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We report our recent development of a conceptually new generation of exceptionally potent non-peptidic HIV-1 protease inhibitors that displayed excellent pharmacological and drug-resistance profiles. Our X-ray structural studies of darunavir and other designed inhibitors from our laboratories led us to create a variety of inhibitors incorporating fused ring polycyclic ethers and aromatic heterocycles to promote hydrogen bonding interactions with the backbone atoms of HIV-1 protease as well as van der Waals interactions with residues in the S2 and S2' subsites. We have also incorporated specific functionalities to enhance van der Waals interactions in the S1 and S1' subsites. The combined effects of these structural templates are critical to the inhibitors' exceptional potency and drug-like properties. We highlight here our molecular design strategies to promote backbone hydrogen bonding interactions to combat drug-resistance and specific design of polycyclic ether templates to mimic peptide-like bonds in the HIV-1 protease active site. Our medicinal chemistry and drug development efforts led to the development of new generation inhibitors significantly improved over darunavir and displaying unprecedented antiviral activity against multidrug-resistant HIV-1 variants.

The emergence of multidrug resistant (MDR) HIV strains severely reduces the effectiveness of antiretroviral therapy. Clinical inhibitor darunavir (1) has picomolar binding affinity for HIV-1 protease (PR), however, drug resistant variants like PR(S17) show poor inhibition by 1, despite the presence of only two mutated residues in the inhibitor-binding site. Antiviral inhibitors that target MDR proteases like PR(S17) would be valuable as therapeutic agents. Inhibitors 2 and 3 derived from 1 through substitutions at P1, P2 and P2ʹ positions exhibit 3.4- to 500-fold better inhibition than clinical inhibitors for PR(S17) with the exception of amprenavir. Crystal structures of PR(S17)/2 and PR(S17)/3 reveal how these inhibitors target the two active site mutations of PR(S17). The substituted methoxy P2 group of 2 forms new interactions with G48V mutation, while the modified bis-fluoro-benzyl P1 group of 3 forms a halogen interaction with V82S mutation, contributing to improved inhibition of PR(S17).

Antiretroviral drug resistance is a therapeutic obstacle for people with HIV. HIV protease inhibitors darunavir and lopinavir are recommended for resistant infections. We characterized a protease mutant (PR10x) derived from a highly resistant clinical isolate including 10 mutations associated with resistance to lopinavir and darunavir. Compared to the wild-type protease, PR10x exhibits ∼3-fold decrease in catalytic efficiency and K

Coronaviruses infect many animals, including humans, due to interspecies transmission. Three of the known human coronaviruses: MERS, SARS-CoV-1, and SARS-CoV-2, the pathogen for the COVID-19 pandemic, cause severe disease. Improved methods to predict host specificity of coronaviruses will be valuable for identifying and controlling future outbreaks. The coronavirus S protein plays a key role in host specificity by attaching the virus to receptors on the cell membrane. We analyzed 1238 spike sequences for their host specificity. Spike sequences readily segregate in t-SNE embeddings into clusters of similar hosts and/or virus species. Machine learning with SVM, Logistic Regression, Decision Tree, Random Forest gave high average accuracies, F1 scores, sensitivities and specificities of 0.95–0.99. Importantly, sites identified by Decision Tree correspond to protein regions with known biological importance. These results demonstrate that spike sequences alone can be used to predict host specificity., Highlights • Coronavirus spike protein sequences can predict host specificity. • Machine learning had accuracies of >0.98 for classification of human vs non-human hosts. • Decision Tree classifier identified spike regions with important biological roles . • Clustering with t-SNE embeddings correctly segregated sequences by virus genus and host .

The design, synthesis, X-ray structural, and biological evaluation of a series of highly potent HIV-1 protease inhibitors are reported herein. These inhibitors incorporated novel cyclohexane-fused tricyclic bis-tetrahydrofuran as P2 ligands in combination with a variety of P1 and P2’-ligands. The inhibitor with a difluoromethylphenyl P1 ligand and a cyclopropylaminobenzothiazole P2’ ligand exhibited the most potent antiviral activity. Also, it maintained potent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The corresponding inhibitor with an enantiomeric ligand was significantly less potent in these antiviral assays. The new P2 ligands were synthesized in optically active form using enzymatic desymmetrization of meso-diols as the key step. To obtain molecular insight, two high resolution X-ray structures of inhibitor-bound HIV-1 protease were determined and structural analyses have been highlighted.

