Exploration of imatinib and nilotinib-derived templates as the P2-Ligand for HIV-1 protease inhibitors: Design, synthesis, protein X-ray structural studies, and biological evaluation.

Structure-based design, synthesis, X-ray structural studies, and biological evaluation of a new series of potent HIV-1 protease inhibitors are described. These inhibitors contain various pyridyl-pyrimidine, aryl thiazole or alkylthiazole derivatives as the P2 ligands in combination with darunavir-like hydroxyethylamine sulfonamide isosteres. These heterocyclic ligands are inherent to kinase inhibitor drugs, such as nilotinib and imatinib. These ligands are designed to make hydrogen bonding interactions with the backbone atoms in the S2 subsite of HIV-1 protease. Various benzoic acid derivatives have been synthesized and incorporation of these ligands provided potent inhibitors that exhibited subnanomolar level protease inhibitory activity and low nanomolar level antiviral activity. Two high resolution X-ray structures of inhibitor-bound HIV-1 protease were determined. These structures provided important ligand-binding site interactions for further optimization of this class of protease inhibitors.

Evaluation of darunavir-derived HIV-1 protease inhibitors incorporating P2' amide-derivatives: Synthesis, biological evaluation and structural studies.

We report here the synthesis and biological evaluation of darunavir derived HIV-1 protease inhibitors and their functional effect on enzyme inhibition and antiviral activity in MT-2 cell lines. The P2' 4-amino functionality was modified to make a number of amide derivatives to interact with residues in the S2' subsite of the HIV-1 protease active site. Several compounds exhibited picomolar enzyme inhibitory and low nanomolar antiviral activity. The X-ray crystal structure of the chloroacetate derivative bound to HIV-1 protease was determined. Interestingly, the active chloroacetate group converted to the acetate functionality during X-ray exposure. The structure revealed that the P2' carboxamide functionality makes enhanced hydrogen bonding interactions with the backbone atoms in the S2'-subsite.

HIV-1 protease with 10 lopinavir and darunavir resistance mutations exhibits altered inhibition, structural rearrangements and extreme dynamics.

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 Ki values of 2-3 orders of magnitude worse for darunavir, lopinavir, and potent investigational inhibitor GRL-519. Crystal structures of the mutant were solved in a ligand-free form and in complex with GRL-519. The structures show altered interactions in the active site, flap-core interface, hydrophobic core, hinge region, and 80s loop compared to the corresponding wild-type protease structures. The ligand-free crystal structure exhibits a highly curled flap conformation which may amplify drug resistance. Molecular dynamics simulations performed for 1 µs on ligand-free dimers showed extremely large fluctuations in the flaps for PR10x compared to equivalent simulations on PR with a single L76V mutation or wild-type protease. This analysis offers insight about the synergistic effects of mutations in highly resistant variants.

Design, Synthesis and X-Ray Structural Studies of Potent HIV-1 Protease Inhibitors Containing C-4 Substituted Tricyclic Hexahydro-Furofuran Derivatives as P2 Ligands.

The design, synthesis, X-ray structural, and biological evaluation of a series of highly potent HIV-1 protease inhibitors are reported herein. These inhibitors incorporate 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.

Revertant mutation V48G alters conformational dynamics of highly drug resistant HIV protease PRS17.

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 PRS17V48G 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-1min-1 compared to 6.3 µM-1min-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 PRS17V48G 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 PRS17V48G 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 PRS17V48G 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.

HIV Protease: Historical Perspective and Current Research.

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.

Vision-Language Navigation Policy Learning and Adaptation.

Vision-language navigation (VLN) is the task of navigating an embodied agent to carry out natural language instructions inside real 3D environments. In this paper, we study how to address three critical challenges for this task: the cross-modal grounding, the ill-posed feedback, and the generalization problems. First, we propose a novel Reinforced Cross-Modal Matching (RCM) approach that enforces cross-modal grounding both locally and globally via reinforcement learning (RL). Particularly, a matching critic is used to provide an intrinsic reward to encourage global matching between instructions and trajectories, and a reasoning navigator is employed to perform cross-modal grounding in the local visual scene. Evaluation on a VLN benchmark dataset shows that our RCM model significantly outperforms baseline methods by 10 percent on Success Rate weighted by Path Length (SPL) and achieves the state-of-the-art performance. To improve the generalizability of the learned policy, we further introduce a Self-Supervised Imitation Learning (SIL) method to explore and adapt to unseen environments by imitating its own past, good decisions. We demonstrate that SIL can approximate a better and more efficient policy, which tremendously minimizes the success rate performance gap between seen and unseen environments (from 30.7 to 11.7 percent).

Design, Synthesis, and X-ray Studies of Potent HIV-1 Protease Inhibitors with P2-Carboxamide Functionalities.

