) of IN in complex with the IN binding domain (IBD) of LEDGF has furthermore revealed essential protein-protein contacts. However, mutagenic studies indicated that interactions between the full-length proteins were more extensive than the contacts observed in the co-crystal structure of the isolated domains. Therefore, we have conducted detailed biochemical characterization of the interactions between full-length IN and LEDGF. Our results reveal a highly dynamic nature of IN subunit-subunit interactions. LEDGF strongly stabilized these interactions and promoted IN tetramerization. Mass spectrometric protein footprinting and molecular modeling experiments uncovered novel intra- and inter-protein-protein contacts in the full-length IN-LEDGF complex that lay outside of the observable IBD-CCD structure. In particular, our studies defined the IN tetramer interface important for enzymatic activities and high affinity LEDGF binding. These findings provide new insight into how LEDGF modulates HIV-1 IN structure and function, and highlight the potential for exploiting the highly dynamic structure of multimeric IN as a novel therapeutic target.
We studied human immunodeficiency virus, type 1 (HIV-1) integrase (IN) complexes derived from nuclei of human cells stably expressing the viral protein from a synthetic gene. We show that in the nuclear extracts IN exists as part of a large distinct complex with an apparent Stokes radius of 61 A, which dissociates upon dilution yielding a core molecule of 41 A. We isolated the IN complexes from cells expressing FLAG-tagged IN and demonstrated that the 41 A core is a tetramer of IN, whereas 61 A molecules are composed of IN tetramers associated with a cellular protein with an apparent molecular mass of 76 kDa. This novel integrase interacting protein was found to be identical to lens epithelium-derived growth factor (LEDGF/p75), a protein implicated in regulation of gene expression and cellular stress response. HIV-1 IN and LEDGF co-localized in the nuclei of human cells stably expressing IN. Furthermore, recombinant LEDGF robustly enhanced strand transfer activity of HIV-1 IN in vitro. Our findings indicate that the minimal IN molecule in human cells is a homotetramer, suggesting that at least an octamer of IN is required to accomplish coordinated integration of both retroviral long terminal repeats and that LEDGF is a cellular factor involved in this process.
Human lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF/p75) protein was recently identified as a binding partner for HIV-1 integrase (IN) in human cells. In this work, we used biochemical and bioinformatic approaches to define the domain organization of LEDGF/p75. Using limited proteolysis and deletion mutagenesis we show that the protein contains a pair of evolutionarily conserved domains, assuming about 35% of its sequence. Whereas the N-terminal PWWP domain had been recognized previously, the second domain is novel. It is comprised of approximately 80 amino acid residues and is both necessary and sufficient for binding to HIV-1 IN. Strikingly, the integrase binding domain (IBD) is not unique to LEDGF/p75, as a second human protein, hepatoma-derived growth factor-related protein 2 (HRP2), contains a homologous sequence. LEDGF/p75 and HRP2 IBDs avidly bound HIV-1 IN in an in vitro GST pull-down assay and each full-length protein potently stimulated HIV-1 IN activity in vitro. LEDGF/p75 and HRP2 are predicted to share a similar domain organization and have an evident evolutionary and likely functional relationship.
Integrase (IN) is an essential retroviral enzyme, and human transcriptional coactivator p75, which is also referred to as lens epithelium-derived growth factor (LEDGF), is the dominant cellular binding partner of HIV-1 IN. Here, we report the crystal structure of the dimeric catalytic core domain of HIV-1 IN complexed to the IN-binding domain of LEDGF. Previously identified LEDGF hotspot residues anchor the protein to both monomers at the IN dimer interface. The principal structural features of IN that are recognized by the host factor are the backbone conformation of residues 168–171 from one monomer and a hydrophobic patch that is primarily comprised of α-helices 1 and 3 of the second IN monomer. Inspection of diverse retroviral primary and secondary sequence elements helps to explain the apparent lentiviral tropism of the LEDGF-IN interaction. Because the lethal phenotypes of HIV-1 mutant viruses unable to interact with LEDGF indicate that IN function is highly sensitive to perturbations of the structure around the LEDGF-binding site, we propose that small molecule inhibitors of the protein–protein interaction might similarly disrupt HIV-1 replication.
