RESUMO
Two new X-ray structures of an HIV-1 protease mutant (A71V, V82T, I84V) in complex with inhibitors SE and SQ, pseudotetrapeptide inhibitors with an acyclic S-hydroxyethylamine isostere, were determined. Comparison of eight structures exploring the binding of four similar inhibitors--SE, SQ (S-hydroxyethylamine isostere), OE (ethyleneamine), and QF34 (hydroxyethylene)--to wild-type and A71V/V82T/I84V HIV-1 protease elucidates the principles of altered interaction with changing conditions. The A71V mutation, which is distant from the active site, causes changes in the structure of the enzyme detectable by the means of X-ray structure analysis, and a route of propagation of the effect toward the active site is proposed.
Assuntos
Etanolaminas/química , Inibidores da Protease de HIV/química , Protease de HIV/química , Protease de HIV/genética , Modelos Moleculares , Oligopeptídeos/química , Sítios de Ligação , Cristalografia por Raios X , Ligação de Hidrogênio , Ligantes , Estrutura Molecular , Mutação , Relação Estrutura-AtividadeRESUMO
Peptidomimetic inhibitors of human immunodeficiency virus-1 protease are successful lead substances for the development of virostatic drugs against HIV as the causative agent of acquired immunodeficiency syndrome (AIDS). The hydroxyethylamine isostere of the proteolytic cleavage intermediate provides a suitable replacement for the peptide bond. A series of acyclic pseudopeptide inhibitors with the hydroxyethylamine isostere varying in chiral carbon configuration and P'2 residue type were structurally analysed by single-crystal X-ray crystallography. The compounds inhibit HIV protease with subnanomolar inhibition constants and block viral replication in tissue cultures. Here, the structure of such a complex with the R configuration of the isosteric group (PDB code 1zsf) is presented together with newly available synchrotron data for a complex with the S stereoisomer of the inhibitor (PDB code 1zsr). Comparison of the structure and binding with other complexes of HIV-1 protease and similar inhibitors contributes to the understanding of how these molecules bind to the wild-type form of this enzyme. The hydroxy group of the R stereoisomer interacts with one of the catalytic aspartic acids by a short hydrogen bond with rather extreme geometry. The change of configuration of the chiral carbon bearing the hydroxyl from S to R does not influence the inhibition efficiency in this case.
Assuntos
Inibidores da Protease de HIV/química , Protease de HIV/química , HIV-1/enzimologia , Modelos Moleculares , Oligopeptídeos/química , Sítios de Ligação , Cristalografia por Raios X , Etanolaminas/química , Protease de HIV/metabolismo , Ligação de Hidrogênio , Ligação Proteica , EstereoisomerismoRESUMO
The crystal structure of the complex between human immunodeficiency virus type 1 (HIV-1) protease and a peptidomimetic inhibitor of ethyleneamine type has been refined to R factor of 0.178 with diffraction limit 2.5 A. The peptidomimetic inhibitor Boc-Phe-Psi[CH2CH2NH]-Phe-Glu-Phe-NH2 (denoted here as OE) contains the ethyleneamine replacement of the scissile peptide bond. The inhibitor lacks the hydroxyl group which is believed to mimic tetrahedral transition state of proteolytic reaction and thus is suspected to be necessary for good properties of peptidomimetic HIV-1 protease inhibitors. Despite the missing hydroxyl group the inhibition constant of OE is 1.53 nm and it remains in the nanomolar range also towards several available mutants of HIV-1 protease. The inhibitor was found in the active site of protease in an extended conformation with a unique hydrogen bond pattern different from hydroxyethylene and hydroxyethylamine inhibitors. The isostere nitrogen forms a hydrogen bond to one catalytic aspartate only. The other aspartate forms two weak hydrogen bridges to the ethylene group of the isostere. A comparison with other inhibitors of this series containing isostere hydroxyl group in R or S configuration shows different ways of accommodation of inhibitor in the active site. Special attention is devoted to intermolecular contacts between neighbouring dimers responsible for mutual protein adhesion and for a special conformation of Met46 and Phe53 side chains not expected for free protein in water solution.