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.

Structure-Based Design of Highly Potent HIV-1 Protease Inhibitors Containing New Tricyclic Ring P2-Ligands: Design, Synthesis, Biological, and X-ray Structural Studies.

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.

Potent HIV-1 Protease Inhibitors Containing Carboxylic and Boronic Acids: Effect on Enzyme Inhibition and Antiviral Activity and Protein-Ligand X-ray Structural Studies.

We report 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 relative to carboxamide-derived inhibitors which showed good antiviral IC50 value. Boronic acid derived inhibitor with bis-THF as the P2 ligand showed very potent enzyme inhibitory activity, but it showed lower antiviral activity than darunavir in the same assay. Boronic acid containing inhibitor with a P2-Crn-THF ligand also showed potent enzyme Ki but significantly decreased 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 HIVDRVRP20 and HIVDRVRP30 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.

Highly Drug-Resistant HIV-1 Protease Mutant PRS17 Shows Enhanced Binding to Substrate Analogues.

We report the structural analysis of highly drug-resistant human immunodeficiency virus protease (PR) variant PRS17, rationally selected by machine learning, in complex with substrate analogues. Crystal structures were solved of inhibitor-free inactive PRS17-D25N, wild-type PR/CA-p2 complex, and PRS17 in complex with substrate analogues, CA-p2 and p2-NC. Peptide analogues p2-NC and CA-p2 exhibit inhibition constants of 514 and 22 nM, respectively, for PRS17 or approximately 3-fold better than for PR. CA-p2 is a better inhibitor of PRS17 than are clinical inhibitors (K i = 50-8390 nM) except for amprenavir (K i = 11 nM). G48V resistance mutation induces curled flap tips in PRS17-D25N structure. The inner P2-P2' residues of substrate analogues in PRS17 complexes maintain similar conformations to those of wild-type complex, while significant conformational changes are observed in the peripheral residues P3, P4' of CA-p2 and P3, P4, and P3' of p2-NC. The loss of ß-branched side chain by V82S mutation initiates a shift in 80's loop and reshapes the S3/S3' subsite, which enhances substrate binding with new hydrogen bonds and van der Waals interactions that are absent in the wild-type structures. The steric hindrance caused by G48V mutation in the flap of PRS17 contributes to altered binding interactions of P3 Arg, P4' norleucine of CA-p2, and P4 and P3' of p2-NC with the addition of new hydrogen bonds and van der Waals contacts. The enhanced interaction of PRS17 with substrate analogues agrees with their relative inhibition, suggesting that this mutant improves substrate binding while decreasing affinity for clinical inhibitors.

Structural studies of antiviral inhibitor with HIV-1 protease bearing drug resistant substitutions of V32I, I47V and V82I.

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 Šresolution 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.

Drug Resistance Mutation L76V Alters Nonpolar Interactions at the Flap-Core Interface of HIV-1 Protease.

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 K i value of 1.9 nM for PRL76V, whereas the other three inhibitors had K i 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.

Design, synthesis, and X-ray studies of potent HIV-1 protease inhibitors incorporating aminothiochromane and aminotetrahydronaphthalene carboxamide derivatives as the P2 ligands.

We describe the design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors with carboxamide derivatives as the P2 ligands. We have specifically designed aminothiochromane and aminotetrahydronaphthalene-based carboxamide ligands to promote hydrogen bonding and van der Waals interactions in the active site of HIV-1 protease. Inhibitors 4e and 4j have shown potent enzyme inhibitory and antiviral activity. High resolution X-ray crystal structures of 4d- and 4k-bound HIV-1 protease revealed molecular insights into the ligand-binding site interactions.

Design and Synthesis of Potent HIV-1 Protease Inhibitors Containing Bicyclic Oxazolidinone Scaffold as the P2 Ligands: Structure-Activity Studies and Biological and X-ray Structural Studies.

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 Ki of 40 pM and antiviral IC50 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.

Design and Synthesis of Highly Potent HIV-1 Protease Inhibitors Containing Tricyclic Fused Ring Systems as Novel P2 Ligands: Structure-Activity Studies, Biological and X-ray Structural Analysis.

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.

[Effects of Tongbi capsule on joint lesions in rabbits with rheumatoid arthritis].

