Discovery, characterization, and lead optimization of 7-azaindole non-nucleoside HIV-1 reverse transcriptase inhibitors.

A library of 585 compounds built off a 7-azaindole core was evaluated for anti-HIV-1 activity, and ten hits emerged with submicromolar potency and therapeutic index >100. Of these, three were identified as non-nucleoside reverse transcriptase (RT) inhibitors and were assayed against relevant resistant mutants. Lead compound 8 inhibited RT with submicromolar potency (IC50=0.73µM) and also maintained some activity against the clinically important RT mutants K103N and Y181C (IC50=9.2, 3.5µM) in cell-free assays. Free energy perturbation guided lead optimization resulted in the development of a compound with a two-fold increase in potency against RT (IC50=0.36µM). These data highlight the discovery of a unique scaffold with the potential to move forward as next-generation anti-HIV-1 agents.

Design, synthesis and evaluation of novel anti-HCV molecules that deliver intracellularly three highly potent NS5A inhibitors.

The design and synthesis of new non-symmetrical NS5A inhibitors with sulfur containing amino acids is reported along with their ability to block HCV replication in an HCV 1b replicon system. These compounds display EC50 values in the picomolar range with a large therapeutic index (>10(6)). Moreover, cellular pharmacology studies show that our preferred compounds intracellularly deliver three potent NS5A inhibitors.

Substrate mimicry: HIV-1 reverse transcriptase recognizes 6-modified-3'-azido-2',3'-dideoxyguanosine-5'-triphosphates as adenosine analogs.

ß-D-3'-Azido-2',3'-dideoxyguanosine (3'-azido-ddG) is a potent inhibitor of HIV-1 replication with a superior resistance profile to zidovudine. Recently, we identified five novel 6-modified-3'-azido-ddG analogs that exhibit similar or superior anti-HIV-1 activity compared to 3'-azido-ddG in primary cells. To gain insight into their structure-activity-resistance relationships, we synthesized their triphosphate (TP) forms and assessed their ability to inhibit HIV-1 reverse transcriptase (RT). Steady-state and pre-steady-state kinetic experiments show that the 6-modified-3'-azido-ddGTP analogs act as adenosine rather than guanosine mimetics in DNA synthesis reactions. The order of potency of the TP analogs against wild-type RT was: 3'-azido-2,6-diaminopurine >3'-azido-6-chloropurine; 3'-azido-6-N-allylaminopurine > 2-amino-6-N,N-dimethylaminopurine; 2-amino-6-methoxypurine. Molecular modeling studies reveal unique hydrogen-bonding interactions between the nucleotide analogs and the template thymine base in the active site of RT. Surprisingly, the structure-activity relationship of the analogs differed in HIV-1 RT ATP-mediated excision assays of their monophosphate forms, suggesting that it may be possible to rationally design a modified base analog that is efficiently incorporated by RT but serves as a poor substrate for ATP-mediated excision reactions. Overall, these studies identify a promising strategy to design novel nucleoside analogs that exert profound antiviral activity against both WT and drug-resistant HIV-1.

Synthesis and evaluation of non-dimeric HCV NS5A inhibitors.

Based on the symmetrical bidentate structure of the NS5A inhibitor BMS-790052, a series of new monodentate molecules were designed. The synthesis of 36 new non-dimeric NS5A inhibitors is reported along with their ability to block HCV replication in an HCV 1b replicon system. Among them compound 5a showed picomolar range activity along with an excellent selectivity index (SI > 90,000).

Drugs that target dynamic microtubules: a new molecular perspective.

Microtubules have long been considered an ideal target for anticancer drugs because of the essential role they play in mitosis, forming the dynamic spindle apparatus. As such, there is a wide variety of compounds currently in clinical use and in development that act as antimitotic agents by altering microtubule dynamics. Although these diverse molecules are known to affect microtubule dynamics upon binding to one of the three established drug domains (taxane, vinca alkaloid, or colchicine site), the exact mechanism by which each drug works is still an area of intense speculation and research. In this study, we review the effects of microtubule-binding chemotherapeutic agents from a new perspective, considering how their mode of binding induces conformational changes and alters biological function relative to the molecular vectors of microtubule assembly or disassembly. These "biological vectors" can thus be used as a spatiotemporal context to describe molecular mechanisms by which microtubule-targeting drugs work.