Drug-resistance is a serious problem for treatment of the HIV/AIDS pandemic. Potent clinical inhibitors of HIV-1 protease show several orders of magnitude worse inhibition of highly drug-resistant variants. Hence, the structure and enzyme activities were analyzed for HIV protease mutant HIV-1 protease (EC 3.4.23.16) (PR) with 22 mutations (PRS5B) from a clinical isolate that was selected by machine learning to represent high-level drug-resistance. PRS5B has 22 mutations including only one (I84V) in the inhibitor binding site; however, clinical inhibitors had poor inhibition of PRS5B activity with kinetic inhibition value (Ki ) values of 4-1000 nm or 18- to 8000-fold worse than for wild-type PR. High-resolution crystal structures of PRS5B complexes with the best inhibitors, amprenavir (APV) and darunavir (DRV) (Ki ~ 4 nm), revealed only minor changes in protease-inhibitor interactions. Instead, two distinct clusters of mutations in distal regions induce coordinated conformational changes that decrease favorable internal interactions across the entire protein subunit. The largest structural rearrangements are described and compared to other characterized resistant mutants. In the protease hinge region, the N83D mutation eliminates a hydrogen bond connecting the hinge and core of the protease and increases disorder compared to highly resistant mutants PR with 17 mutations and PR with 20 mutations with similar hinge mutations. In a distal β-sheet, mutations G73T and A71V coordinate with accessory mutations to bring about shifts that propagate throughout the subunit. Molecular dynamics simulations of ligand-free dimers show differences consistent with loss of interactions in mutant compared to wild-type PR. Clusters of mutations exhibit both coordinated and antagonistic effects, suggesting PRS5B may represent an intermediate stage in the evolution of more highly resistant variants. DATABASES: Structural data are available in Protein Data Bank under the accession codes 6P9A and 6P9B for PRS5B/DRV and PRS5B/APV, respectively.

d-Arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) catalyzes the flavin-dependent oxidation of d-arginine and other d-amino acids. Here, we report the crystal structure at 1.29 Å resolution for PaDADH-Y249F expressed and co-crystallized with d-arginine. The overall structure of PaDADH-Y249F resembled PaDADH-WT, but the electron density for the flavin cofactor was ambiguous, suggesting the presence of modified flavins. Electron density maps and mass spectrometric analysis confirmed the presence of both N5-(4-guanidino-oxobutyl)-FAD and 6-OH-FAD in a single crystal of PaDADH-Y249F and helped with the further refinement of the X-ray crystal structure. The versatility of the reduced flavin is apparent in the PaDADH-Y249F structure and is evidenced by the multiple functions it can perform in the same active site.

Drug-resistance threatens effective treatment of HIV/AIDS. Clinical inhibitors, including darunavir (1), are ineffective for highly resistant protease mutant PR20, however, antiviral compound 2 derived from 1 with fused tricyclic group at P2, extended amino-benzothiazole P2’ ligand and two fluorine atoms on P1 shows 16-fold better inhibition of PR20 enzyme activity. Crystal structures of PR20 and wild-type PR complexes reveal how the extra groups of 2 counteract the expanded ligand-binding pocket, dynamic flaps, and faster dimer dissociation of PR20.