• integration
• structure
• retrovirus
• transcription
• host factor
LEDGF/p75 directly interacts with lentiviral integrase proteins and can modulate their enzymatic activities and chromosomal association. A novel genetic knockout model was established that allowed us for the first time to analyze HIV-1 integration in the absence of LEDGF/p75 protein. Supporting a crucial role for the cofactor in viral replication, HIV-1 vector integration and reporter gene expression were significantly reduced in LEDGF-null cells. Yet, integrase processed the viral cDNA termini normally and maintained its local target DNA sequence preference during integration. Preintegration complexes extracted from knockout cells moreover supported normal levels of DNA strand transfer activity in vitro. In contrast, HIV-1 lost its strong bias toward integrating into transcription units, displaying instead increased affinity for promoter regions and CpG islands. Our results reveal LEDGF/p75 as a critical targeting factor, commandeering lentiviruses from promoter- and/or CpG island-proximal pathways that are favored by other members of Retroviridae. Akin to yeast retrotransposons, disrupting the lentiviral targeting mechanism significantly perturbs overall integration.
To replicate, lentiviruses such as HIV must integrate DNA copies of their RNA genomes into host cell chromosomes. Lentiviral integration is favored in active transcription units, which allows efficient viral gene expression after integration, but the mechanisms directing integration targeting are incompletely understood. A cellular protein, PSIP1/LEDGF/p75, binds tightly to the lentiviral-encoded integrase protein (IN), and has been reported to be important for HIV infectivity and integration targeting.
Here we report studies of lentiviral integration targeting in 1) human cells with intensified RNAi knockdowns of PSIP1/LEDGF/p75, and 2) murine cells with homozygous gene trap mutations in the PSIP1/LEDGF/p75 locus. Infections with vectors derived from equine infections anemia virus (EIAV) and HIV were compared. Integration acceptor sites were analyzed by DNA bar coding and pyrosequencing.
In both PSIP1/LEDGF/p75-depleted cell lines, reductions were seen in lentiviral infectivity compared to controls. For the human cells, integration was reduced in transcription units in the knockdowns, and this reduction was greater than in our previous studies of human cells less completely depleted for PSIP1/LEDGF/p75. For the homozygous mutant mouse cells, similar reductions in integration in transcription units were seen, paralleling a previous study of a different mutant mouse line. Integration did not become random, however-integration in transcription units in both cell types was still favored, though to a reduced degree. New trends also appeared, including favored integration near CpG islands. In addition, we carried out a bioinformatic study of 15 HIV integration site data sets in different cell types, which showed that the frequency of integration in transcription units was correlated with the cell-type specific levels of PSIP1/LEDGF/p75 expression.
During systematic analysis of nonbonded contacts in protein-ligand complexes derived from crystal structures in the Protein Data Bank, Cl-pi interactions have been found, not only in the well-documented serine proteases but also, to a lesser extent, in other proteins. From geometric analysis of such Cl-pi interactions in the crystal structures, two distinct geometries were found: the "edge-on" approach of a Cl atom to a ring atom or C-C bond and the "face-on" approach toward the ring centroid with an average interatomic distance of 3.6 A. High-level ab initio calculations using benzene-chlorohydrocarbon model systems elucidated that the calculated Cl-pi interaction energy is -2.01 kcal/mol, and the dispersion force is the major source of attraction. We also discussed the geometric flexibility in Cl-pi interactions and a relationship between the intensity of the pi density in an aromatic ring and the interaction position of the Cl atom.
Allosteric integrase inhibitors (ALLINIs) bind to the lens epithelial-derived growth factor (LEDGF) pocket on HIV-1 integrase (IN) and possess potent antiviral effects. Rather than blocking proviral integration, ALLINIs trigger IN conformational changes that have catastrophic effects on viral maturation, rendering the virions assembled in the presence of ALLINIs non-infectious. A high-throughput screen for compounds that disrupt the IN·LEDGF interaction was executed, and extensive triage led to the identification of a t-butylsulfonamide series, as exemplified by 1. The chemical, biochemical and virological characterization of this series revealed that 1 and its analogs produce an ALLINI-like phenotype through engagement of IN sites distinct from the LEDGF pocket. Key to demonstrating target engagement and differentiating this new series from the existing ALLINIs was the development of a fluorescence polarization probe of IN (FLIPPIN) based on the t-butylsulfonamide series. These findings further solidify the late antiviral of mechanism of ALLINIs, and point towards opportunities to develop structurally and mechanistically novel antiretroviral agents with unique resistance patterns.
While an essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosome is established, studies with IN mutants and allosteric IN inhibitors (ALLINIs) have suggested that IN can also influence viral particle maturation. However, it has remained enigmatic as to how IN contributes to virion morphogenesis. Here, we demonstrate that IN directly binds the viral RNA genome in virions. These interactions have specificity, as IN exhibits distinct preference for select viral RNA structural elements. We show that IN substitutions that selectively impair its binding to viral RNA result in eccentric, non-infectious virions without affecting nucleocapsid-RNA interactions. Likewise, ALLINIs impair IN binding to viral RNA in virions of wild-type, but not escape mutant, virus. These results reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate the mode of action of ALLINIs.