Assuntos
Protease de HIV/metabolismo , HIV-1/enzimologia , Oligopeptídeos/química , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Sítios de Ligação , Domínio Catalítico , Protease de HIV/química , HIV-1/efeitos dos fármacos , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Ligação Proteica , Conformação Proteica , Eletricidade Estática , Difração de Raios XRESUMO
Depending on the excess of ligand used for complex formation, the HIV-1 protease complexed with a novel phenylnorstatine inhibitor forms crystals of either hexagonal (P6(1)) or orthorhombic (P2(1)2(1)2(1)) symmetry. The orthorhombic form shows an unusual complexity of crystal packing: in addition to one inhibitor molecule that is bound to the enzyme active site, the second inhibitor molecule is bound as an outer ligand at the protein interface. Binding of the outer ligand apparently increases the crystal-quality parameters so that the diffraction data allow solution of the structure of the complex at 1.03 A, the best resolution reported to date. The outer ligand interacts with all four surrounding HIV-1 protease molecules and has a bent conformation owing to its accommodation in the intermolecular space. The parameters of the solved structures of the orthorhombic and hexagonal forms are compared.
Assuntos
Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/metabolismo , Protease de HIV/química , Protease de HIV/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Conformação Proteica , Sensibilidade e EspecificidadeRESUMO
HIV protease represents a major target for development of antiviral therapeutics. The introduction of HIV protease (PR) inhibitors (PIs) to clinical practice and the application of highly active antiretroviral therapy resulted in decreased mortality and prolonged life expectancy of HIV-positive patients. However, the high polymorphism of HIV leads to rapid selection of viral variants resistant towards the inhibitors. Such resistant PR variants have developed in HIV-positive patients after treatment with any of the eight PIs approved for clinical use. In this review we overview (i) the methods for the identification and assessment of viral resistance in HIV positive patients, and (ii) the approaches medicinal chemists take to overcome it. Rational antiviral therapy brings about the need for quantitative assessment of the level of drug resistance development in the course of the treatment. At present, two main approaches are taken: in genotypic assays the viral sequences are PCR amplified, sequenced and changes in the viral gene sequence known to be associated with reduced drug sensitivity are identified, while phenotypic assays test the ability of a virus to grow in the presence of a drug or combination of drugs. The advantages and drawbacks of these methods, as well as their relevance for the therapy are discussed. We also review the efforts to design second-generation PIs, capable of potently inhibiting multi-resistant HIV-1 PR species, using structure-assisted design of the compounds targeted to the active site, as well as alternative approaches with compounds binding to other domains of the PR molecule.
Assuntos
Desenho de Fármacos , Farmacorresistência Viral/efeitos dos fármacos , Inibidores da Protease de HIV/química , Dimerização , Farmacorresistência Viral/genética , Genótipo , HIV/efeitos dos fármacos , HIV/genética , HIV/metabolismo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Inibidores da Protease de HIV/farmacologia , Humanos , Fenótipo , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
The X-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH(2) has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibitor shows subnanomolar K(i) values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance development. The structure comprising the phenylnorstatine moiety of (2R,3S)-chirality displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. This high resolution makes it possible to assess the donor and acceptor relations of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. A structural mechanism for the unimpaired inhibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.
Assuntos
Inibidores da Protease de HIV/química , Protease de HIV/química , Fenilbutiratos/química , Sítios de Ligação , Cristalografia por Raios X , Farmacorresistência Viral/genética , Protease de HIV/genética , Modelos Moleculares , Mutação , Ligação Proteica , Estereoisomerismo , TermodinâmicaRESUMO
An X-ray structure (resolution 2.2 A) of mutant HIV-1 protease (A71V, V82T, I84V) complexed with a newly developed peptidomimetic inhibitor with an ethylenamine isostere Boc-Phe-Psi[CH(2)CH(2)NH]-Phe-Glu-Phe-NH(2), denoted as OE, is described and compared with the complex of wild-type HIV-1 protease with the same inhibitor (resolution 2.5 A). OE shows tight binding to the wild type (K(i) = 1.5 nM) as well as mutant (K(i) = 4.1 nM) protease. The hydrogen bonds formed, in the case of hydroxyethylamine inhibitors, by a hydroxyl group are, in the case of OE inhibitors, replaced by a bifurcated hydrogen bond from the isosteric NH group to both catalytic aspartates Asp 25 and Asp 125. The binding modes of OE inhibitor to the wild type and mutant protease are similar. However, in the mutant protease, weaker van der Waals interactions of the mutated residues Val 84 and Val 184 with OE were found. This lack of interaction energy is compensated by a new aromatic hydrogen bond between the phenyl ring of the inhibitor in position P1 and the mutated residue Thr 182. Energy analysis based on molecular mechanics has been performed to distinguish between the static and dynamic backgrounds of disorder observed at the mutation sites Thr 82, Val 84, Thr 182, and Val 184.