Selection and characterization of HIV-1 with a novel S68 deletion in reverse transcriptase.

Resistance to human immunodeficiency virus type 1 (HIV-1) represents a significant problem in the design of novel therapeutics and the management of treatment regimens in infected persons. Resistance profiles can be elucidated by defining modifications to the viral genome conferred upon exposure to novel nucleoside reverse transcriptase (RT) inhibitors (NRTI). In vitro testing of HIV-1LAI-infected primary human lymphocytes treated with ß-D-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine (DFC; Dexelvucitabine; Reverset) produced a novel deletion of AGT at codon 68 (S68Δ) alone and in combination with K65R that differentially affects drug response. Dual-approach clone techniques utilizing TOPO cloning and pyrosequencing confirmed the novel S68Δ in the HIV-1 genome. The S68Δ HIV-1 RT was phenotyped against various antiviral agents in a heteropolymeric DNA polymerase assay and in human lymphocytes. Drug susceptibility results indicate that the S68Δ displayed a 10- to 30-fold increase in resistance to DFC, lamivudine, emtricitabine, tenofovir, abacavir, and amdoxovir and modest resistance to stavudine, ß-d-2',3'-oxa-5-fluorocytidine, or 9-(ß-D-1,3-dioxolan-4-yl)guanine and remained susceptible to 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine (ddI), 1-(ß-D-dioxolane)thymine (DOT) and lopinavir. Modeling revealed a central role for S68 in affecting conformation of the ß3-ß4 finger region and provides a rational for the selective resistance. These data indicate that the novel S68Δ is a previously unrecognized deletion that may represent an important factor in NRTI multidrug resistance treatment strategies.

The tubulin binding mode of microtubule stabilizing agents studied by electron crystallography.

Since tubulin was discovered in 1967, drug probes have been used to manipulate mechanisms of microtubule polymerization and disassembly. In parallel, advances in optical imagery, electron microscopy, along with both electron and X-ray diffraction have provided ability to "see" the molecular underpinning of these machines. Nanoscale mapping of different tubulin polymers formed in the presence of different drugs and cofactors provide a context for examining the dynamic features relevant to their biological activity. Models built from EM maps have been used to understand the binding of stabilizing drugs such as taxanes and epothilones, to predict more effective molecules, and to explain mutation based resistance. Here, we discuss drug binding in the context of different polymeric forms and propose a trigger mechanism associated with microtubules' dynamic instability.

Approaches for the development of antiviral compounds: the case of hepatitis C virus.

Traditional methods for general drug discovery typically include evaluating random compound libraries for activity in relevant cell-free or cell-based assays. Success in antiviral development has emerged from the discovery of more focused libraries that provide clues about structure activity relationships. Combining these with more recent approaches including structural biology and computational modeling can work efficiently to hasten discovery of active molecules, but that is not enough. There are issues related to biology, toxicology, pharmacology, and metabolism that have to be addressed before a hit compound becomes nominated for clinical development. The objective of gaining early preclinical knowledge is to reduce the risk of failure in Phases 1, 2, and 3, leading to the goal of approved drugs that benefit the infected individual. This review uses hepatitis C virus (HCV), for which we still do not have an ideal therapeutic modality, as an example of the multidisciplinary efforts needed to discover new antiviral drugs for the benefit of humanity.

Rational design of the microtubule-targeting anti-breast cancer drug EM015.