Proteins are inherently dynamic, and proper enzyme function relies on conformational flexibility. In this study, we demonstrated how an active site residue changes an enzyme's reactivity by modulating fluctuations between conformational states. Replacement of tyrosine 249 (Y249) with phenylalanine in the active site of the flavin-dependent d-arginine dehydrogenase yielded an enzyme with both an active yellow FAD (Y249F-y) and an inactive chemically modified green FAD, identified as 6-OH-FAD (Y249F-g) through various spectroscopic techniques. Structural investigation of Y249F-g and Y249F-y variants by comparison to the wild-type enzyme showed no differences in the overall protein structure and fold. A closer observation of the active site of the Y249F-y enzyme revealed an alternative conformation for some active site residues and the flavin cofactor. Molecular dynamics simulations probed the alternate conformations observed in the Y249F-y enzyme structure and showed that the enzyme variant with FAD samples a metastable conformational state, not available to the wild-type enzyme. Hybrid quantum/molecular mechanical calculations identified differences in flavin electronics between the wild type and the alternate conformation of the Y249F-y enzyme. The computational studies further indicated that the alternate conformation in the Y249F-y enzyme is responsible for the higher spin density at the C6 atom of flavin, which is consistent with the formation of 6-OH-FAD in the variant enzyme. The observations in this study are consistent with an alternate conformational space that results in fine-tuning the microenvironment around a versatile cofactor playing a critical role in enzyme function.

We describe here design, synthesis, and biological evaluation of a series of highly potent HIV-1 protease inhibitors containing stereochemically defined and unprecedented tricyclic furanofuran derivatives as P2 ligands in combination with a variety of sulfonamide derivatives as P2’ ligands. These inhibitors were designed to enhance the ligand-backbone binding and van der Waals interactions in the protease active site. A number of inhibitors containing the new P2 ligand, an aminobenzothiazole as the P2’ ligand and a difluorophenylmethyl as the P1 ligand, displayed very potent enzyme inhibitory potency and also showed excellent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The tricyclic P2 ligand has been synthesized efficiently in an optically active form using enzymatic desymmetrization of meso-1,2-(dihydroxymethyl)cyclohex-4-ene as the key step. We determined high-resolution X-ray structures of inhibitor-bound HIV-1 protease. These structures revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insights into the binding properties of these new inhibitors.

HIV-1 protease is an essential therapeutic target for the design and development of antiviral inhibitors to treat AIDS. We used room temperature neutron crystallography to accurately determine hydrogen atom positions in several protease complexes with clinical drugs, amprenavir and darunavir. Hydrogen bonding interactions were carefully mapped to provide an unprecedented picture of drug binding to the protease target. We demonstrate that hydrogen atom positions within the enzyme catalytic site can be altered by introducing drug resistant mutations and by protonating surface residues that trigger proton transfer reactions between the catalytic Asp residues and the hydroxyl group of darunavir. When protein perdeuteration is not feasible, we validate the use of initial H/D exchange with unfolded protein and partial deuteration in pure D

HIV-1 protease is an essential therapeutic target for the design and development of antiviral inhibitors to treat AIDS. We used room temperature neutron crystallography to accurately determine hydrogen atom positions in several protease complexes with clinical drugs, amprenavir and darunavir. Hydrogen bonding interactions were carefully mapped to provide an unprecedented picture of drug binding to the protease target. We demonstrate that hydrogen atom positions within the enzyme catalytic site can be altered by introducing drug resistant mutations and by protonating surface residues that trigger proton transfer reactions between the catalytic Asp residues and the hydroxyl group of darunavir. When protein perdeuteration is not feasible, we validate the use of initial H/D exchange with unfolded protein and partial deuteration in pure D2O with hydrogenous glycerol to maximize deuterium incorporation into the protein, with no detrimental effects on the growth of quality crystals suitable for neutron diffraction experiments.

We have designed, synthesized, and evaluated a new class of potent HIV-1 protease inhibitors with novel bicyclic oxazolidinone derivatives as the P2 ligand. We have developed an enantioselective synthesis of these bicyclic oxazolidinones utilizing a key o-iodoxybenzoic acid mediated cyclization. Several inhibitors displayed good to excellent activity toward HIV-1 protease and significant antiviral activity in MT-4 cells. Compound 4k has shown an enzyme K(i) of 40 pM and antiviral IC(50) of 31 nM. Inhibitors 4k and 4l were evaluated against a panel of highly resistant multidrug-resistant HIV-1 variants, and their fold-changes in antiviral activity were similar to those observed with darunavir. Additionally, two X-ray crystal structures of the related inhibitors 4a and 4e bound to HIV-1 protease were determined at 1.22 and 1.30 Å resolution, respectively, and revealed important interactions in the active site that have not yet been explored.

