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ABSTRACT: A novel strategy for targeting the pathogenic organisms Candida albicans and Candida glabrata focuses on the development of potent and selective antifolates effective against dihydrofolate reductase. Crystal structure analysis suggested that an essential loop at the active site (Thr 58-Phe 66) differs from the analogous residues in the human enzyme, potentially providing a mechanism for achieving selectivity. In order to probe the role of this loop, we employed chemical synthesis, crystal structure determination and molecular dynamics simulations. The results of these analyses show that the loop residues undergo ligand-induced conformational changes that are similar among the fungal and human species.
Bioorganic & medicinal chemistry letters 01/2013; · 2.65 Impact Factor
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Pablo Gainza,
Kyle E Roberts,
Ivelin Georgiev,
Ryan H Lilien,
Daniel A Keedy,
Cheng-Yu Chen,
Faisal Reza, Amy C Anderson,
David C Richardson,
Jane S Richardson,
Bruce R Donald
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ABSTRACT: We have developed a suite of protein redesign algorithms that improves realistic in silico modeling of proteins. These algorithms are based on three characteristics that make them unique: (1) improved flexibility of the protein backbone, protein side-chains, and ligand to accurately capture the conformational changes that are induced by mutations to the protein sequence; (2) modeling of proteins and ligands as ensembles of low-energy structures to better approximate binding affinity; and (3) a globally optimal protein design search, guaranteeing that the computational predictions are optimal with respect to the input model. Here, we illustrate the importance of these three characteristics. We then describe osprey, a protein redesign suite that implements our protein design algorithms. osprey has been used prospectively, with experimental validation, in several biomedically relevant settings. We show in detail how osprey has been used to predict resistance mutations and explain why improved flexibility, ensembles, and provability are essential for this application. Availability: osprey is free and open source under a Lesser GPL license. The latest version is osprey 2.0. The program, user manual, and source code are available at www.cs.duke.edu/donaldlab/software.php. Contact: osprey@cs.duke.edu.
Methods in enzymology 01/2013; 523:87-107. · 1.90 Impact Factor
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ABSTRACT: Naturally occurring furanosteroids such as viridin and wortmannin have long been known as potent inhibitors of the lipid kinase PI-3K. We have been interested in directly accessing analogs of these complex natural products from abundant steroid feedstock materials. In this communication, we describe the synthesis of viridin/wortmannin hybrid molecules from readily available building blocks that function as PI-3K inhibitors and maintain their electrophilic properties. The compounds also show anti-proliferative effects against a breast cancer line.
Bioorganic & medicinal chemistry letters 09/2012; 22(22):6919-22. · 2.65 Impact Factor
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ABSTRACT: Resistance to trimethoprim (TMP) resulting from point mutations in the enzyme drug target dihydrofolate reductase (DHFR) drives the development of new antifolate inhibitors effective against methicillin-resistant Staphylococcus aureus (MRSA). For the past several years we have used structure-based design to create propargyl-linked antifolates that are highly potent antibacterial agents. In order to focus priority on the development of lead compounds with a low propensity to induce resistance, we prospectively evaluated resistance profiles for two of these inhibitors in an MRSA strain. By selection with the lead inhibitors, we generated resistant strains that contain single point mutations F98Y and H30N associated with TMP resistance and one novel mutation, F98I, in DHFR. Encouragingly, the pyridyl propargyl-linked inhibitor selects mutants at low frequency (6.85 × 10(-10) to 1.65 × 10(-9)) and maintains a low MIC (2.5 μg/ml) and a low mutant prevention concentration (1.25 μg/ml), strongly supporting its position as a lead compound. Results from this prospective screening method inform the continued design of antifolates effective against mutations at the Phe 98 position. Furthermore, the method can be used broadly to incorporate ideas for overcoming resistance early in the development process.
