Structural basis of HIV-1 maturation inhibitor binding and activity

Abstract

HIV-1 maturation inhibitors (MIs), Bevirimat (BVM) and its analogs interfere with the catalytic cleavage of spacer peptide 1 (SP1) from the capsid protein C-terminal domain (CACTD), by binding to and stabilizing the CACTD-SP1 region. MIs are under development as alternative drugs to augment current antiretroviral therapies. Although promising, their mechanism of action and associated virus resistance pathways remain poorly understood at the molecular, biochemical, and structural levels. We report atomic-resolution magic-angle-spinning NMR structures of microcrystalline assemblies of CACTD-SP1 complexed with BVM and/or the assembly cofactor inositol hexakisphosphate (IP6). Our results reveal a mechanism by which BVM disrupts maturation, tightening the 6-helix bundle pore and quenching the motions of SP1 and the simultaneously bound IP6. In addition, BVM-resistant SP1-A1V and SP1-V7A variants exhibit distinct conformational and binding characteristics. Taken together, our study provides a structural explanation for BVM resistance as well as guidance for the design of new MIs.

Sub-stoichiometric Modulation of Viral Targets─Potent Antiviral Agents That Exploit Target Vulnerability

“…These observations collectively point to an induction of asymmetry within the binding pocket (Figures and ). The binding of 3 to the 6-HB reduced the dynamic mobility of IP 6 and the side chains of residues within SP-1, and the MI did not appear to rotate freely within the binding pocket, an observation that contrasted with the results of a detailed molecular dynamics study. , Further experimentation indicated that the binding of 3 to the Ala 364 Val-resistant virus was attenuated, while the Val 370 Ala mutation reduced the conformational stability of SP-1, presumably allowing for a more facile transition to the conformation that exposes the cleavage site to HIV-1 protease. ,, The deduced binding pose suggests that MIs like 1 and 2 , which introduce larger substituents at C-17 of the core, extend interactions of the molecule with elements of SP-1, including projecting into the region defined by the QVT motif, thereby reducing the mobility of the 6-HB complex in a fashion that prevents access to the conformation recognized by the protease. ,, Some of the mutations that confer resistance to MIs, notably Val 370 , are involved in interstrand interactions that presumably destabilize the 6-HB complex and enhance conformational dynamics, which promotes cleavage site exposure. Collectively, these observations are consistent with the binding dissociation data developed for 1 and 2 in virus-like particles (VLPs) expressing WT and mutated sequences that are compiled in Table .…”

“…Virions produced in the presence of 3 were non-infectious and exhibited a morphology in which the capsid core of the virion adopted a spherical disposition rather than the classic cone shape that is associated with correctly functioning HIV-1 particles. However, 3 was not a direct inhibitor of HIV-1 protease, pointing to a unique mechanism that began to be more fully understood at the molecular level from structural studies conducted long after the compound had entered clinical trials and which continue to provide critical insights. − In clinical study, 3 had demonstrated an effective reduction in viral load in HIV-1-infected individuals susceptible to the drug; however, approximately 50% of the patients failed to experience a decline in viremia . The dichotomous response was traced to the presence of baseline polymorphisms in the Gag protein in a subset of the patient population that represented a source of inherent resistance and which meant that patients would need to be pre-screened for susceptibility prior to being prescribed the drug. − This ultimately led to the compound being abandoned by its sponsors on June 8, 2010. , …”

“…The HIV-1 MIs 1 – 3 interfere with viral protease-mediated processing of the Gag polyprotein and specifically block the final and rate-determining step in which the 14-residue SP-1 is cleaved from the C-terminus of the virus CA protein. − ,,− This processing step is essential to the formation of an authentic and mature HIV-1 capsid with correct folding and functioning that is infectious. This is particularly the case with respect to capsid protection of the viral RNA during the reverse transcription to double-stranded viral cDNA that occurs in the subsequent infection cycle, where a carefully choreographed dismantling of the CA complex is essential to the timely release of the viral genetic template.…”

“…This is particularly the case with respect to capsid protection of the viral RNA during the reverse transcription to double-stranded viral cDNA that occurs in the subsequent infection cycle, where a carefully choreographed dismantling of the CA complex is essential to the timely release of the viral genetic template. Structural studies with 3 that have emerged only recently have revealed that the compound binds to the core of the assembled 6-helix bundle (6-HB) form of CA-SP-1 and stabilizes the complex in a fashion that interferes with the exposure of the site of protease-mediated cleavage, which lies between Leu 363 and Ala 364 . − The most contemporary structural analysis studied microcrystalline assemblies of the C-terminal domain of CA-SP-1 complexed with 3 using magic-angle-spinning NMR and demonstrated that the compound bound to the 6-HB in conjunction with inositol hexakis­phosphate (IP 6 ), which has been identified as an assembly cofactor that is critical to virus replication (Figure ). ,− The equatorial phosphates of IP 6 coordinate with the basic side chains of Lys 290 that are located close to the junction between the base of the goblet shape created by CA and the top of the goblet stem, which is mostly defined by SP-1 residues, and which project in a ring-like arrangement around the central core of the bundle.…”

“… ,− The equatorial phosphates of IP 6 coordinate with the basic side chains of Lys 290 that are located close to the junction between the base of the goblet shape created by CA and the top of the goblet stem, which is mostly defined by SP-1 residues, and which project in a ring-like arrangement around the central core of the bundle. The immature 6-HB assembly is convened by the Lys 290 residues engaging IP 6 , a phenomenon that extends to other retroviruses, while the axial phosphate associates with Lys 359 , which is proximal to the cleavage site between Leu 363 and Ala 364 . − ,− In the absence of IP 6 , inositol pentakis­phosphate (IP 5 ) is an effective substitute, while a Thr 371 Ile mutation in the SP-1 protein that appears in response to the passaging of viruses incorporating a Lys 359 Ala mutation, which compromises IP 6 binding, is able to rescue infectivity. − , In WT virus, the Thr 371 Ile mutation mimics the effects of 3 by suspending CA-SP-1 processing, while, interestingly, 3 itself was found to be capable of rescuing a mutant virus defective in IP 6 binding by acting as a functional surrogate . After cleavage of the CA-SP-1 junction, IP 6 stabilizes the mature lattice by engaging with the circle of side chains presented by Arg 18 in the goblet element of the 6-HB structure.…”