Background Drug resistance in HIV is the major problem limiting effective antiviral therapy. Computational techniques for predicting drug resistance profiles from genomic data can accelerate the appropriate choice of therapy. These techniques can also be used to select protease mutants for experimental studies of resistance and thereby assist in the development of next-generation therapies. Results The machine learning produced highly accurate and robust classification of HIV protease resistance. Genotype data were mapped to the enzyme structure and encoded using Delaunay triangulation. Generative machine learning models trained on one inhibitor could classify resistance from other inhibitors with varying levels of accuracy. Generally, the accuracy was best when the inhibitors were chemically similar. Conclusions Restricted Boltzmann Machines are an effective machine learning tool for classification of genomic and structural data. They can also be used to compare resistance profiles of different protease inhibitors.

Four HIV-1 protease (PR) inhibitors, clinical inhibitors lopinavir and tipranavir, and two investigational compounds 4 and 5, were studied for their effect on the structure and activity of PR with drug-resistant mutation L76V (PRL76V). Compound 5 exhibited the best Ki value of 1.9 nM for PRL76V, whereas the other three inhibitors had Ki values of 4.5–7.6 nM, 2–3 orders of magnitude worse than for wild-type enzymes. Crystal structures showed only minor differences in interactions of inhibitors with PRL76V compared to wild-type complexes. The shorter side chain of Val76 in the mutant lost hydrophobic interactions with Lys45 and Ile47 in the flap, and with Asp30 and Thr74 in the protein core, consistent with decreased stability. Inhibitors forming additional polar interactions with the flaps or dimer interface of PRL76V were unable to compensate for the decrease in internal hydrophobic contacts. These structures provide insights for inhibitor design.

[Image: see text] The design, synthesis, biological evaluation, and X-ray structural studies are reported for a series of highly potent HIV-1 protease inhibitors. The inhibitors incorporated stereochemically defined amide-based bicyclic and tricyclic ether derivatives as the P2 ligands with (R)-hydroxyethylaminesulfonamide transition-state isosteres. A number of inhibitors showed excellent HIV-1 protease inhibitory and antiviral activity; however, ligand combination is critical for potency. Inhibitor 4h with a difluorophenylmethyl as the P1 ligand, crown-THF-derived acetamide as the P2 ligand, and a cyclopropylaminobenzothiazole P2′-ligand displayed very potent antiviral activity and maintained excellent antiviral activity against selected multidrug-resistant HIV-1 variants. A high resolution X-ray structure of inhibitor 4h-bound HIV-1 protease provided molecular insight into the binding properties of the new inhibitor.

The design, synthesis, and biological evaluation of a new class of HIV-1 protease inhibitors containing stereochemically defined fused tricyclic polyethers as the P2 ligands and a variety of sulfonamide derivatives as the P2′ ligands, are described. A number of ring sizes and various substituent effects were investigated to enhance the ligand-backbone interactions in the protease active site. Inhibitors 5c and 5d containing this unprecedented fused 6-5-5 ring system as the P2 ligand, an aminobenzothiazole as the P2′ ligand and a difluorophenylmethyl as the P1 ligand exhibited exceptional enzyme inhibitory potency and maintained excellent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The umbrella-like P2 ligand for these inhibitors has been synthesized efficiently in an optically active form using a Pauson-Khand cyclization reaction as the key step. The racemic alcohols were resolved efficiently using a lipase catalyzed enzymatic resolution. Two high resolution X-ray structures of inhibitor-bound HIV-1 protease revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insight into the binding properties of these new inhibitors.

Nitronate monooxygenase (NMO) is an FMN-dependent enzyme that oxidizes the neurotoxin propionate 3-nitronate (P3N) and represents the best-known system for P3N detoxification in different organisms. The crystal structure of the first eukaryotic Class I NMO from Cyberlindnera saturnus (CsNMO) has been solved at 1.65 Å resolution and refined to an R-factor of 14.0%. The three-dimensional structures of yeast CsNMO and bacterial PaNMO are highly conserved with the exception of three additional loops on the surface in the CsNMO enzyme and differences in four active sites residues. A PEG molecule was identified in the structure and formed extensive interactions with CsNMO, suggesting a specific binding site; however, 8% PEG showed no significant effect on the enzyme activity. This new crystal structure of a eukaryotic NMO provides insight into the function of this class of enzymes.

