Molecular Recognition of Macrocyclic Peptidomimetic Inhibitors by HIV1 Protease † , ‡ (original) (raw)
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Proceedings of the National Academy of Sciences, 2006
HIV-1 protease is an effective target for designing drugs against AIDS, and structural information about the true transition state and the correct mechanism can provide important inputs. We present here the three-dimensional structure of a bi-product complex between HIV-1 protease and the two cleavage product peptides AETF and YVDGAA. The structure, refined against synchrotron data to 1.65 Å resolution, shows the occurrence of the cleavage reaction in the crystal, with the product peptides still held in the enzyme active site. The separation between the scissile carbon and nitrogen atoms is 2.67 Å, which is shorter than a normal van der Waal separation, but it is much longer than a peptide bond length. The substrate is thus in a stage just past the G'Z intermediate described in Northrop's mechanism [Northrop DB (2001) Acc Chem Res 34:790-797]. Because the products are generated in situ, the structure, by extrapolation, can give insight into the mechanism of the cleavage reaction. Both oxygens of the generated carboxyl group form hydrogen bonds with atoms at the catalytic center: one to the OD2 atom of a catalytic aspartate and the other to the scissile nitrogen atom. The latter hydrogen bond may have mediated protonation of scissile nitrogen, triggering peptide bond cleavage. The inner oxygen atoms of the catalytic aspartates in the complex are 2.30 Å apart, indicating a low-barrier hydrogen bond between them at this stage of the reaction, an observation not included in Northrop's proposal. This structure forms a template for designing mechanism-based inhibitors.
Proceedings of the National Academy of Sciences, 1993
The crystal structure of HIV-2 protease in complex with a reduced amide inhibitor [BI-LA-398; Phe-Val-Phe-psi (CH2NH)-Leu-Glu-Ile-amide] has been determined at 2.2-A resolution and refined to a crystallographic R factor of 17.6%. The rms deviation from ideality in bond lengths is 0.018 A and in bond angles is 2.8 degrees. The largest structural differences between HIV-1 and HIV-2 proteases are located at residues 15-20, 34-40, and 65-73, away from the flap region and the substrate binding sites. The rms distance between equivalent C alpha atoms of HIV-1 and HIV-2 protease structures excluding these residues is 0.5 A. The shapes of the S1 and S2 pockets in the presence of this inhibitor are essentially unperturbed by the amino acid differences between HIV-1 and HIV-2 proteases. The interaction of the inhibitor with HIV-2 protease is similar to that observed in HIV-1 protease structures. The unprotected N terminus of the inhibitor interacts with the side chains of Asp-29 and Asp-30. The glutamate side chain of the inhibitor forms hydrogen bonds with the main-chain amido groups of residues 129 and 130.
General physiology and biophysics, 1998
HIV protease has become one of possible targets of anti AIDS treatment The complexmg abihtv of subnanomolar K, tetrapeptide inhibitors Boc-Phe-<r[(S/R)-CH(OH)CH2NH]-Phe-Glu/Gln-Phe-NH 2 (slashes denote alternatives, the four inhibitors are coded as SE, SQ, RE, RQ) (Konvalinka et al 1997) is a subject of in vestigation by X-ray structure analysis of inhibitor-protease complexes to elucidate high affinity of the inhibitors to the protease dimer, the change of affinity as a result of Glu/Gln alteration at the P2' position and of chirahtv of the tetrahedrally co-ordinated transitionstate-analogue carbon Details of the binding mode of these inhibitors are expected to explain the differences in affinities measured by K, , K^E = 0 15 nM, Kf Q = 33 0 nM, K, RE = 0 12 nM, K RQ = 14 0 nM
Scientific Reports
The HIV-2 protease (PR2) is a homodimer of 99 residues with asymmetric assembly and binding various ligands. We propose an exhaustive study of the local structural asymmetry between the two monomers of all available PR2 structures complexed with various inhibitors using a structural alphabet approach. On average, PR2 exhibits asymmetry in 31% of its positions-i.e., exhibiting different backbone local conformations in the two monomers. This asymmetry was observed all along its structure, particularly in the elbow and flap regions. We first differentiated structural asymmetry conserved in most PR2 structures from the one specific to some PR2. Then, we explored the origin of the detected asymmetry in PR2. We localized asymmetry that could be induced by PR2's flexibility, allowing transition from the semi-open to closed