Hironori Hayashi

Dimerization of HIV-1 protease occurs through two steps relating to the mechanism of protease dimerization inhibition by darunavir

Significance Dimerization of HIV-1 protease (PR) plays a critical role in the replication of HIV-1. Darunavir (DRV) inhibits not only proteolytic activity but also PR dimerization. The present study shows that PR dimerization process undergoes two steps and that DRV inhibits the first step of PR dimerization by binding to PR monomers in a one-to-one molar ratio. The present study also demonstrates that DRV binds to a transframe precursor PR protein, indicating that DRV’s monomer binding is involved in the Gag-Pol autoprocessing inhibition. To our knowledge, the present report represents the first demonstration of the two-step PR dimerization dynamics and the mechanism of dimerization inhibition by DRV, which should help design further, more potent novel PR inhibitors. Dimerization of HIV-1 protease (PR) subunits is an essential process for PR’s acquisition of proteolytic activity, which plays a critical role in the maturation of HIV-1. Recombinant wild-type PR (PRWT) proved to dimerize, as examined with electrospray ionization mass spectrometry; however, two active site interface PR mutants (PRT26A and PRR87K) remained monomeric. On the other hand, two termini interface PR mutants (PR1-C95A and PR97/99) took both monomeric and dimeric forms. Differential scanning fluorimetry indicated that PR1-C95A and PR97/99 dimers were substantially less stable than PRWT dimers. These data indicate that intermolecular interactions of two monomers occur first at the active site interface, generating unstable or transient dimers, and interactions at the termini interface subsequently occur, generating stable dimers. Darunavir (DRV), an HIV-1 protease inhibitor, inhibits not only proteolytic activity but also PR dimerization. DRV bound to protease monomers in a one-to-one molar ratio, inhibiting the first step of PR dimerization, whereas conventional protease inhibitors (such as saquinavir) that inhibit enzymatic activity but not dimerization failed to bind to monomers. DRV also bound to mutant PRs containing the transframe region-added PR (TFR-PRD25N and TFR-PRD25N-7AA), whereas saquinavir did not bind to TFR-PRD25N or TFR-PRD25N-7AA. Notably, DRV failed to bind to mutant PR containing four amino acid substitutions (V32I, L33F, I54M, and I84V) that confer resistance to DRV on HIV-1. To our knowledge, the present report represents the first demonstration of the two-step PR dimerization dynamics and the mechanism of dimerization inhibition by DRV, which should help design further, more potent novel PIs.

Design of HIV-1 Protease Inhibitors with Amino-bis-tetrahydrofuran Derivatives as P2-Ligands to Enhance Backbone-Binding Interactions: Synthesis, Biological Evaluation, and Protein-Ligand X-ray Studies.

Structure-based design, synthesis, and biological evaluation of a series of very potent HIV-1 protease inhibitors are described. In an effort to improve backbone ligand-binding site interactions, we have incorporated basic-amines at the C4 position of the bis-tetrahydrofuran (bis-THF) ring. We speculated that these substituents would make hydrogen bonding interactions in the flap region of HIV-1 protease. Synthesis of these inhibitors was performed diastereoselectively. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 25f, 25i, and 25j were evaluated against a number of highly-PI-resistant HIV-1 strains, and they exhibited improved antiviral activity over darunavir. Two high resolution X-ray structures of 25f- and 25g-bound HIV-1 protease revealed unique hydrogen bonding interactions with the backbone carbonyl group of Gly48 as well as with the backbone NH of Gly48 in the flap region of the enzyme active site. These ligand-binding site interactions are possibly responsible for their potent activity.

C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir.

ABSTRACT We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1WT), with 50% effective concentrations (EC50s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC50) for GRL-015, -085, and -097 of 80, >100, and >100 μM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multidrug-resistant HIV-1 variants (HIVMDR). Importantly, darunavir (DRV) was >1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1DRVR P51); the three compounds remained active against HIV-1DRVR P51 with only a 6.8- to 68-fold reduction. Moreover, the emergence of HIV-1 variants resistant to the three compounds was considerably delayed compared to the case of DRV. In particular, HIV-1 variants resistant to GRL-085 and -097 did not emerge even when two different highly DRV-resistant HIV-1 variants were used as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings indicate that the three compounds warrant further study as possible therapeutic agents for treating individuals harboring wild-type HIV and/or HIVMDR. IMPORTANCE Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1 strains and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1 strains (HIVDRVR ) has recently been observed in vivo and in vitro. Here, we identified three novel HIV-1 protease inhibitors (PIs) containing a tetrahydropyrano-tetrahydrofuran (Tp-THF) moiety with a C-5 hydroxyl (GRL-015, -085, and -097) which potently suppress the replication of HIVDRVR . Moreover, the emergence of HIV-1 strains resistant to the three compounds was considerably delayed compared to the case of DRV. The C-5 hydroxyl formed a strong hydrogen bonding interaction with the carbonyl oxygen atom of Gly48 of protease as examined in the structural analyses. Interestingly, a compound with Tp-THF lacking the hydroxyl moiety substantially decreased activity against HIVDRVR . The three novel compounds should be further developed as potential drugs for treating individuals harboring wild-type and multi-PI-resistant HIV variants as well as HIVDRVR .

