Drug Design Strategies to Avoid Resistance in Direct-Acting Antivirals and Beyond.

Drug resistance is prevalent across many diseases, rendering therapies ineffective with severe financial and health consequences. Rather than accepting resistance after the fact, proactive strategies need to be incorporated into the drug design and development process to minimize the impact of drug resistance. These strategies can be derived from our experience with viral disease targets where multiple generations of drugs had to be developed to combat resistance and avoid antiviral failure. Significant efforts including experimental and computational structural biology, medicinal chemistry, and machine learning have focused on understanding the mechanisms and structural basis of resistance against direct-acting antiviral (DAA) drugs. Integrated methods show promise for being predictive of resistance and potency. In this review, we give an overview of this research for human immunodeficiency virus type 1, hepatitis C virus, and influenza virus and the lessons learned from resistance mechanisms of DAAs. These lessons translate into rational strategies to avoid resistance in drug design, which can be generalized and applied beyond viral targets. While resistance may not be completely avoidable, rational drug design can and should incorporate strategies at the outset of drug development to decrease the prevalence of drug resistance.

Evaluation of Free Energy Calculations for the Prioritization of Macrocycle Synthesis.

A tremendous research and development effort was exerted toward combating chronic hepatitis C, ultimately leading to curative oral treatments, all of which are targeting viral proteins. Despite the advantage of numerous targets allowing for broad hepatitis C virus (HCV) genotype coverage, the only host target inhibitors that advanced into clinical development were Cyclosporin A based cyclophilin inhibitors. While cyclosporin-based molecules typically require a fermentation process, Gilead successfully pursued a fully synthetic, oral program based on Sanglifehrin A. The drug discovery process, though greatly helped by facile crystallography, was still hampered by the limitations in the accuracy of predictive computational methods for prioritizing compound ideas. Recent advances in accuracy and speed of free energy perturbation (FEP) methods, however, are attractive for prioritizing and derisking synthetically challenging molecules and potentially could have had a significant impact on the speed of the development of this program. Here in our simulated prospective study, the binding free energies of 26 macrocyclic cyclophilin inhibitors were blindly predicted using FEP+ to test this hypothesis. The predictions had a low mean unsigned error (MUE) (1.1 kcal/mol) and accurately reproduced many design decisions from the program, suggesting that FEP+ has the potential to drive synthetic chemistry efforts by more accurately ranking compounds with nonintuitive structure-activity relationships (SARs).

Probing Structural Changes among Analogous Inhibitor-Bound Forms of HIV-1 Protease and a Drug-Resistant Mutant in Solution by Nuclear Magnetic Resonance.

In the era of state-of-the-art inhibitor design and high-resolution structural studies, detection of significant but small protein structural differences in the inhibitor-bound forms is critical to further developing the inhibitor. Here, we probed differences in HIV-1 protease (PR) conformation among darunavir and four analogous inhibitor-bound forms and compared them with a drug-resistant mutant using nuclear magnetic resonance chemical shifts. Changes in amide chemical shifts of wild-type (WT) PR among these inhibitor-bound forms, ΔCSP, were subtle but detectable and extended >10 Å from the inhibitor-binding site, asymmetrically between the two subunits of PR. Molecular dynamics simulations revealed differential local hydrogen bonding as the molecular basis of this remote asymmetric change. Inhibitor-bound forms of the drug-resistant mutant also showed a similar long-range ΔCSP pattern. Differences in ΔCSP values of the WT and the mutant (ΔΔCSPs) were observed at the inhibitor-binding site and in the surrounding region. Comparing chemical shift changes among highly analogous inhibitors and ΔΔCSPs effectively eliminated local environmental effects stemming from different chemical groups and enabled exploitation of these sensitive parameters to detect subtle protein conformational changes and to elucidate asymmetric and remote conformational effects upon inhibitor interaction.

Structural analysis of the active sites of dihydrofolate reductase from two species of Candida uncovers ligand-induced conformational changes shared among species.

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.

Scoring ensembles of docked protein:ligand interactions for virtual lead optimization.

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.

In vitro biological activity and structural analysis of 2,4-diamino-5-(2'-arylpropargyl)pyrimidine inhibitors of Candida albicans.

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.

Interdependence of Inhibitor Recognition in HIV-1 Protease.

Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1' subsite highly influences other subsites: the extension of the hydrophobic P1' moiety results in 1) reduced van der Waals contacts in the P2' subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.

Citrullination of NF-κB p65 promotes its nuclear localization and TLR-induced expression of IL-1ß and TNFα.