conformations and the asymmetry potentially induced by ligand binding. This latter could be important for the PR2's adaptation to diverse ligands. Our results highlighted some differences between asymmetry of PR2 bound to darunavir and amprenavir that could explain their differences of affinity. This knowledge is critical for a better description of PR2's recognition and adaptation to various ligands and for a better understanding of the resistance of PR2 to most PR2 inhibitors, a major antiretroviral class. The human immunodeficiency virus (HIV) of type 2 is a retrovirus that was isolated in 1985 from Western African patients presenting AIDS (acquired immune deficiency syndrome) but that were HIV of type 1 (HIV-1) seronegative. The HIV of type 2 (HIV-2) therapeutic arsenal is limited compared to HIV-1. Indeed, among the antiretroviral classes targeting several viral enzymes, such as reverse transcriptase, fusion protein, integrase and protease (PR) inhibitors, HIV-2 naturally presents resistance to all non-nucleosidic inhibitors of reverse transcriptase, the fusion inhibitor and most of the protease inhibitors (PIs) 1-6. Among the latter, the potency of FDA (Food and Drug Administration)-approved PIs for HIV-2 protease (PR2) compared to HIV-1 protease (PR1) is decreased by factors ranging from 2 to 80, resulting in only 3 usable PIs for HIV-2: saquinavir, lopinavir, and darunavir (DRV) 1,7. Recent in vivo studies also showed that HIV-2 does not present a stronger virological response to the more recently available class of integrase inhibitors than previously observed response to PIs 8 , underlying the need for a third strong antiretroviral agent that will prevail against HIV-2 infection. Thus, it is still necessary to develop new molecules designed for HIV-2 today. HIV PR is essential for hydrolysing the viral Gag and the Gag-Pol precursor polyproteins during the maturation of infectious viral particles. PR is an aspartic protease corresponding to a C2-symmetric homodimer of 99 residues in each monomer. The binding site is located at the interface between the two monomers and includes the catalytic triplet, Asp-Thr-Gly, conserved in all aspartic proteases. The PR recognizes various non-homologous substrates (Gag and Pol polyproteins) at several cleavage sites and PIs 9. All these ligands are often asymmetric, and their binding is associated with large conformational changes resulting in a transition from a semi-open
Conformationally constrained HIV-1 protease inhibitors
Bioorganic & Medicinal Chemistry Letters, 1994
The synthesis and structure activity relationships of conformationally constrained analogs of the HIV-1 protease inhibitor L-685,434 are described. In addition, the X-ray crystal structure of a complex between L700,497 and the HIV-l protease is shown. The human immun~e~ciency virus type-l (HIV-l) protease plays a key role in the HIV viral life cycle by posttranslational processing of gag and gag-pal polypmteins into viral core components.1 Genetic inactivation of the protease resulted in the production of non-infectious virions in cell culture.2
Journal of Molecular Biology, 2004
The compound UIC-94017 (TMC-114) is a second-generation HIV protease inhibitor with improved pharmacokinetics that is chemically related to the clinical inhibitor amprenavir. UIC-94017 is a broad-spectrum potent inhibitor active against HIV-1 clinical isolates with minimal cytotoxicity. We have determined the high-resolution crystal structures of UIC-94017 in complexes with wild-type HIV-1 protease (PR) and mutant proteases PR V82A and PR I84V that are common in drug-resistant HIV. The structures were refined at resolutions of 1.10-1.53 Å . The crystal structures of PR and PR I84V with UIC-94017 ternary complexes show that the inhibitor binds to the protease in two overlapping positions, while the PR V82A complex had one ordered inhibitor. In all three structures, UIC-94017 forms hydrogen bonds with the conserved main-chain atoms of Asp29 and Asp30 of the protease. These interactions are proposed to be critical for the potency of this compound against HIV isolates that are resistant to multiple protease inhibitors. Other small differences were observed in the interactions of the mutants with UIC-94017 as compared to PR. PR V82A showed differences in the position of the main-chain atoms of residue 82 compared to PR structure that better accommodated the inhibitor. Finally, the 1.10 Å resolution structure of PR V82A with UIC-94017 showed an unusual distribution of electron density for the catalytic aspartate residues, which is discussed in relation to the reaction mechanism.