Drug resistance is a serious problem for controlling the HIV/AIDS pandemic. Current antiviral drugs show several orders of magnitude worse inhibition of highly resistant clinical variant PRS17 of HIV-1 protease compared with wild-type protease. We have analyzed the effects of a common resistance mutation G48V in the flexible flaps of the protease by assessing the revertant PRS17(V48G) for changes in enzyme kinetics, inhibition, structure, and dynamics. Both PRS17 and the revertant showed about 10-fold poorer catalytic efficiency than wild-type enzyme (0.55 and 0.39 μM(−1)min(−1) compared to 6.3 μM(−1)min(−1)). Clinical inhibitors, amprenavir and darunavir, showed 2-fold and 8-fold better inhibition, respectively, of the revertant than of PRS17, although the inhibition constants for PRS17(V48G) were still 25 to 1,200-fold worse than for wild-type protease. Crystal structures of inhibitor-free revertant and amprenavir complexes with revertant and PRS17 were solved at 1.3-1.5 Å resolution. The amprenavir complexes of PRS17(V48G) and PRS17 showed no significant differences in the interactions with inhibitor, although changes were observed in the conformation of Phe53 and the interactions of the flaps. The inhibitor-free structure of the revertant showed flaps in an open conformation, however, the flap tips do not have the unusual curled conformation seen in inhibitor-free PRS17. Molecular dynamics simulations were run for 1 μs on the two inhibitor-free mutants and wild-type protease. PRS17 exhibited higher conformational fluctuations than the revertant, while the wild-type protease adopted the closed conformation and showed the least variation. The second half of the simulations captured the transition of the flaps of PRS17 from a closed to a semi-open state, whereas the flaps of PRS17(V48G) tucked into the active site and the wild-type protease retained the closed conformation. These results suggest that mutation G48V contributes to drug resistance by altering the conformational dynamics of the flaps.

We report here the synthesis and biological evaluation of phenylcarboxylic acid and phenylboronic acid containing HIV-1 protease inhibitors and their functional effect on enzyme inhibition and antiviral activity in MT-2 cell lines. Inhibitors bearing bis-THF ligand as P2 ligand and phenylcarboxylic acids and carboxamide as the P2’ ligands, showed very potent HIV-1 protease inhibitory activity. However, carboxylic acid containing inhibitors showed very poor antiviral activity compared to carboxamide-derived inhibitors which showed good antiviral IC(50) value. Boronic acid-derived inhibitor with bis-THF as the P2 ligand showed very potent enzyme inhibitory activity, but it showed relatively reduced antiviral activity compared to darunavir in the same assay. Boronic acid-containing inhibitor with a P2-Crn-THF ligand also showed potent enzyme K(i) but significantly reduced antiviral activity. We have evaluated antiviral activity against a panel of highly drug-resistant HIV-1 variants. One of the inhibitors maintained good antiviral activity against HIV(DRV)(R)(P20) and HIV(DRV)(R)(P30) viruses. We have determined high resolution X-ray structures of two synthetic inhibitors bound to HIV-1 protease and obtained molecular insight into the ligand-binding site interactions.

HIV-1 protease inhibitors are effective in HIV/AIDS therapy, although drug resistance is a severe problem. This study examines the effects of four investigational inhibitors against HIV-1 protease with drug resistant mutations of V32I, I47V and V82I (PRTri) that model the inhibitor-binding site of HIV-2 protease. These inhibitors contain diverse chemical modifications on the darunavir scaffold and form new interactions with wild type protease, however, the measured inhibition constants for PRTri mutant range from 17 to 40 nM or significantly worse than picomolar values reported for wild type enzyme. The X-ray crystal structure of PRTri mutant in complex with inhibitor 1 at 1.5 A resolution shows minor changes in interactions with inhibitor compared with the corresponding wild type PR complex. Instead, the basic amine at P2 of inhibitor together with mutation V82I induces two alternate conformations for the side chain of Arg8 with new interactions with inhibitor and Leu10. Hence, inhibition is influenced by small coordinated changes in hydrophobic interactions.