Many citrullinated proteins are known autoantigens in rheumatoid arthritis, a disease mediated by inflammatory cytokines, such as tumor necrosis factor-α (TNFα). Citrullinated proteins are generated by converting peptidylarginine to peptidylcitrulline, a process catalyzed by the peptidylarginine deiminases (PADs), including PAD1 to PAD4 and PAD6. Several major risk factors for rheumatoid arthritis are associated with heightened citrullination. However, the physiological role of citrullination in immune cells is poorly understood. We report that suppression of PAD activity attenuates Toll-like receptor-induced expression of interleukin-1ß (IL-1ß) and TNFα by neutrophils in vivo and in vitro but not their global transcription activity. Mechanistically, PAD4 directly citrullinates nuclear factor κB (NF-κB) p65 and enhances the interaction of p65 with importin α3, which brings p65 into the nucleus. The citrullination-enhanced interaction of p65 with importin α3 and its nuclear translocation and transcriptional activity can be attributed to citrullination of four arginine residues located in the Rel homology domain of p65. Furthermore, a rheumatoid arthritis-prone variant of PAD4, carrying three missense mutations, is more efficient in interacting with p65 and enhancing NF-κB activity. Together, these data not only demonstrate a critical role of citrullination in an NF-κB-dependent expression of IL-1ß and TNFα but also provide a molecular mechanism by which heightened citrullination propagates inflammation in rheumatoid arthritis. Accordingly, attenuating p65-mediated production of IL-1ß and TNFα by blocking the citrullination of p65 has great therapeutic potential in rheumatoid arthritis.

Structure-guided design of a high affinity inhibitor to human CtBP.

Oncogenic transcriptional coregulators C-terminal Binding Protein (CtBP) 1 and 2 possess regulatory d-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domains that provide an attractive target for small molecule intervention. Findings that the CtBP substrate 4-methylthio 2-oxobutyric acid (MTOB) can interfere with CtBP oncogenic activity in cell culture and in mice confirm that such inhibitors could have therapeutic benefit. Recent crystal structures of CtBP 1 and 2 revealed that MTOB binds in an active site containing a dominant tryptophan and a hydrophilic cavity, neither of which are present in other D2-HDH family members. Here, we demonstrate the effectiveness of exploiting these active site features for the design of high affinity inhibitors. Crystal structures of two such compounds, phenylpyruvate (PPy) and 2-hydroxyimino-3-phenylpropanoic acid (HIPP), show binding with favorable ring stacking against the CtBP active site tryptophan and alternate modes of stabilizing the carboxylic acid moiety. Moreover, ITC experiments show that HIPP binds to CtBP with an affinity greater than 1000-fold over that of MTOB, and enzymatic assays confirm that HIPP substantially inhibits CtBP catalysis. These results, thus, provide an important step, and additional insights, for the development of highly selective antineoplastic CtBP inhibitors.

Drug Resistance Mutations Alter Dynamics of Inhibitor-Bound HIV-1 Protease.

Under the selective pressure of therapy, HIV-1 protease mutants resistant to inhibitors evolve to confer drug resistance. Such mutations can impact both the dynamics and structures of the bound and unbound forms of the enzyme. Flap+ is a multidrug-resistant variant of HIV-1 protease with a combination of primary and secondary resistance mutations (L10I, G48V, I54V, V82A) and a strikingly altered thermodynamic profile for darunavir (DRV) binding relative to the wild-type protease. We elucidated the impact of these mutations on protein dynamics in the DRV-bound state using molecular dynamics simulations and NMR relaxation experiments. Both methods concur in that the conformational ensemble and dynamics of protease are impacted by the drug resistance mutations in Flap+ variant. Surprisingly this change in ensemble dynamics is different from that observed in the unliganded form of the same variant (Cai, Y. et al. J. Chem. Theory Comput.2012, 8, 3452-3462). Our comparative analysis of both inhibitor-free and bound states presents a comprehensive picture of the altered dynamics in drug-resistant mutant HIV-1 protease and underlies the importance of incorporating dynamic analysis of the whole system, including the unliganded state, into revealing drug resistance mechanisms.

Propargyl-linked antifolates are dual inhibitors of Candida albicans and Candida glabrata.

Species of Candida, primarily C. albicans and with increasing prevalence, C. glabrata, are responsible for the majority of fungal bloodstream infections that cause morbidity, especially among immune compromised patients. While the development of new antifungal agents that target the essential enzyme, dihydrofolate reductase (DHFR), in both Candida species would be ideal, previous attempts have resulted in antifolates that exhibit inconsistencies between enzyme inhibition and antifungal properties. In this article, we describe the evaluation of pairs of propargyl-linked antifolates that possess similar physicochemical properties but different shapes. All of these compounds are effective at inhibiting the fungal enzymes and the growth of C. glabrata; however, the inhibition of the growth of C. albicans is shape-dependent with extended para-linked compounds proving more effective than compact, meta-linked compounds. Using crystal structures of DHFR from C. albicans and C. glabrata bound to lead compounds, 13 new para-linked compounds designed to inhibit both species were synthesized. Eight of these compounds potently inhibit the growth of both fungal species with three compounds displaying dual MIC values less than 1 µg/mL. Analysis of the active compounds shows that shape and distribution of polar functionality is critical in achieving dual antifungal activity.

Crystal structures of Candida albicans dihydrofolate reductase bound to propargyl-linked antifolates reveal the flexibility of active site loop residues critical for ligand potency and selectivity.

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.

2,4-Diamino-5-(2'-arylpropargyl)pyrimidine derivatives as new nonclassical antifolates for human dihydrofolate reductase inhibition.

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.