We studied in silico docking of noscapine onto tubulin, combined with calculations of surface charge, pi-pi, van der Waals, and hydrogen bonding interactions, to rationally design a new compound, EM015. This tubulin-binding semisynthetic compound is a selective and potent anti-breast cancer agent and displays a 20-fold lower IC(50) against many tumor cells compared with our founding compound, (S)-6,7-dimethoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro[1,3]-dioxolo-[4,5-g]isoquinolin-5-yl)isobenzo-furan-1(3H)-one (noscapine). Furthermore, EM015 is also effective against a variety of drug-resistant cells. Surprisingly, the cell cycle profile of nontumorigenic normal cells is not affected. Many antimicrotubule cancer drugs in clinic today, particularly taxanes and Vincas, face challenges including frequent visits to the hospital for prolonged i.v. infusions, toxicities, and tumor recurrences due to drug resistance. EM015, on the other hand, is orally available, regresses breast tumor xenografts in nude mice models, and increases longevity. Furthermore, we have failed to observe any detectable toxicity in tissues, such as liver, kidney, spleen, lung, heart, and brain, as well as neurons, which are common targets of antimicrotubule drug therapy. Thus, EM015 has a great promise in the clinic.

Farnesyl transferase inhibitors impair chromosomal maintenance in cell lines and human tumors by compromising CENP-E and CENP-F function.

Farnesyl transferase inhibitors (FTI) exhibit anticancer activity as a single agent in preclinical studies and show promise in combination with other therapeutics in clinical trials. Previous studies show that FTIs arrest cancer cells in mitosis; however, the mechanism by which this occurs is unclear. Here, we observed that treatment of various cancer cell lines with the FTI lonafarnib caused mitotic chromosomal alignment defects, leaving cells in a pseudometaphase state, whereby both aligned chromosomes and chromosomes juxtaposed to the spindle poles (termed "lagging chromosomes") were observed in the same cell. To determine how this occurs, we investigated the functionality of two farnesylated mitotic proteins, CENP-E and CENP-F, which mediate chromosomal capture and alignment. The data show that lonafarnib in proliferating cancer cells depletes CENP-E and CENP-F from metaphase but not prometaphase kinetochores. Loss of CENP-E and CENP-F metaphase localization triggered aberrant chromosomal maintenance, causing aligned chromosomes to be prematurely released from the spindle equator and become lagging chromosomes, resulting in a mitotic delay. Furthermore, lonafarnib treatment reduces sister kinetochore tension and activates the BubR1 spindle checkpoint, suggesting that farnesylation of CENP-E and CENP-F is critical for their functionality in maintaining kinetochore-microtubule interactions. Importantly, apparently similar chromosomal alignment defects were observed in head and neck tumors samples from a phase I trial with lonafarnib, providing support that lonafarnib disrupts chromosomal maintenance in human cancers. Lastly, to examine how farnesylation could regulate CENP-E in mediating kinetochore-microtubule attachments, we examined possible docking motifs of a farnesyl group on the outer surface of the microtubule. This analysis revealed three hydrophobic patches on the tubulin dimer for insertion of a farnesyl group, alluding to the possibility of an association between a farnesyl group and the microtubule.

"Plate cherry picking": a novel semi-sequential screening paradigm for cheaper, faster, information-rich compound selection.

This work describes a novel semi-sequential technique for in silico enhancement of high-throughput screening (HTS) experiments now employed at Novartis. It is used in situations in which the size of the screen is limited by the readout (e.g., high-content screens) or the amount of reagents or tools (proteins or cells) available. By performing computational chemical diversity selection on a per plate basis (instead of a per compound basis), 25% of the 1,000,000-compound screening was optimized for general initial HTS. Statistical models are then generated from target-specific primary results (percentage inhibition data) to drive the cherry picking and testing from the entire collection. Using retrospective analysis of 11 HTS campaigns, the authors show that this method would have captured on average two thirds of the active compounds (IC(50) < 10 microM) and three fourths of the active Murcko scaffolds while decreasing screening expenditure by nearly 75%. This result is true for a wide variety of targets, including G-protein-coupled receptors, chemokine receptors, kinases, metalloproteinases, pathway screens, and protein-protein interactions. Unlike time-consuming "classic" sequential approaches that require multiple iterations of cherry picking, testing, and building statistical models, here individual compounds are cherry picked just once, based directly on primary screening data. Strikingly, the authors demonstrate that models built from primary data are as robust as models built from IC(50) data. This is true for all HTS campaigns analyzed, which represent a wide variety of target classes and assay types.

Flexible 3D pharmacophores as descriptors of dynamic biological space.