Allosteric HIV-1 integrase inhibitors (ALLINIs) have recently emerged as a promising class of antiretroviral agents and are currently in clinical trials. In infected cells ALLINIs potently inhibit viral replication by impairing virus particle maturation but surprisingly exhibit a reduced EC50 for inhibiting HIV-1 integration in target cells. To better understand the reduced antiviral activity of ALLINIs during the early stage of HIV-1 replication, we investigated the competitive interplay between a potent representative ALLINI, BI/D, and LEDGF/p75 with HIV-1 integrase. While the principal binding sites of BI/D and LEDGF/p75 overlap at the integrase catalytic core domain dimer interface, we show that the inhibitor and the cellular cofactor induce markedly different multimerization patterns of full-length integrase. LEDGF/p75 stabilizes an integrase tetramer through additional interactions with the integrase N-terminal domain, whereas BI/D induces protein-protein interactions in C-terminal segments that lead to aberrant, higher-order integrase multimerization. We demonstrate that LEDGF/p75 binds HIV-1 integrase with significantly higher affinity than BI/D and that the cellular protein is able to reverse the inhibitor induced aberrant, higher-order integrase multimerization in a dose-dependent manner in vitro. Consistent with these observations, alterations of the cellular levels of LEDGF/p75 markedly affected BI/D EC50 values during the early steps of HIV-1 replication. Furthermore, genome-wide sequencing of HIV-1 integration sites in infected cells demonstrate that LEDGF/p75-dependent integration site selection is adversely affected by BI/D treatment. Taken together, our studies elucidate structural and mechanistic details of the interplay between LEDGF/p75 and BI/D during the early stage of HIV-1 replication.
HIV-1 integrase (IN) is an important therapeutic target as its function is essential for the viral lifecycle. The discovery of multifunctional allosteric IN inhibitors or ALLINIs, which potently impair viral replication by promoting aberrant, higher order IN multimerization as well as inhibit IN interactions with its cellular cofactor, LEDGF/p75, has opened new venues to exploit IN multimerization as a therapeutic target. Furthermore, the recent discovery of multimerization selective IN inhibitors or MINIs, has provided new investigational probes to study the direct effects of aberrant IN multimerization in vitro and in infected cells. Here we describe three complementary methods designed to detect and quantify the effects of these new classes of inhibitors on IN multimerization. These methods include a homogenous time-resolved fluorescence-based assay which allows for measuring EC50 values for the inhibitor-induced aberrant IN multimerization, a dynamic light scattering-based assay which allows for monitoring the formation and sizes of oligomeric IN particles in a time-dependent manner, and a chemical cross-linking-based assay of interacting IN subunits which allows for the determination of IN oligomers in viral particles.
HIV integration. A tetramer of HIV integrase (B) assembles on viral DNA (A) ends and mediates its integration into host cell chromatin. Cellular protein LEDGF/p75 (C) binds IN tetramer in the nucleoprotein complex and navigates HIV-1 integration in active genes
Multimeric HIV-1 integrase (IN) plays an essential, multifunctional role in virus replication and serves as an important therapeutic target. Structural and biochemical studies have revealed the importance of the ordered interplay between IN molecules for its function. In the presence of viral DNA ends, individual IN subunits assemble into a tetramer and form a stable synaptic complex (SSC), which mediates integration of the reverse transcribed HIV-1 genome into chromatin. Cellular chromatin-associated protein LEDGF/p75 engages the IN tetramer in the SSC and directs HIV-1 integration into active genes. A mechanism to deregulate the productive interplay between IN subunits with small molecule inhibitors has recently received considerable attention. Most notably, allosteric IN inhibitors (ALLINIs) have been shown to bind to the IN dimer interface at the LEDGF/p75 binding pocket, stabilize interacting IN subunits, and promote aberrant, higher order IN multimerization. Consequently, these compounds impair formation of the SSC and associated LEDGF/p75-independent IN catalytic activities as well as inhibit LEDGF/p75 binding to the SSC in vitro. However, in infected cells, ALLINIs more potently impaired correct maturation of virus particles than the integration step. ALLINI treatments induced aberrant, higher order IN multimerization in virions and resulted in eccentric, non-infectious virus particles. These studies have suggested that the correctly ordered IN structure is important for virus particle morphogenesis and highlighted IN multimerization as a plausible therapeutic target for developing new inhibitors to enhance treatment options for HIV-1-infected patients.
