The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed.

• Klíčová slova
• HAART, HIV protease, alternative inhibitors, pharmacokinetic boosting, protease dimerization, protease inhibitors, resistance development,
• Publikační typ
• časopisecké články MeSH

Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2' binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

• MeSH
• darunavir MeSH
• genové produkty env - virus lidské imunodeficience metabolismus   MeSH
• genové produkty gag - virus lidské imunodeficience metabolismus   MeSH
• HIV infekce virologie   MeSH
• HIV-1 účinky léků   izolace a purifikace   MeSH
• HIV-proteasa chemie   genetika   metabolismus   MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• missense mutace MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA MeSH
• polyproteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• substituce aminokyselin MeSH
• sulfonamidy farmakologie   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virová léková rezistence * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• darunavir MeSH
• genové produkty env - virus lidské imunodeficience MeSH
• genové produkty gag - virus lidské imunodeficience MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• p16 protease, Human immunodeficiency virus 1 MeSH Prohlížeč
• polyproteiny MeSH
• sulfonamidy MeSH

Lopinavir (LPV) is a second-generation HIV protease inhibitor (PI) designed to overcome resistance development in patients undergoing long-term antiviral therapy. The mutation of isoleucine at position 47 of the HIV protease (PR) to alanine is associated with a high level of resistance to LPV. In this study, we show that recombinant PR containing a single I47A substitution has the inhibition constant (K(i) ) value for lopinavir by two orders of magnitude higher than for the wild-type PR. The addition of the I47A substitution to the background of a multiply mutated PR species from an AIDS patient showed a three-order-of-magnitude increase in K(i) in vitro relative to the patient PR without the I47A mutation. The crystal structure of I47A PR in complex with LPV showed the loss of van der Waals interactions in the S2/S2' subsites. This is caused by the loss of three side-chain methyl groups due to the I47A substitution and by structural changes in the A47 main chain that lead to structural changes in the flap antiparallel beta-strand. Furthermore, we analyzed possible interaction of the I47A mutation with secondary mutations V32I and I54V. We show that both mutations in combination with I47A synergistically increase the relative resistance to LPV in vitro. The crystal structure of the I47A/I54V PR double mutant in complex with LPV shows that the I54V mutation leads to a compaction of the flap, and molecular modeling suggests that the introduction of the I54V mutation indirectly affects the strain of the bound inhibitor in the PR binding cleft.

• MeSH
• alanin metabolismus   MeSH
• Escherichia coli genetika   MeSH
• HIV-proteasa chemie   genetika   izolace a purifikace   metabolismus   MeSH
• inhibitory HIV-proteasy chemie   metabolismus   farmakologie   MeSH
• katalýza MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• Lopinavir MeSH
• molekulární modely MeSH
• náchylnost k nemoci * MeSH
• pyrimidinony chemie   metabolismus   farmakologie   MeSH
• rekombinantní proteiny antagonisté a inhibitory   chemie   izolace a purifikace   MeSH
• sekundární struktura proteinů MeSH
• substituce aminokyselin * MeSH
• virová léková rezistence genetika   MeSH
• vodíková vazba MeSH
• výpočetní biologie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanin MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• Lopinavir MeSH
• pyrimidinony MeSH
• rekombinantní proteiny MeSH

While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development.

• MeSH
• buněčné linie MeSH
• chemické modely MeSH
• difrakce rentgenového záření MeSH
• HIV-1 enzymologie   genetika   fyziologie   MeSH
• HIV-proteasa chemie   genetika   izolace a purifikace   metabolismus   MeSH
• inhibitory reverzní transkriptasy chemie   MeSH
• inzerční mutageneze * MeSH
• katalýza MeSH
• kinetika MeSH
• konsenzuální sekvence MeSH
• látky proti HIV terapeutické užití   MeSH
• ledviny cytologie   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• rekombinantní proteiny chemie   izolace a purifikace   metabolismus   MeSH
• replikace viru MeSH
• RNA virová analýza   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virová léková rezistence * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HIV-proteasa MeSH
• inhibitory reverzní transkriptasy MeSH
• látky proti HIV MeSH
• rekombinantní proteiny MeSH
• RNA virová MeSH

HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a K(i) value of 2.2 nM and a submicromolar EC(50) in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 A resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3' subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition.

• MeSH
• aspartátové endopeptidasy chemie   MeSH
• borany chemická syntéza   chemie   farmakologie   MeSH
• HIV-proteasa chemie   MeSH
• inhibitory HIV-proteasy chemická syntéza   chemie   farmakologie   MeSH
• krystalografie rentgenová MeSH
• racionální návrh léčiv * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aspartátové endopeptidasy MeSH
• borany MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• p16 protease, Human immunodeficiency virus 2 MeSH Prohlížeč