Antimicrobial Agents and Chemotherapy 04/2012; 56(7):3556-62. · 4.84 Impact Factor
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Amy C Anderson
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ABSTRACT: Proteomic and genomic discoveries have identified vast numbers of new drug targets for investigation. In the quest to discover drugs that modulate the function of these targets, identification of small-molecule drug leads is one of the earliest steps. Structure-based drug design has emerged as a valuable, inexpensive, and rapid computational resource that identifies lead compounds that are complementary to the structure of the target. Leads identified through this process are biologically evaluated and "hit compounds" with affinity and activity are further optimized. This chapter introduces the process of structure-based drug design, including preparation of the ligand database, preparation of the target structure, docking and scoring, and evaluation.
Methods in molecular biology (Clifton, N.J.) 01/2012; 823:359-66.
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ABSTRACT: Hospital- and community-acquired, complicated skin and soft tissue infections, often attributed to Staphylococcus aureus and Streptococcus pyogenes, present a significant health burden that is associated with increased health care costs and mortality. As these two species are difficult to discern on diagnosis and are associated with differential profiles of drug resistance, the development of an efficacious antibacterial agent that targets both organisms is a high priority. Herein we describe a structure-based drug development effort that has produced highly potent inhibitors of dihydrofolate reductase from both species. Optimized propargyl-linked antifolates containing a key pyridyl substituent display antibacterial activity against both methicillin-resistant S. aureus and S. pyogenes at MIC values below 0.1 µg/mL and minimal cytotoxicity against mammalian cells. Further evaluation against a panel of clinical isolates shows good efficacy against a range of important phenotypes such as hospital- and community-acquired strains as well as strains resistant to vancomycin.
PLoS ONE 01/2012; 7(2):e29434. · 4.09 Impact Factor
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ABSTRACT: Drug resistance is problematic in microbial disease, viral disease and cancer. Understanding at the outset that resistance will impact the effectiveness of any new drug that is developed for these disease categories is imperative. In this Feature, we detail approaches that have been taken with selected drug targets to reduce the susceptibility of new drugs to resistance mechanisms. We will also define the concepts of robust drugs and resilient targets, and discuss how the design of robust drugs and the selection of resilient targets can lead to successful strategies for combating resistance.
Drug discovery today 07/2011; 16(17-18):755-61. · 6.63 Impact Factor
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ABSTRACT: Candida albicans and Candida glabrata cause fungal bloodstream infections that are associated with significant mortality. As part of an effort to develop potent and selective antifolates that target dihydrofolate reductase (DHFR) from Candida species, we report three ternary crystal structures of C. albicans DHFR (CaDHFR) bound to novel propargyl-linked analogs. Consistent with earlier modeling results, these structures show that hydrophobic pockets in the binding site may be exploited to increase ligand potency. The crystal structures also confirm that loop residues Thr 58- Phe 66, which flank the active site and influence ligand potency and selectivity, adopt multiple conformations. To aid the development of a dual Candida spp. inhibitor, three new crystal structures of C. glabrata DHFR (CgDHFR) bound to similar ligands as those bound in the ternary structures of CaDHFR are also reported here. Loop residues 58-66 in CgDHFR and human DHFR are 1 and 3 Å closer to the folate binding site, respectively, than loop residues in CaDHFR, suggesting that a properly size ligand could be a potent and selective dual inhibitor of CaDHFR and CgDHFR.
Chemical Biology & Drug Design 07/2011; 78(4):505-12. · 2.28 Impact Factor
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ABSTRACT: INTRODUCTION: For > 50 years, drugs targeting the folate pathway have significantly impacted disease treatment as anticancer, antimicrobial and immunomodulatory agents. The discovery of novel antifolate agents with improved properties and superior activities remains an attractive strategy, both in academia and the pharmaceutical industry. AREAS COVERED: This review surveys the patent literature from 2006 to 2010 for small molecule inhibitors of enzymatic targets in the folate biosynthetic pathway. EXPERT OPINION: The pursuit of antifolates as anticancer and antimicrobial agents continues to be an active area of research. New patent disclosures reveal novel antifolate scaffolds, antifolates with improved drug-like properties and new strategies to effectively target cancer cells. The continued use of high resolution structural information has guided the discovery of several compounds. Owing to the need for high levels of potency and selectivity, especially in targeting pathogenic species, the use of high resolution crystal structures remains an important tool to guide the design of novel antifolates. Interestingly, the patents disclosing novel compounds were ones where X-ray crystallography was an integral component of the design process. Finally, a variety of new structures have been reported that may play an important role in the future development of therapeutic antifolates.