Development of a pharmacophore hypothesis related to small-molecule activity is pivotal to chemical optimization of a series, since it defines features beneficial or detrimental to activity. Although crystal structures may provide detailed 3D interaction information for one molecule with its receptor, docking a different ligand to that model often leads to unreliable results due to protein flexibility. Graham Richards' lab was one of the first groups to utilize "fuzzy" pattern recognition algorithms taken from the field of image processing to solve problems in protein modeling. Thus, descriptor "fuzziness" was partly able to emulate conformational flexibility of the target while simultaneously enhancing the speed of the search. In this work, we extend these developments to a ligand-based method for describing and aligning molecules in flexible chemical space termed FEature POint PharmacophoreS (FEPOPS), which allows exploration of dynamic biological space. We develop a novel, combinatorial algorithm for molecular comparisons and evaluate it using the WOMBAT dataset. The new approach shows superior retrospective virtual screening performance than earlier shape-based or charge-based algorithms. Additionally, we use target prediction to evaluate how FEPOPS alignments match the molecules biological activity by identifying the atoms and features that make the key contributions to overall chemical similarity. Overall, we find that FEPOPS are sufficiently fuzzy and flexible to find not only new ligand scaffolds, but also challenging molecules that occupy different conformational states of dynamic biological space as from induced fits.

Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of beta-tubulin (Asp26Glu) and less stable microtubules.

Resistance to paclitaxel-based therapy is frequently encountered in the clinic. The mechanisms of intrinsic or acquired paclitaxel resistance are not well understood. We sought to characterize the resistance mechanisms that develop upon chronic exposure of a cancer cell line to paclitaxel in the presence of the P-glycoprotein reversal agent, CL-347099. The epidermoid tumor line KB-3-1 was exposed to increasing concentrations of paclitaxel and 5 micromol/L CL-347099 for up to 1 year. Cells grown in 15 nmol/L paclitaxel plus CL-347099 (KB-15-PTX/099) developed 18-fold resistance to paclitaxel and were dependent upon paclitaxel for maximal growth. They grew well and retained resistance to paclitaxel when grown in athymic mice. Cross-resistance (3- to 5-fold) was observed in tissue culture to docetaxel, the novel taxane MAC-321, and epothilone B. Collateral sensitivity (approximately 3-fold) was observed to the depolymerizing agents vinblastine, dolastatin-10, and HTI-286. KB-15-PTX/099-resistant cells did not overexpress P-glycoprotein nor did they have an alteration of [14C]paclitaxel accumulation compared with parental cells. However, a novel point mutation (T to A) resulting in Asp26 to glutamate substitution in class I (M40) beta-tubulin was found. Based on an electron crystallography structure of Zn-stabilized tubulin sheets, the phenyl ring of C-3' NHCO-C6H5 of paclitaxel makes contact with Asp26 of beta-tubulin, suggesting a ligand-induced mutation. Optimized model complexes of paclitaxel, docetaxel, and MAC-321 in beta-tubulin show a novel hydrogen bonding pattern for the glutamate mutant and rationalize the observed resistance profiles. However, a mutation in the paclitaxel binding pocket does not explain the phenotype completely. KB-15-PTX/099 cells have impaired microtubule stability as determined by a reduced percentage of tubulin in microtubules and reflected by less acetylated tubulin. These results suggest that a mutation in tubulin might affect microtubule stability as well as drug binding and contribute to the observed resistance profile.

Substrates and Inhibitors of SAMHD1.