A novel central nervous system-penetrating protease inhibitor overcomes human immunodeficiency virus 1 resistance with unprecedented aM to pM potency

Antiretroviral therapy for HIV-1 infection/AIDS has significantly extended the life expectancy of HIV-1-infected individuals and reduced HIV-1 transmission at very high rates. However, certain individuals who initially achieve viral suppression to undetectable levels may eventually suffer treatment failure mainly due to adverse effects and the emergence of drug-resistant HIV-1 variants. Here, we report GRL-142, a novel HIV-1 protease inhibitor containing an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran, which has extremely potent activity against all HIV-1 strains examined with IC50 values of attomolar-to-picomolar concentrations, virtually no effects on cellular growth, extremely high genetic barrier against the emergence of drug-resistant variants, and favorable intracellular and central nervous system penetration. GRL-142 forms optimum polar, van der Waals, and halogen bond interactions with HIV-1 protease and strongly blocks protease dimerization, demonstrating that combined multiple optimizing elements significantly enhance molecular and atomic interactions with a target protein and generate unprecedentedly potent and practically favorable agents.

Early phase dynamics of traceable mCherry fluorescent protein-carrying HIV-1 infection in human peripheral blood mononuclear cells-transplanted NOD/SCID/Jak3-/- mice

Abstract We attempted to elucidate early‐phase dynamics of HIV‐1 infection using replication‐competent, red‐fluorescent‐protein (mCherry)‐labeled HIV‐1JR‐FL (HIVJR‐FLmC) and NOD/SCID/Jak3‐/‐ mice transplanted with Individual‐As human peripheral blood mononuclear cells (hPBMC)(hNOJ mice). On day 7 following HIVJR‐FLmC inoculation, mCherry‐signal‐emitting infection foci were readily identified in the subserosa of 10 of 10 HIVJR‐FLmC‐inoculated hNOJ mice, although infection foci were not located without the mCherry signal in unlabeled HIV‐1JR‐FL‐inoculated mice (n = 6). Even on day 14, infection foci were hardly located in the unlabeled HIV‐1JR‐FL‐inoculated mice, while in all of 7 HIVJR‐FLmC‐inoculated hNOJ mice examined, mCherry‐signal‐emitting lymph nodes were easily identified, in which active viral replication was present. On day 14, a significantly larger number of mesenteric lymph nodes were seen in HIVJR‐FLmC‐exposed hNOJ mice than in HIVJR‐FLmC‐unexposed mice (P = 0.0025). The weights of mesenteric lymph nodes of those HIVJR‐FLmC‐exposed hNOJ mice were also greater than those of HIVJR‐FLmC‐unexposed mice (P = 0.0005). When hNOJ mice were inoculated with HIVJR‐FLmC‐exposed hPBMC from Individual‐B, significantly greater viremia was seen than in cell‐free HIVJR‐FLmC‐inoculated hNOJ mice as examined on day 7. In the lymph nodes of those mice inoculated with HIVJR‐FLmC‐exposed hPBMC from Individual‐B, a substantial number of Individual‐Bs HIVJR‐FLmC‐infected cells were identified together with Individual‐As cells as examined on day 14. The present HIVJR‐FLmC‐infected mouse model represents the first system reported using traceable HIVJR‐FLmC and human target cells, not using SIV or simian cells, which should be of utility in studies of early‐phases of HIV‐1 transmission and in evaluating the effects of potential agents for post‐exposure and pre‐exposure prophylaxis. HighlightsFirst‐reported visualization of HIV foci using mCherry fluorescent protein to examine initial phases of HIV infection.First report that mCherry‐labeled HIVJR‐FL exposure produces greater numbers of and larger lymph nodes than when unexposed.Exposure of mCherry‐labeled HIVJR‐FL virions plus cell‐associated virions cause more efficient and rapid HIV propagation.The present mCherry‐labeled HIVJR‐FL mouse model should be in utility in studies of HIV transmission and drug evaluation.