An assay recapitulating the 3′ processing activity of HIV-1 integrase (IN) was used to screen the Boehringer Ingelheim compound collection. Hit-to-lead and lead optimization beginning with compound 1 established the importance of the C3 and C4 substituent to antiviral potency against viruses with different aa124/aa125 variants of IN. The importance of the C7 position on the serum shifted potency was established. Introduction of a quinoline substituent at the C4 position provided a balance of potency and metabolic stability. Combination of these findings ultimately led to the discovery of compound 26 (BI 224436), the first NCINI to advance into a phase Ia clinical trial.
This year marks the thirtieth anniversary of the publication of the study that first reported the isolation of HIV-1. In this Timeline article, we provide a historical perspective of some of the major milestones in HIV science, highlighting how translational research has affected treatment and prevention of HIV. Finally, we discuss some of the current research directions and the scientific challenges ahead, in particular in the search for a cure for HIV.
HIV integrase (IN) catalyzes the insertion into the genome of the infected human cell of viral DNA produced by retrotranscription process. The discovery of raltegravir validated the existence of the IN, which is a new target in the field of anti-HIV drug research. The mechanism of catalysis of IN is depicted and the characteristics of the inhibitors of the catalytic site of this viral enzyme are reported. The role played by the resistance is elucidated, as well as the possibility to bypass this problem. New approaches aimed to block the integration process are depicted as future perspectives, such as development of allosteric IN inhibitors, dual inhibitors targeting both IN and other enzymes, inhibitors of enzymes that activate IN, activators of IN activity, as well as a gene therapy approach.
Chemoselective cross-coupling reactions of monoesters of dicarboxylic acid chlorides with organocopper reagents derived from Grignard reagents, cuprous bromide, and lithium bromide, provide a simple and straightforward method for synthesizing a variety of ketoesters.
HIV-1 integrase is an important therapeutic target in the fight against HIV/AIDS. Integrase strand transfer inhibitors (INSTIs), which target the enzyme active site, have witnessed clinical success over the past 5 years, but the generation of drug resistance poses challenges to INSTI-based therapies moving forward. Integrase is a dynamic protein, and its ordered multimerization is critical to enzyme activity. The integrase tetramer, bound to viral DNA, interacts with host LEDGF/p75 protein to tether integration to active genes. Allosteric integrase inhibitors (ALLINIs) that compete with LEDGF/p75 for binding to integrase disrupt integrase assembly with viral DNA and allosterically inhibit enzyme function. ALLINIs display steep dose response curves and synergize with INSTIs ex vivo, highlighting this novel inhibitor class for clinical development.
Integrase strand transfer inhibitors (INSTIs) have become a key component of antiretroviral therapy since the approval of twice-daily raltegravir in 2007 and the more recent approval of elvitegravir in 2012. At the same time, a third compound, dolutegravir, is in late-phase clinical trials, being tested as part of a multidrug once-daily formulation comprising this INSTI and two other antiretroviral (ARV) drugs. This review focuses on the factors leading to the development of drug resistance mutations (DRMs) against INSTIs, evidence of cross-resistance among them, and the results of regimen simplification in regard to this topic.
Sequencing data show that DRMs are highly dynamic in patients failing INSTI therapy. Considerations of viral fitness and drug resistance can together determine the evolution of drug resistance mutations, and in this regard the Y143 and Q148 pathways are superior to the N155 pathway in the promotion of resistance. Preventing the emergence of DRMs requires that effective reverse transcriptase or other inhibitors be used together with INSTIs and that high-level adherence to treatment be maintained.
Because of the susceptibility to drug resistance, INSTIs should always be used together with other effective ARV drugs.
Retroviral replication depends on successful integration of the viral genetic material into a host cell chromosome. Virally encoded integrase, an enzyme from the DDE(D) nucleotidyltransferase superfamily, is responsible for the key DNA cutting and joining steps associated with this process. Insights into the structural and mechanistic aspects of integration are directly relevant for the development of antiretroviral drugs. Recent breakthroughs have led to biochemical and structural characterization of the principal integration intermediates revealing the tetramer of integrase that catalyzes insertion of both 3' viral DNA ends into a sharply bent target DNA. This review discusses the mechanism of retroviral DNA integration and the mode of action of HIV-1 integrase strand transfer inhibitors in light of the recent visualization of the prototype foamy virus intasome, target DNA capture and strand transfer complexes.