The retroviral protease of human immunodeficiency virus (HIV) is an excellent target for antiviral inhibitors for treating HIV/AIDS. Despite the efficacy of therapy, current efforts to control the disease are undermined by the growing threat posed by drug resistance. This review covers the historical background of studies on the structure and function of HIV protease, the subsequent development of antiviral inhibitors, and recent studies on drug-resistant protease variants. We highlight the important contributions of Dr. Stephen Oroszlan to fundamental knowledge about the function of the HIV protease and other retroviral proteases. These studies, along with those of his colleagues, laid the foundations for the design of clinical inhibitors of HIV protease. The drug-resistant protease variants also provide an excellent model for investigating the molecular mechanisms and evolution of resistance.

The virally-encoded HIV-1 protease is an effective target for antiviral drugs, however, treatment for HIV infections is limited by the prevalence of drug resistant viral mutants. In this review, we describe our three-pronged approach to analyze and combat drug resistance. Understanding the molecular basis for resistance due to protease inhibitors is a key initial step in this approach. This knowledge is being employed for the design of new, improved inhibitors with high affinity for resistant mutants as well as wild type enzyme. In parallel with experimental studies of diverse mutants and inhibitory compounds, we are developing efficient algorithms to predict drug resistance phenotype from genotype data. This approach has important practical applications in the clinic where genotyping is recommended for individuals with new infections.

In Group A streptococcus (GAS), the metallorepressor MtsR regulates iron homeostasis. Here we describe a new MtsR-repressed gene, which we named hupZ (heme utilization protein). A recombinant HupZ protein was purified bound to heme from Escherichia coli grown in the presence of 5-aminolevulinic acid and iron. HupZ specifically binds heme with stoichiometry of 1:1. The addition of NADPH to heme-bound HupZ (in the presence of cytochrome P450 reductase, NADPH-regeneration system and catalase) triggered progressive decrease of the HupZ Soret band and the appearance of an absorption peak at 660 nm that was resistance to hydrolytic conditions. No spectral changes were observed when ferredoxin and ferredoxin reductase were used as redox partners. Differential spectroscopy with myoglobin or with the ferrous chelator, ferrozine, confirmed that carbon monoxide and free iron are produced during the reaction. ApoHupZ was crystallized as a homodimer with a split β-barrel conformation in each monomer comprising six β strands and three α helices. This structure resembles the split β-barrel domain shared by the members of a recently described family of heme degrading enzymes. However, HupZ is smaller and lacks key residues found in the proteins of the latter group. Phylogenetic analysis places HupZ on a clade separated from those for previously described heme oxygenases. In summary, we have identified a new GAS enzyme-containing split β-barrel and capable of heme biotransformation in vitro; to the best of our knowledge, this is the first enzyme among Streptococcus species with such activity.

The crystal structure of the NADH:quinone oxidoreductase PA1024 has been solved in complex with NAD(+) to 2.2 Å resolution. The nicotinamide C4 is 3.6 Å from the FMN N5 atom, with a suitable orientation for facile hydride transfer. NAD(+) binds in a folded conformation at the interface of the TIM‐barrel domain and the extended domain of the enzyme. Comparison of the enzyme‐NAD(+) structure with that of the ligand‐free enzyme revealed a different conformation of a short loop (75–86) that is part of the NAD(+)‐binding pocket. P78, P82, and P84 provide internal rigidity to the loop, whereas Q80 serves as an active site latch that secures the NAD(+) within the binding pocket. An interrupted helix consisting of two α‐helices connected by a small three‐residue loop binds the pyrophosphate moiety of NAD(+). The adenine moiety of NAD(+) appears to π–π stack with Y261. Steric constraints between the adenosine ribose of NAD(+), P78, and Q80, control the strict specificity of the enzyme for NADH. Charged residues do not play a role in the specificity of PA1024 for the NADH substrate.