Mechanism of Darunavir (DRV)’s High Genetic Barrier to HIV-1 Resistance: A Key V32I Substitution in Protease Rarely Occurs, but Once It Occurs, It Predisposes HIV-1 To Develop DRV Resistance

ABSTRACT Darunavir (DRV) has bimodal activity against HIV-1 protease, enzymatic inhibition and protease dimerization inhibition, and has an extremely high genetic barrier against development of drug resistance. We previously generated a highly DRV-resistant HIV-1 variant (HIVDRVRP51). We also reported that four amino acid substitutions (V32I, L33F, I54M, and I84V) identified in the protease of HIVDRVRP51 are largely responsible for its high-level resistance to DRV. Here, we attempted to elucidate the role of each of the four amino acid substitutions in the development of DRV resistance. We found that V32I is a key substitution, which rarely occurs, but once it occurs, it predisposes HIV-1 to develop high-level DRV resistance. When two infectious recombinant HIV-1 clones carrying I54M and I84V (rHIVI54M and rHIVI84V, respectively) were selected in the presence of DRV, V32I emerged, and the virus rapidly developed high-level DRV resistance. rHIVV32I also developed high-level DRV resistance. However, wild-type HIVNL4-3 (rHIVWT) failed to acquire V32I and did not develop DRV resistance. Compared to rHIVWT, rHIVV32I was highly susceptible to DRV and had significantly reduced fitness, explaining why V32I did not emerge upon selection of rHIVWT with DRV. When the only substitution is at residue 32, structural analysis revealed much stronger van der Waals interactions between DRV and I-32 than between DRV and V-32. These results suggest that V32I is a critical amino acid substitution in multiple pathways toward HIV-1’s DRV resistance development and elucidate, at least in part, a mechanism of DRV’s high genetic barrier to development of drug resistance. The results also show that attention should be paid to the initiation or continuation of DRV-containing regimens in people with HIV-1 containing the V32I substitution. IMPORTANCE Darunavir (DRV) is the only protease inhibitor (PI) recommended as a first-line therapeutic and represents the most widely used PI for treating HIV-1-infected individuals. DRV possesses a high genetic barrier to development of HIV-1’s drug resistance. However, the mechanism(s) of the DRV’s high genetic barrier remains unclear. Here, we show that the preexistence of certain single amino acid substitutions such as V32I, I54M, A71V, and I84V in HIV-1 protease facilitates the development of high-level DRV resistance. Interestingly, all in vitro-selected highly DRV-resistant HIV-1 variants acquired V32I but never emerged in wild-type HIV (HIVWT), and V32I itself rendered HIV-1 more sensitive to DRV and reduced viral fitness compared to HIVWT, strongly suggesting that the emergence of V32I plays a critical role in the development of HIV-1’s resistance to DRV. Our results would be of benefit in the treatment of HIV-1-infected patients receiving DRV-containing regimens. Darunavir (DRV) is the only protease inhibitor (PI) recommended as a first-line therapeutic and represents the most widely used PI for treating HIV-1-infected individuals. DRV possesses a high genetic barrier to development of HIV-1’s drug resistance. However, the mechanism(s) of the DRV’s high genetic barrier remains unclear. Here, we show that the preexistence of certain single amino acid substitutions such as V32I, I54M, A71V, and I84V in HIV-1 protease facilitates the development of high-level DRV resistance. Interestingly, all in vitro-selected highly DRV-resistant HIV-1 variants acquired V32I but never emerged in wild-type HIV (HIVWT), and V32I itself rendered HIV-1 more sensitive to DRV and reduced viral fitness compared to HIVWT, strongly suggesting that the emergence of V32I plays a critical role in the development of HIV-1’s resistance to DRV. Our results would be of benefit in the treatment of HIV-1-infected patients receiving DRV-containing regimens.

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.