Three-dimensional macromolecular structures shed critical light on biological mechanism and facilitate development of small molecule inhibitors. Clinical success of raltegravir, a potent inhibitor of HIV-1 integrase, demonstrated the utility of this viral DNA recombinase as an antiviral target. A variety of partial integrase structures reported in the past 16 years have been instrumental and very informative to the field. Nonetheless, because integrase protein fragments are unable to functionally engage the viral DNA substrate critical for strand transfer inhibitor binding, the early structures did little to materially impact drug development efforts. However, recent results based on prototype foamy virus integrase have fully reversed this trend, as a number of X-ray crystal structures of active integrase-DNA complexes revealed key mechanistic details and moreover established the foundation of HIV-1 integrase strand transfer inhibitor action. In this review we discuss the landmarks in the progress of integrase structural biology during the past 17 years.
Laser light scattering comes in two major 'flavors': dynamic and static. This noninvasive technique provides a means for investigating key size and shape properties of macromolecules in solution. Light scattering has long been an indispensable tool to the polymer physical chemist, and is seeing increased use in exploring properties of biological macromolecules, alone and in association. As examples, recent investigations using light scattering have clearly demonstrated the relationship between the self-association and activity of important regulatory enzymes, and examined conformational properties of DNA and polysaccharides.
HIV-1 integrase is an essential enzyme in the life cycle of the virus, responsible for catalyzing the insertion of the viral genome into the host cell chromosome; it provides an attractive target for antiviral drug design. The previously reported crystal structure of the HIV-1 integrase core domain revealed that this domain belongs to the superfamily of polynucleotidyltransferases. However, the position of the conserved catalytic carboxylic acids differed from those observed in other enzymes of the class, and attempts to crystallize in the presence of the cofactor, Mg2+, were unsuccessful. We report here three additional crystal structures of the core domain of HIV-1 integrase mutants, crystallized in the presence and absence of cacodylate, as well as complexed with Mg2+. These three crystal forms, containing between them seven independent core domain structures, demonstrate the unambiguous extension of the previously disordered helix alpha4 toward the amino terminus from residue M154 and show that the catalytic E152 points in the general direction of the two catalytic aspartates, D64 and D116. In the vicinity of the active site, the structure of the protein in the absence of cacodylate exhibits significant deviations from the previously reported structures. These differences can be attributed to the modification of C65 and C130 by cacodylate, which was an essential component of the original crystallization mixture. We also demonstrate that in the absence of cacodylate this protein will bind to Mg2+, and could provide a satisfactory platform for binding of inhibitors.
HIV DNA integration is favored in active genes, but the underlying mechanism is unclear. Cellular lens epithelium-derived growth factor (LEDGF/p75) binds both chromosomal DNA and HIV integrase, and might therefore direct integration by a tethering interaction. We analyzed HIV integration in cells depleted for LEDGF/p75, and found that integration was (i) less frequent in transcription units, (ii) less frequent in genes regulated by LEDGF/p75 and (iii) more frequent in GC-rich DNA. LEDGF is thus the first example of a cellular protein controlling the location of HIV integration in human cells.
In this work, physicochemical properties of two globular proteinsbovine serum albumin (BSA) having a molecular weight of 67 kDa and human serum albumin (HSA) having a molecular weight of 69 kDawere characterized. The bulk characteristics of these proteins involved the diffusion coefficient (hydrodynamic radius), electrophoretic mobility, and dynamic viscosity as a function of protein solution concentration for various pH values. The hydrodynamic radius data suggested an association of protein molecules, most probably forming compact dimers. Using the hydrodynamic diameter and the electropheretic mobility data allowed the determination of the number of uncompensated (electrokinetic) charges on protein surfaces. The electrophoretic mobility data were converted to zeta potential values, which allowed one to determine the isoelectric point (iep) of these proteins. It was found to be at pH 5.1 for both proteins, in accordance with previous experimental data and theoretical estimations derived from amino acid composition and p K values. To determine further the stability of protein solutions, dynamic viscosity measurements were carried out as a function of their bulk volume concentration for various pH values. The intrinsic viscosity derived from these measurements was interpreted in terms of the Brenner model, which is applicable to hard spheroidal particles. It was found that the experimental values of the intrinsic viscosity of these proteins were in good agreement with this model when assuming protein dimensions of 9.5 x 5 x 5 nm3 (prolate spheroid). The possibility of forming linear aggregates of association degree higher than 2 was excluded by these measurements. It was concluded that the combination of dynamic viscosity and dynamic light scattering can be exploited as a convenient tool for detecting not only the onset of protein aggregation in suspensions but also the form and composition of these aggregates.