Expert Opinion on Therapeutic Patents 05/2011; 21(9):1293-308. · 3.57 Impact Factor
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ABSTRACT: Dihydrofolate reductase (DHFR) has been a well-recognized target for the development of therapeutics for human cancers for several decades. Classical inhibitors of DHFR use an active transport mechanism to gain access to the cell; disabling this mechanism creates a pathway for resistance. In response, recent research focuses on nonclassical lipid-soluble DHFR inhibitors that are designed to passively diffuse through the membrane. Here, a new series of propargyl-linked antifolates are investigated as potential nonclassical human DHFR inhibitors. Several of these compounds exhibit potent enzyme inhibition with 50% inhibition concentration values under 500 nM. Molecular docking investigations show that the compounds maintain conserved hydrogen bonds between the pyrimidine ring and the enzyme as well as form van der Waals interactions with critical residues in the active site. Interestingly, the most potent compound, 2,4-diamino-5-(3-(3,4,5-trimethoxyphenyl)prop-1-ynyl)-6-ethylpyrimidine (compound 35), is 3500-fold more potent than trimethoprim, a potent inhibitor of bacterial DHFR but weak inhibitor of human DHFR. The two structural differences between compound 35 and trimethoprim show that the propargyl linkage and the substitution at C6 of the pyrimidine ring are critical to the formation of contacts with Thr 56, Ser 59, Ile 60, Leu 22, Phe 31 and Phe 34 and hence, to enhancing potency. The propargyl-linked antifolates are efficient ligands with a high ratio of potency to the number of non-hydrogen atoms and represent a potentially fruitful avenue for future development of antineoplastic agents.
Journal of molecular graphics & modelling 02/2011; 29(5):608-13. · 2.17 Impact Factor
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ABSTRACT: Several antifolates, including trimethoprim (TMP) and a series of propargyl-linked analogues, bind dihydrofolate reductase from Bacillus anthracis (BaDHFR) with lower affinity than is typical in other bacterial species. To guide lead optimization for BaDHFR, we explored a new approach to determine structure-activity relationships whereby the enzyme is altered and the analogues remain constant, essentially reversing the standard experimental design. Active site mutants of the enzyme, Ba(F96I)DHFR and Ba(Y102F)DHFR, were created and evaluated with enzyme inhibition assays and crystal structures. The affinities of the antifolates increase up to 60-fold with the Y102F mutant, suggesting that interactions with Tyr 102 are critical for affinity. Crystal structures of the enzymes bound to TMP and propargyl-linked inhibitors reveal the basis of TMP resistance and illuminate the influence of Tyr 102 on the lipophilic linker between the pyrimidine and aryl rings. Two new inhibitors test and validate these conclusions and show the value of the technique for providing new directions during lead optimization.
Journal of Medicinal Chemistry 09/2010; 53(20):7327-36. · 4.80 Impact Factor
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ABSTRACT: Drug resistance resulting from mutations to the target is an unfortunate common phenomenon that limits the lifetime of many of the most successful drugs. In contrast to the investigation of mutations after clinical exposure, it would be powerful to be able to incorporate strategies early in the development process to predict and overcome the effects of possible resistance mutations. Here we present a unique prospective application of an ensemble-based protein design algorithm, K*, to predict potential resistance mutations in dihydrofolate reductase from Staphylococcus aureus using positive design to maintain catalytic function and negative design to interfere with binding of a lead inhibitor. Enzyme inhibition assays show that three of the four highly-ranked predicted mutants are active yet display lower affinity (18-, 9-, and 13-fold) for the inhibitor. A crystal structure of the top-ranked mutant enzyme validates the predicted conformations of the mutated residues and the structural basis of the loss of potency. The use of protein design algorithms to predict resistance mutations could be incorporated in a lead design strategy against any target that is susceptible to mutational resistance.