SAMHD1 hydrolyzes 2'-deoxynucleoside-5'-triphosphates (dNTPs) into 2'-deoxynucleosides and inorganic triphosphate products. In this paper, we evaluated the impact of 2' sugar moiety substitution for different nucleotides on being substrates for SAMHD1 and mechanisms of actions for the results. We found that dNTPs ((2'R)-2'-H) are only permissive in the catalytic site of SAMHD1 due to L150 exclusion of (2'R)-2'-F and (2'R)-2'-OH nucleotides. However, arabinose ((2'S)-2'-OH) nucleoside-5'-triphosphates analogs are permissive to bind in the catalytic site and be hydrolyzed by SAMHD1. Moreover, when the (2'S)-2' sugar moiety is increased to a (2'S)-2'-methyl as with the SMDU-TP analog, we detect inhibition of SAMHD1's dNTPase activity. Our computational modeling suggests that (2'S)-2'-methyl sugar moiety clashing with the Y374 of SAMHD1. We speculate that SMDU-TP mechanism of action requires that the analog first docks in the catalytic pocket of SAMHD1 but prevents the A351-V378 helix conformational change from being completed, which is needed before hydrolysis can occur. Collectively we have identified stereoselective 2' substitutions that reveal nucleotide substrate specificity for SAMHD1, and a novel inhibitory mechanism for the dNTPase activity of SAMHD1. Importantly, our data is beneficial for understanding if FDA-approved antiviral and anticancer nucleosides are hydrolyzed by SAMHD1 in vivo.

Bridging chemical and biological space: "target fishing" using 2D and 3D molecular descriptors.

Bridging chemical and biological space is the key to drug discovery and development. Typically, cheminformatics methods operate under the assumption that similar chemicals have similar biological activity. Ideally then, one could predict a drug's biological function(s) given only its chemical structure by similarity searching in libraries of compounds with known activities. In practice, effectively choosing a similarity metric is case dependent. This work compares both 2D and 3D chemical descriptors as tools for predicting the biological targets of ligand probes, on the basis of their similarity to reference molecules in a 46,000 compound, biologically annotated chemical database. Overall, we found that the 2D methods employed here outperform the 3D (88% vs 67% success) in correct target prediction. However, the 3D descriptors proved superior in cases of probes with low structural similarity to other compounds in the database (singletons). Additionally, the 3D method (FEPOPS) shows promise for providing pharmacophoric alignment of the small molecules' chemical features consistent with those seen in experimental ligand/ receptor complexes. These results suggest that querying annotated chemical databases with a systematic combination of both 2D and 3D descriptors will prove more effective than employing single methods.

Ligand similarity guided receptor selection enhances docking accuracy and recall for non-nucleoside HIV reverse transcriptase inhibitors.

Non-nucleoside reverse transcriptase inhibitors (NNRTI) are allosteric inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a viral polymerase essential to infection. Despite the availability of >150 NNRTI-bound RT crystal structures, rational design of new NNRTI remains challenging because of the variability of their induced fit, hydrophobic binding patterns. Docking NNRTI yields inconsistent results that vary markedly depending on the receptor structure used, as only 27% of the >20k cross-docking calculations we performed using known NNRTI were accurate. In order to determine if a hospitable receptor for docking could be selected a priori, we evaluated more than 40 chemical descriptors for their ability to pre-select a best receptor for NNRTI cross-docking. The receptor selection was based on similarity scores between the bound- and target-ligands generated by each descriptor. The top descriptors were able to double the probability of cross-docking accuracy over random receptor selection. Additionally, recall of known NNRTI from a large library of similar decoys was increased using the same approach. The results demonstrate the utility of pre-selecting receptors when docking into difficult targets. Graphical Abstract Cross-docking accuracy increases when using chemical descriptors to determine the NNRTI with maximum similarity to the new compound and then docking into its respective receptor.

Approaches to hepatitis C treatment and cure using NS5A inhibitors.

Recent progress in the understanding of hepatitis C virus (HCV) biology and the availability of in vitro models to study its replication have facilitated the development of direct-acting antiviral agents (DAAs) that target specific steps in the viral replication cycle. Currently, there are three major classes of DAA in clinical development: NS3/4A protease inhibitors, NS5B polymerase inhibitors, and NS5A directed inhibitors. Several compounds thought to bind directly with NS5A are now in various clinical trial phases, including the most advanced, daclatasvir (BMS-790052), ledipasvir (GS-5885), and ABT-267. While many NS5A-targeted compounds demonstrate picomolar potency, the exact mechanism(s) of their action is still unclear. In the clinic, NS5A HCV inhibitors show promise as important components in DAA regimens and have multifunctionality. In addition to inhibiting viral replication, they may synergize with other DAAs, possibly by modulating different viral proteins, to help suppress the emergence of resistant viruses. Structure-based models have identified target interaction domains and spatial interactions that explain drug resistance for mutations at specific positions (eg, residues 93 and 31) within NS5A and potential binding partners. This review provides, insights into the unique complexity of NS5A as a central platform for multiple viral/host protein interactions, and possible mechanism(s) for the NS5A inhibitors currently undergoing clinical trials that target this nonstructural viral protein.