Assuntos
Etilaminas/química , Inibidores da Protease de HIV/química , Protease de HIV/química , HIV-1/enzimologia , Oligopeptídeos/química , Cristalografia por Raios X , Protease de HIV/genética , Modelos Moleculares , Conformação Molecular , Mimetismo Molecular , Estrutura Molecular , Ligação Proteica , TermodinâmicaRESUMO
The yeasts of the genus Candida are opportunistic pathogens associated with the rising incidence of life-threatening infections in immunocompromised individuals. Secretion of aspartic proteinases has been determined to be one of the virulence factors of the pathogenic Candida species. To analyze the extracellular proteolytic activities of a large number of Candida clinical isolates, we developed a screening system based on a solid medium containing hemoglobin as the sole nitrogen source. The cleavage of hemoglobin by the secreted proteinases results in formation of clearance zones. The visibility of such zones was enhanced by addition of an acid-base indicator. Using this system, we assessed 245 clinical isolates of Candida from patients in the hospital of the Faculty of Medicine, Palacky University, Olomouc, Czech Republic, for the presence of secreted aspartic proteases (Saps). We also used the test plates for rapid semiquantitative testing of Sap inhibitors. Most of the pepstatin analogs affected the formation of the zones of clearance as well as the growth of Candida albicans, C. tropicalis, and C. parapsilosis colonies. By contrast, the human immunodeficiency virus proteinase inhibitors saquinavir, ritonavir, nelfinavir, and indinavir had no effect on the Candida strains tested. These results are in agreement with the inhibition constants obtained for the individual inhibitors with purified Saps. Thus, the plates containing hemoglobin proved to be an appropriate tool for the rapid and reliable assessment of Sap production and inhibition.
Assuntos
Candida/isolamento & purificação , Endopeptidases/análise , Técnicas Microbiológicas/métodos , Candida/enzimologia , Meios de Cultura , Endopeptidases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismoRESUMO
Protease inhibitors (PIs) are an important class of drugs for the treatment of HIV infection. However, in the course of treatment, resistant viral variants with reduced sensitivity to PIs often emerge and become a major obstacle to successful control of viral load. On the basis of a compound equipotently inhibiting HIV-1 and 2 proteases (PR), we have designed a pseudopeptide inhibitor, QF34, that efficiently inhibits a wide variety of PR variants. In order to analyze the potency of the inhibitor, we constructed PR species harboring the typical (signature) mutations that confer resistance to commercially available PIs. Kinetic analyses showed that these mutated PRs were inhibited up to 1,000-fold less efficiently by the clinically approved PIs. In contrast, all PR species were effectively inhibited by QF34. In a clinical study, we have monitored 30 HIV-positive patients in the Czech Republic undergoing highly active antiretroviral therapy, and have identified highly PI resistant variants. Kinetic analyses revealed that QF34 retained its subnanomolar potency against multi-drug resistant PR variants. X-ray crystallographic analysis and molecular modeling experiments explained the wide specificity of QF34: this inhibitor binds to the PR in an unusual manner, thus avoiding contact sites that are mutated upon resistance development, and the unusual binding mode and consequently the binding energy is therefore preserved in the complex with a resistant variant. These results suggest a promising route for the design of second-generation PIs that are active against a variety of resistant PR variants.
Assuntos
Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Substituição de Aminoácidos , Terapia Antirretroviral de Alta Atividade , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Resistência Microbiana a Medicamentos/genética , Resistência a Múltiplos Medicamentos/genética , Feminino , Genótipo , Infecções por HIV/virologia , Inibidores da Protease de HIV/química , HIV-1/genética , HIV-1/metabolismo , Humanos , Cinética , Masculino , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Mutação , Oligopeptídeos/química , Conformação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
A complex structure of HIV-1 protease with a hydroxyethylamine-containing inhibitor Boc-Phe-Psi[(S)-CH(OH)CH2NH]-Phe-Gln-Phe-NH2 has been determined by X-ray diffraction to 1.8 A resolution. The inhibitor is bound in the active site of the protease dimer with its hydroxyethylamine isostere participating in hydrogen bonds to the catalytic aspartates 25 and 25' and glycine 27' of the active site triads via five hydrogen bonds. The isostere amine interactions with the catalytic aspartates result in a displacement of the isostere hydroxy group in comparison with the common position known for analogous hydroxyethylamine containing inhibitors. A comparison with another inhibitor of this series shows that the change of one atom of the P2' side chain (Glu/Gln) leads to an altered ability of creating hydrogen bonds to the active site and within the inhibitor molecule. The diffraction data collected at a synchrotron radiation source enabled a detailed analysis of the complex solvation and of alternative conformations of protein side chains.