Bioorganic & Medicinal Chemistry Letters, 1994
Conformationally-constrained macrocyclic peptide-based hydroxyethylamines, with 17-to 19membered ring systems, have been designed and synthesized as HIV protease inhibitors. Structure-activity relationships were consistent with molecular modeling studies, and certain cyclic inhibitors were developed with HIV protease IC50 values of-1 nM, and antiviral activities (HIV-l/RF infected MT-2 cells) of EC50 4-8 nM. Introduction. The aspartic proteinase encoded by the Human Immunodeficiency Virus (HIV), essential for the processing of the gag and gag-pal polyprotein gene products, has been identified as a viable chemotherapeutic target for the Acquired Immune Deficiency Syndrome (AIDS)l. As a result, this proteinase has been the subject of intensive research, and a great number of inhibitors have been developed for this enzyme213. Several transition-state analog inhibitors293, including reduced amides, statines, hydroxyethylenes, dihydroxyethylenes, hydroxyethylamines, and hydroxyamides (norstatines)4 have been reported, with many examples expressing low nanomolar or subnanomolar HIV protease inhibitory activity. Of particular note, potent C2-symmetric and pseudosymmetric inhibitors, including linear peptide-based molecules5 and non-peptide cyclic ureas6, have been developed through the aid of molecular modeling studies of HIV-proteaselinhibitor complex X-ray structures. The incorporation of conformationally-constrained macrocyclic ring structures in peptide-based enzyme inhibitors has the potential to provide a number of advantages relative to the acyclic analogs. These include increased binding affinity resulting from an entropic advantage, enhanced stabilization toward proteolytic enzymes, and specificity for the target enzyme. Several examples of macrocyclic peptide-based inhibitors of the aspartic proteinase renin have been reported 7. In efforts to develop novel and potent HIV protease inhibitors with such favorable properties, we have therefore designed and synthesized macrocyclic peptide-based inhibitors, as analogs of the linear hydroxyethylamine inhibitors reported by Rich and coworkers8 and by the Roche group9. Results and Discussion. Examination 10 of the X-ray crystal structure of the heptapeptide-derived hydroxyethylamine inhibitor 'JG-365' (Ac-Ser-Leu-Asn-PheY[CH(OH)CH2N]Pro-Ile-Val-OMe) complexed to
Capturing the Reaction Pathway in Near-Atomic-Resolution Crystal Structures of HIV-1 Protease
Biochemistry, 2012
Snapshots of three consecutive steps in the proteolytic reaction of HIV-1 protease (PR) were obtained in crystal structures at resolutions of 1.2−1.4 Å. Structures of wild-type protease and two mutants (PR V32I and PR I47V ) with V32I and I47V substitutions, which are common in drug resistance, reveal the gem-diol tetrahedral intermediate, the separating N-and C-terminal products, and the C-terminal product of an autoproteolytic peptide. These structures represent three stages in the reaction pathway and shed light on the reaction mechanism. The near-atomic-resolution geometric details include a short hydrogen bond between the intermediate and the outer carboxylate oxygen of one catalytic Asp25 that is conserved in all three structures. The two products in the complex with mutant PR I47V have a 2.2 Å separation of the amide and carboxyl carbon of the adjacent ends, suggesting partial cleavage prior to product release. The complex of mutant PR V32I with a single C-terminal product shows density for water molecules in the other half of the binding site, including a partial occupancy water molecule interacting with the product carboxylate end and the carbonyl oxygen of one conformation of Gly27, which suggests a potential role of Gly27 in recycling from the product complex to the ligand-free enzyme. These structural details at near-atomic resolution enhance our understanding of the reaction pathway and will assist in the design of mechanism-based inhibitors as antiviral agents.