Asymmetric binding to NS5A by daclatasvir (BMS-790052) and analogs suggests two novel modes of HCV inhibition.

Symmetric, dimeric daclatasvir (BMS-790052) is the clinical lead for a class of picomolar inhibitors of HCV replication. While specific, resistance-bearing mutations at positions 31 and 93 of domain I strongly suggest the viral NS5A as target, structural mechanism(s) for the drugs' activities and resistance remains unclear. Several previous models suggested symmetric binding modes relative to the homodimeric target; however, none can fully explain SAR details for this class. We present semiautomated workflows to model potential receptor conformations for docking. Surprisingly, ranking docked hits with our library-derived 3D-pharmacophore revealed two distinct asymmetric binding modes, at a conserved poly-proline region between 31 and 93, consistent with SAR. Interfering with protein-protein interactions at this membrane interface can explain potent inhibition of replication-complex formation, resistance, effects on lipid droplet distribution, and virion release. These detailed interaction models and proposed mechanisms of action will allow structure-based design of new NS5A directed compounds with higher barriers to HCV resistance.

Variation of human immunodeficiency virus type-1 reverse transcriptase within the simian immunodeficiency virus genome of RT-SHIV.

RT-SHIV is a chimera of simian immunodeficiency virus (SIV) containing the reverse transcriptase (RT)-encoding region of human immunodeficiency virus type 1 (HIV-1) within the backbone of SIVmac239. It has been used in a non-human primate model for studies of non-nucleoside RT inhibitors (NNRTI) and highly active antiretroviral therapy (HAART). We and others have identified several mutations that arise in the "foreign" HIV-1 RT of RT-SHIV during in vivo replication. In this study we catalogued amino acid substitutions in the HIV-1 RT and in regions of the SIV backbone with which RT interacts that emerged 30 weeks post-infection from seven RT-SHIV-infected rhesus macaques. The virus set points varied from relatively high virus load, moderate virus load, to undetectable virus load. The G196R substitution in RT was detected from 6 of 7 animals at week 4 post-infection and remained in virus from 4 of 6 animals at week 30. Virus from four high virus load animals showed several common mutations within RT, including L74V or V75L, G196R, L214F, and K275R. The foreign RT from high virus load isolates exhibited as much variation as that of the highly variable envelope surface glycoprotein, and 10-fold higher than that of the native RT of SIVmac239. Isolates from moderate virus load animals showed much less variation in the foreign RT than the high virus load isolates. No variation was found in SIVmac239 genes known to interact with RT. Our results demonstrate substantial adaptation of the foreign HIV-1 RT in RT-SHIV-infected macaques, which most likely reflects selective pressure upon the foreign RT to attain optimal activity within the context of the chimeric RT-SHIV and the rhesus macaque host.

Synthesis, antiviral activity, cytotoxicity and cellular pharmacology of l-3'-azido-2',3'-dideoxypurine nucleosides.

Microwave-assisted optimized transglycosylation reactions were used to prepare eleven modified l-3'-azido-2',3'-dideoxypurine nucleosides. These l-nucleoside analogs were evaluated against HIV and hepatitis B virus. The l-3'-azido-2',3'-dideoxypurines nucleosides were metabolized to nucleoside 5'-triphosphates in primary human lymphocytes, but exhibited weak or no antiviral activity against HIV-1. The nucleosides were also inactive against HBV in HepG2 cells. Pre-steady state kinetic experiments demonstrated that the l-3'-azido-2',3'-dideoxypurine triphosphates could be incorporated by purified HIV-1 reverse transcriptase, although their catalytic efficiency (k(pol)/K(d)) of incorporation was low. Interestingly, a phosphoramidate prodrug of l-3'-azido-2',3'-dideoxyadenosine exhibited anti-HIV-1 activity without significant toxicity.