Proceedings of the National Academy of Sciences 08/2010; 107(31):13707-12. · 9.68 Impact Factor
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ABSTRACT: Resistance to therapeutics such as trimethoprim-sulfamethoxazole has become an increasing problem in strains of methicillin-resistant Staphylococcus aureus (MRSA). Clinically isolated trimethoprim-resistant strains reveal a double mutation, H30N/F98Y, in dihydrofolate reductase (DHFR). In order to develop novel and effective therapeutics against these resistant strains, we evaluated a series of propargyl-linked antifolate lead compounds for inhibition of the mutant enzyme. For the propargyl-linked antifolates, the F98Y mutation generates minimal (between 1.2- and 6-fold) losses of affinity and the H30N mutation generates greater losses (between 2.4- and 48-fold). Conversely, trimethoprim affinity is largely diminished by the F98Y mutation (36-fold) and is not affected by the H30N mutation. In order to elucidate a mechanism of resistance, we determined a crystal structure of a complex of this double mutant with a lead propargyl-linked antifolate. This structure suggests a resistance mechanism consistent both for the propargyl-linked class of antifolates and for trimethoprim that is based on the loss of a conserved water-mediated hydrogen bond.
Journal of Structural Biology 12/2009; 170(1):93-7. · 3.41 Impact Factor
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ABSTRACT: Ensembles of protein structures to simulate protein flexibility are widely used throughout several applications including virtual lead optimization where they have been shown to improve ligand ranking. Yet, there is no established convention for weighting individual scores generated from ensemble members. To investigate the best method for weighting ensemble scores for proper ligand ranking, a series of dihydrofolate reductase inhibitors was docked to ensembles of Candida albicans dihydrofolate reductase (CaDHFR) structures created from a molecular dynamics (MD) simulation. From a single MD simulation, two ensemble collections were generated, one of which was subjected to a minimization procedure to create a group of structures of equal probability. As expected, ligand ranking accuracy was significantly improved when Boltzmann weighting was applied to the energies of the ensemble without structural minimization (60%), relative to that achieved with averaging (36%). However, accuracy was further improved (72%) by averaging docking scores across a minimized ensemble. To examine whether this accuracy results from structural variation in the single trajectory versus the possibility that error is minimized by averaging, a third collection of receptor structures was created in which each member was taken from an independent molecular dynamics simulation after minimization. Comparison of the docking accuracy results from the single trajectory (72%) to this third collection (61%) showed decreased accuracy, suggesting that ligands are more accurately oriented and assessed when docked to the minimized ensemble from a single MD trajectory, an effect that is more than simply error minimization. Averaging docking scores over a minimized ensemble of another target, influenza A neuraminidase, yielded a ligand ranking accuracy of 83%, representing a 24% improvement over other methods tested.
Journal of Chemical Information and Modeling 12/2009; 49(12):2813-9. · 4.68 Impact Factor
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ABSTRACT: In order to develop new antifungal agents effective against two species of Candida, we have designed a series of dihydrofolate reductase (DHFR) inhibitors. Here, we explore the structure-activity relationships of these inhibitors toward Candida albicans DHFR by evaluating enzyme inhibition, antifungal activity and toxicity to mammalian cells. Analysis of docked complexes of the enzyme and inhibitors yields the structural basis of relative potency. The meta-biphenyl series of this class exhibits the greatest enzyme inhibition, selectivity and antifungal activity.
Bioorganic & medicinal chemistry 07/2009; 17(14):4866-72. · 2.82 Impact Factor
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ABSTRACT: Phosphoinositide-3-kinase is a pivotal protein involved in a wide variety of signaling cascades and there has been a great deal of interest in the development of potent and selective inhibitors of this enzyme. In this review, the potency and selectivity of the known inhibitors is presented along with key structural information that helps rationalize the observed trends.
Organic & Biomolecular Chemistry 04/2009; 7(5):840-50. · 3.70 Impact Factor
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ABSTRACT: We report a computational, structure-based redesign of the phenylalanine adenylation domain of the nonribosomal peptide synthetase enzyme gramicidin S synthetase A (GrsA-PheA) for a set of noncognate substrates for which the wild-type enzyme has little or virtually no specificity. Experimental validation of a set of top-ranked computationally predicted enzyme mutants shows significant improvement in the specificity for the target substrates. We further present enhancements to the methodology for computational enzyme redesign that are experimentally shown to result in significant additional improvements in the target substrate specificity. The mutant with the highest activity for a noncognate substrate exhibits 1/6 of the wild-type enzyme/wild-type substrate activity, further confirming the feasibility of our computational approach. Our results suggest that structure-based protein design can identify active mutants different from those selected by evolution.
