Structure of the Antiviral Assembly Inhibitor CAP-1 Complex with the HIV-1 CA Protein

Abstract

The CA domain of the HIV-1 Gag polyprotein plays critical roles in both the early and late phases of viral replication and is therefore an attractive antiviral target. Compounds with antiviral activity were recently identified that bind to the N-terminal domain of CA (CA N ) and inhibit core assembly during viral maturation. We have determined the structure of the complex between CA N and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)-N'-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) using a combination of NMR spectroscopy and X-ray crystallography. The protein undergoes a remarkable conformational change upon CAP-1 binding, in which Phe32 is displaced from its buried position in the protein core to open a deep hydrophobic cavity that serves as the ligand binding site. The aromatic ring of CAP-1 inserts into the cavity, with the urea NH groups forming hydrogen bonds with the backbone oxygen of Val59 and the dimethylamino group interacting with the side chains of Glu28 and Glu29. Elements that could be exploited to improve binding affinity are apparent in the structure. The displacement of Phe32 by CAP-1 appears to be facilitated by a strained main chain conformation, which suggests a potential role for a Phe32 conformational switch during normal capsid assembly.The AIDS epidemic continues to be a significant international health problem, with approximately 40 million people living with HIV infection world-wide 1 . In 2006 alone, 4.3 million individuals became infected with HIV, and approximately 3 million deaths were attributed to AIDS. Therapeutic agents currently used to treat HIV infection target the viral reverse transcriptase, protease, and fusion proteins, and drugs that target the integrase enzyme are undergoing clinical trials (www.aidsinfo.nih.gov). Although sustained reductions in viral load can be achieved for many years with combination drug therapies 2; 3; 4 , inadequate suppression due to poor compliance, resistance, and interactions with other drugs or diet can be a significant problem for some patients and can lead to the spread of drug-resistant strains = These authors contributed equally to this work. * To whom correspondence should be addressed: W.I.S.: tel, 801-585-5402; e-mail, wes@biochem.utah.edu. M.F.S: tel 410-455-2880; e-mail summers@hhmi.umbc.edu. C.P.H: tel, 801-585-5536; e-mail, chris@biochem.utah.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The CA protein plays critical roles in the early and late phases of replication and has long been considered an attractive potential therapeuti...

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

“…2(A)]. 13 C NMR linewidths of roughly 0.5 ppm are observed for several isolated crosspeaks, including isoleucine C a /C c2 and C c1 /C d crosspeaks, threonine C a /C c crosspeaks, and asparagine C a /C b crosspeaks (assignments based on typical chemical shift ranges). Linewidths of roughly 1.0 ppm are observed for alanine C a /C b crosspeaks, isoleucine C a /C c1 crosspeaks, proline C c /C d crosspeaks, aspartate C a /C b crosspeaks, and other asparagine C a /C b crosspeaks.…”

“…To reduce spectral congestion, the CA protein was prepared with partial 13 C labeling by expression on a medium that contained 1,3-13 C 2 -glycerol as the sole carbon source. Labeling with 1,3-13 C 2 -glycerol has been shown previously to facilitate resonance assignments and extraction of structural constraints in solid state NMR studies of various protein systems.…”

“…However, a qualitative comparison with the chemical shifts of NTD and CTD determined by solution NMR 23,27 can be performed. In Figure 3(B), a simulated 2D 13 CA 13 C spectrum is overlaid with the experimental spectrum, assuming crosspeaks only between the solution NMR chemical shifts of directly bonded 13 C sites and assuming the ideal residue-specific labeling patterns expected when 1,3-13 C 2 -glycerol is the sole carbon source. 59 Crosspeaks in the simulated 2D spectrum are assumed to have Gaussian shapes with 0.5 ppm linewidths.…”

“…1 The capsid is believed to be formed by a triangular lattice of CA hexamers, with 12 CA pentamers unevenly distributed at the two ends of the cone to produce a closed shell. 2 Although much progress has been made toward understanding the structure, stability, and assembly of the HIV-1 capsid, through electron microscopy, [3][4][5][6][7][8][9][10] X-ray crystallography, [11][12][13][14][15][16][17][18][19][20] solution nuclear magnetic resonance (NMR), 13,[21][22][23][24][25][26][27] and various biophysical techniques, [28][29][30][31][32][33][34][35][36][37][38][39][40] substantial questions remain regarding the details of intermolecular interactions within and between CA hexamers, the structural variations that permit CA to form both hexamers and pentamers, the driving force for curvature and closure of the CA lattice, and the mechanism and pathway for CA self-assembly. In principle, modern solid state NMR techniques, which can provide sitespecific molecular-level structural information about noncrystalline protein assemblies, [41][...…”

Host–rabies virus protein–protein interactions as druggable antiviral targets

“…Host assembly machines that have been optimized for the virus rather than for the host (e.g., by viral manipulation of host-signaling pathways) are promising antiviral targets that likely can be blocked without substantial toxicity; although they are composed of host proteins, their loss should not impair a host function, only propagation of the virus. Perhaps previous efforts to develop anti-capsid therapeutics have been disappointing precisely because they did not target the hypothesized host enzymes of capsid formation but rather attempted to block capsid protein interactions directly (17,18).…”