The purpose of this experiment is to observe the effects of Tongbi capsule on joint lesions in rabbit with rheumatoid arthritis induced by ovalbumin and explore the mechanism in order to provide reference for clinical application of Tongbi capsule. Rheumatoid arthritis in rabbits was induced by subcutaneous injection of emulsions of ovalbumin and Freund's complete adjuvant and intra articular injection of ovalbumin. After successful modeling, 30 New Zealand rabbits with arthritis were randomly divided into model control group, the high, medium and low dose groups of Tongbi capsule (90, 45, 22.5 mg·kg⁻¹) and prednisone group (5 mg·kg⁻¹). Another six normal rabbits were used as normal control group. After 24 hours of modeling, the rabbits in Tongbi capsule groups received intragastric (i.g.) administrations of Tongbi capsule at 90, 45, 22.5 mg·kg⁻¹·d⁻¹, and the rabbits of prednisone group received i.g. administrations of prednisone at 5 mg·kg⁻¹·d⁻¹ for 2 weeks. The rabbits in normal and model groups received the same volume of distilled water at the same time. The swelling degree of rabbit knee joint and local skin temperature were observed daily. After two weeks of administration, pathological changes of rabbit knee joint were examined by magnetic resonance imaging (MRI); the morphological changes of articular cartilage and synovial membrane were observed by microscope; and the contents of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α) in serum were detected by enzyme linked immunosorbent assay (ELISA).The results showed that 24 h after modeling, the knee joints of the rabbits were swollen, with red or dark redlocal skin, and fever, elevated local skin temperature and increased diameters of knee joints. Two weeks after modeling, the swelling of rabbit knee joints was obvious in model group; the joint cavities were filled with purulent fluid; joint synovial membranes were obviously thickened, and even joint cavities were fibrotic and cartilage surfaces showed slight defect; the surface of articular cartilage was obvious fibrosis; synovial epithelial cell proliferation was obvious and accompanied by extensive inflammatory cell infiltration; the levels of IL-1 and TNF-α were significantly higher as compared with those seen in model rabbits (P<0.05, P<0.01). After 1 and 2 weeks of administration, knee joint diameters and local skin temperatures were smaller or lower than thosein model group (P<0.05, P<0.01); The lesions of joint cartilage and synovial of all rabbits in each group were less than those in model group; IL-1 and TNF-α levels in serum were also lower than those in model group (P<0.05, P<0.01). The results reveal that high and medium doses of Tongbi capsule can suppress rheumatoid arthritis induced by ovalbumin in rabbits, reduce joint swelling, inhibit synovial epithelial and fiber hyperplasia and inflammatory cell infiltration, and alleviate articular cartilage damage. The mechanism may be associated with decreasing IL-1 and TNF-α levels in serum.

Crystal structure of yeast nitronate monooxygenase from Cyberlindnera saturnus.

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.

Design of Highly Potent, Dual-Acting and Central-Nervous-System-Penetrating HIV-1 Protease Inhibitors with Excellent Potency against Multidrug-Resistant HIV-1 Variants.

Herein we report the design, synthesis, X-ray structural, and biological studies of an exceptionally potent HIV-1 protease inhibitor, compound 5 ((3S,7aS,8S)-hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-yl ((2S,3R)-4-((2-(cyclopropylamino)-N-isobutylbenzo[d]thiazole)-6-sulfonamido)-1-(3,5-difluorophenyl)-3-hydroxybutan-2-yl)carbamate). Using structure-based design, we incorporated an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran as the P2-ligand, a cyclopropylaminobenzothiazole as the P2'-ligand, and a 3,5-difluorophenylmethyl group as the P1-ligand. The resulting inhibitor 5 exhibited exceptional HIV-1 protease inhibitory and antiviral potency at the picomolar level. Furthermore, it displayed antiviral IC50 values in the picomolar range against a wide panel of highly multidrug-resistant HIV-1 variants. The inhibitor shows an extremely high genetic barrier against the emergence of drug-resistant variants. It also showed extremely potent inhibitory activity toward dimerization as well as favorable central nervous system penetration. We determined a high-resolution X-ray crystal structure of the complex between inhibitor 5 and HIV-1 protease, which provides molecular insight into the unprecedented activity profiles observed.

Design, Synthesis, Biological Evaluation, and X-ray Studies of HIV-1 Protease Inhibitors with Modified P2' Ligands of Darunavir.

The structure-based design, synthesis, and biological evaluation of a series of nonpeptidic HIV-1 protease inhibitors with rationally designed P2' ligands are described. The inhibitors are designed to enhance backbone binding interactions, particularly at the S2' subsite. Synthesis of inhibitors was carried out efficiently. The stereochemistry of alcohol functionalities of the P2' ligands was set by asymmetric reduction of the corresponding ketone using (R,R)- or (S,S)-Noyori catalysts. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 3g and 3h showed enzyme Ki values of 27.9 and 49.7 pm and antiviral activity of 6.2 and 3.9 nm, respectively. These inhibitors also remained quite potent against darunavir-resistant HIV-1 variants. An X-ray structure of inhibitor 3g in complex with HIV-1 protease revealed key interactions in the S2' subsite.