Raltegravir blocks the infectivity of red-fluorescent-protein (mCherry)-labeled HIV-1JR-FL in the setting of post-exposure prophylaxis in NOD/SCID/Jak3−/− mice transplanted with human PBMCs

ABSTRACT Employing NOD/SCID/Jak3−/− mice transplanted with human PBMCs (hNOJ mice) and replication‐competent, red‐fluorescent‐protein (mCherry; mC)‐labeled HIV‐1JR‐FL (HIVmC), we examined whether early antiretroviral treatment blocked the establishment of HIV‐1 infection. The use of hNOJ mice and HIVmC enabled us to visually locate infection foci and to examine the early dynamics of HIVmC infection without using a large amount of antiretroviral unlike in non‐human primate models. Although when raltegravir (RAL) administration was begun 1 day after intraperitoneal (ip) inoculation of HIVmC, no plasma p24 or plasma HIV‐1‐RNA (pRNA) were detected in 10 of 12 hNOJ (hNOJmCRAL+) mice as assessed on the last day of the 14‐day continuous twice‐daily RAL administration, all 10 untreated hNOJmC (hNOJmCRAL−) mice became positive for p24 and pRNA and had significantly swollen lymph nodes in peritoneal cavity and abundant p24+/mC+/CD3+/CD4+ T cells and p24+/mC+/CD68+ monocytes/macrophages were identified in their omenta and mesenteric lymphoid tissues/lymph nodes upon necropsy of the mice on day 14. In 12 hNOJmCRAL+ mice, no significantly swollen lymph nodes were seen compared to hNOJmCRAL− mice; however, in the omentum of the 2 hNOJmCRAL+ mice that were positive for pRNA and in site RNA, mC+/p24+/CD3+/CD83+ cells were identified, suggesting that viral breakthrough occurred later in the observation period. The present data suggest that the use of hNOJ mouse model and HIVmC may shed light on the study of early‐phase dynamics of HIV‐1 infection and cellular events in post‐exposure/pre‐exposure prophylaxis. HighlightsFirst‐report of PEP with RAL using red‐fluorescent protein‐labeled HIVmC and hPBMC‐transplanted hNOJ mice.First report that RAL reduces the numbers and sizes of lymph nodes in HIVmC‐exposed hNOJ mice compared to unexposed hNOJ mice.Study of HIVmC breakthrough in few HIVmC‐exposed hNOJ mice enabled by in vivo imaging technology.Use of hNOJ and HIVmC should be in utility in studies of HIV transmission and evaluation of drugs in PEP and PrEP.

GRL-079, a novel P2-Tp-THF-C5-alkylamine- and P2’ -Abt-containing HIV-1 protease inhibitor, is extremely potent against multi-drug-resistant HIV-1 variants including HIVDRVRp51 and has a high genetic barrier against the emergence of resistant variants

ABSTRACT We identified four novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-078, -079, -077, and -058, containing an alkylamine at the C-5 position of P2 tetrahydropyrano-tetrahydrofuran (Tp-THF) and a P2′ cyclopropyl (Cp) (or isopropyl)-aminobenzothiazole (Abt) moiety. Their 50% effective concentrations (EC50s) were 2.5 to 30 nM against wild-type HIV-1NL4-3, 0.3 to 6.7 nM against HIV-2EHO, and 0.9 to 90 nM against laboratory-selected PI-resistant HIV-1 and clinical HIV-1 variants resistant to multiple FDA-approved PIs (HIVMDR). GRL-078, -079, -077, and -058 also effectively blocked the replication of HIV-1 variants highly resistant to darunavir (DRV) (HIVDRVrp51), with EC50s of 38, 62, 61, and 90 nM, respectively, while four FDA-approved PIs examined (amprenavir, atazanavir, lopinavir [LPV], and DRV) had virtually no activity (EC50s of >1,000 nM) against HIVDRVrp51. Structurally, GRL-078, -079, and -058 form strong hydrogen bond interactions between Tp-THF modified at C-5 and Asp29/Asp30/Gly48 of wild-type protease, while the P2′ Cp-Abt group forms strong hydrogen bonds with Asp30′. The Tp-THF and Cp-Abt moieties also have good nonpolar interactions with protease residues located in the flap region. For selection with LPV and DRV by use of a mixture of 11 HIVMDR strains (HIV11MIX), HIV11MIX became highly resistant to LPV and DRV over 13 to 32 and 32 to 41 weeks, respectively. However, for selection with GRL-079 and GRL-058, HIV11MIX failed to replicate at >0.08 μM and >0.2 μM, respectively. Thermal stability results supported the highly favorable anti-HIV-1 potency of GRL-079 as well as other PIs. The present data strongly suggest that the P2 Tp-THF group modified at C-5 and the P2′ Abt group contribute to the potent anti-HIV-1 profiles of the four PIs against HIV-1NL4-3 and a wide spectrum of HIVMDR strains.