Proceedings of the National Academy of Sciences 03/2009; 106(10):3764-9. · 9.68 Impact Factor
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ABSTRACT: Both hospital- and community-acquired Staphylococcus aureus infections have become major health concerns in terms of morbidity, suffering and cost. Trimethoprim-sulfamethoxazole (TMP-SMZ) is an alternative treatment for methicillin-resistant S. aureus (MRSA) infections. However, TMP-resistant strains have arisen with point mutations in dihydrofolate reductase (DHFR), the target for TMP. A single point mutation, F98Y, has been shown biochemically to confer the majority of this resistance to TMP. Using a structure-based approach, we have designed a series of novel propargyl-linked DHFR inhibitors that are active against several trimethoprim-resistant enzymes. We screened this series against wild-type and mutant (F98Y) S. aureus DHFR and found that several are active against both enzymes and specifically that the meta-biphenyl class of these inhibitors is the most potent. In order to understand the structural basis of this potency, we determined eight high-resolution crystal structures: four each of the wild-type and mutant DHFR enzymes bound to various propargyl-linked DHFR inhibitors. In addition to explaining the structure-activity relationships, several of the structures reveal a novel conformation for the cofactor, NADPH. In this new conformation that is predominantly associated with the mutant enzyme, the nicotinamide ring is displaced from its conserved location and three water molecules complete a network of hydrogen bonds between the nicotinamide ring and the protein. In this new position, NADPH has reduced interactions with the inhibitor. An equilibrium between the two conformations of NADPH, implied by their occupancies in the eight crystal structures, is influenced both by the ligand and the F98Y mutation. The mutation induced equilibrium between two NADPH-binding conformations may contribute to decrease TMP binding and thus may be responsible for TMP resistance.
Journal of Molecular Biology 03/2009; 387(5):1298-308. · 4.00 Impact Factor
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ABSTRACT: Candida glabrata, a fungal strain resistant to many commonly administered antifungal agents, has become an emerging threat to human health. In previous work, we validated that the essential enzyme, dihydrofolate reductase, is a drug target in C. glabrata. Using a crystal structure of dihydrofolate reductase from C. glabrata bound to an initial lead compound, we designed a class of biphenyl antifolates that potently and selectively inhibit both the enzyme and the growth of the fungal culture. In this work, we explore the structure-activity relationships of this class of antifolates with four new high resolution crystal structures of enzyme:inhibitor complexes and the synthesis of four new inhibitors. The designed inhibitors are intended to probe key hydrophobic pockets visible in the crystal structure. The crystal structures and an evaluation of the new compounds reveal that methyl groups at the meta and para positions of the distal phenyl ring achieve the greatest number of interactions with the pathogenic enzyme and the greatest degree of selectivity over the human enzyme. Additionally, antifungal activity can be tuned with substitution patterns at the propargyl and para-phenyl positions.
Chemical Biology & Drug Design 02/2009; 73(1):62-74. · 2.28 Impact Factor
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ABSTRACT: Trimethoprim, an antifolate commonly prescribed in combination with sulfamethoxazole, potently inhibits several prokaryotic species of dihydrofolate reductase (DHFR). However, several eukaryotic pathogenic organisms are resistant to trimethoprim, preventing its effective use as a therapeutic for those infections. We have been building a program to reengineer trimethoprim to more potently and selectively inhibit eukaryotic species of DHFR as a viable strategy for new drug discovery targeting several opportunistic pathogens. We have developed a series of compounds that exhibit potent and selective inhibition of DHFR from the parasitic protozoa Cryptosporidium and Toxoplasma as well as the fungus Candida glabrata. A comparison of the structures of DHFR from the fungal species Candida glabrata and Pneumocystis suggests that the compounds may also potently inhibit Pneumocystis DHFR.
Eukaryotic Cell 02/2009; 8(4):483-6. · 3.60 Impact Factor
