Peptide substrates and inhibitors of the HIV-1 protease

Moore, Michael L.; Bryan, William M.; Fakhoury, Stephen A.; Magaard, Victoria W.; Huffman, William F.; Dayton, Brian D.; Meek, Thomas D.; Hyland, Lawrence J.; Dreyer, Geoffrey B.; Metcalf, Brian W.; Strickler, J. Rudi; Gorniak, Joselina G.; Debouck, Christine

doi:10.1016/0006-291x(89)90008-9

Cited by 160 publications

(77 citation statements)

References 17 publications

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“…A high probability of 1 being converted to products presents a virtually irreversible step prior to chemistry, which can mask expression of the KIE on the chemical step (the intrinsic KIE), the value that reports on the transition state. The reportedly high K m for this peptide (37) and the observation that the 15 N KIE value is within the limit of all calculated transition-state models for this reaction (see discussion below) suggest that the forward commitment can be considered negligible in our analysis, simplifying Eq. 2 of the observed isotope effects to the product of the equilibrium isotope effect (EIE) on formation of 3 and the intrinsic KIE determined by the ratelimiting transition state…”

Section: Resultsmentioning

confidence: 84%

Transition states of native and drug-resistant HIV-1 protease are the same

Kipp¹,

Hirschi²,

Wakata³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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HIV-1 protease is an important target for the treatment of HIV/ AIDS. However, drug resistance is a persistent problem and new inhibitors are needed. An approach toward understanding enzyme chemistry, the basis of drug resistance, and the design of powerful inhibitors is to establish the structure of enzymatic transition states. Enzymatic transition structures can be established by matching experimental kinetic isotope effects (KIEs) with theoretical predictions. However, the HIV-1 protease transition state has not been previously resolved using these methods. We have measured primary 14 at the carbonyl carbon, proton transfer to the amide nitrogen leaving group, and C-N bond cleavage. A transition structure consistent with the KIE values involves proton transfer from the active site Asp-125 (1.32 Å) with partial hydrogen bond formation to the accepting nitrogen (1.20 Å) and partial bond loss from the carbonyl carbon to the amide leaving group (1.52 Å). The KIEs measured for the native and I84V enzyme indicate nearly identical transition states, implying that a true transition-state analogue should be effective against both enzymes.aspartyl protease | protease mechanism | transition-state structure | drug design

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Section: Resultsmentioning

confidence: 84%

Transition states of native and drug-resistant HIV-1 protease are the same

Kipp¹,

Hirschi²,

Wakata³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Among them there are: reverse transcription, which can be efficiently blocked by nucleoside analogues such as AZT, ddl, and ddC (for a review see De Clercq, 1989), and proteolytic processing of viral precursors which can be inhibited efficiently by proteinase inhibitors Moore et al, 1989;Grobelny et al, 1990;Roberts et al, 1990;Tomaselli et et., 1990;Sham et al, 1991;Vacca et al, 1991). Specifically, the nucleoside analogues inhibit the reverse transcriptase of HIV preventing the formation of newly synthethized viral DNA (Mitsuya and Broder, 1987), while the proteinase inhibitors suppress the action of the HIV-encoded proteinase which is responsible for post-translational processing of the polyprotein gene products of gag and gag/pol, thus preventing the maturation of viral progeny (Martin, 1992).…”

Section: Introductionmentioning

confidence: 99%

In vitroSelection of Human Immunodeficiency Virus Type 1 Resistant to Ro 31-8959 Proteinase Inhibitor

Dianzani

Antonelli

Turriziani

et al. 1993

Antivir Chem Chemother

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“…Cleavage of the Gag precursor at these multiple sites also appears to be highly regulated. Peptide substrates of these different sites are cleaved by the viral protease at different rates, and cleavage of the intact Gag precursor in vitro goes through a distinct series of intermediates that are generated due to different rates of cleavage at the various sites (4,7,9,24,30,36,38,43,52). The pattern of intermediates seen in infected cells and in recently budded virus particles is consistent with the ordered cleavage of Gag that has been observed in vitro (11,12,29,36,56,58).…”

mentioning

confidence: 99%

“…The amino acids flanking the target scissile bond are generally hydrophobic (16,33,37). A number of studies have used peptide or Gag substrates in an attempt to define the role of specific amino acids at different positions flanking the scissile bond as they impact the rate of cleavage by the viral protease (3,5,13,14,19,30,34,36,38,44,45,51,(53)(54)(55); recently reviewed in reference 27). In the present study we have examined the ability of approximately 15 different amino acids in the P1 position of the five different Gag cleavage sites to support cleavage by the HIV-1 protease (the P1 position is the amino acid immediately upstream of the scissile bond, and the P1Ј position is the amino acid immediately downstream of the scissile bond).…”

mentioning

confidence: 99%

Replacement of the P1 Amino Acid of Human Immunodeficiency Virus Type 1 Gag Processing Sites Can Inhibit or Enhance the Rate of Cleavage by the Viral Protease

Pettit

Henderson

Schiffer

et al. 2002

J Virol

105

116

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Processing of the human immunodeficiency virus type 1 (HIV-1) Gag precursor is highly regulated, with differential rates of cleavage at the five major processing sites to give characteristic processing intermediates. We examined the role of the P1 amino acid in determining the rate of cleavage at each of these five sites by using libraries of mutants generated by site-directed mutagenesis. Between 12 and 17 substitution mutants were tested at each P1 position in Gag, using recombinant HIV-1 protease (PR) in an in vitro processing reaction of radiolabeled Gag substrate. There were three sites in Gag (MA/CA, CA/p2, NC/p1) where one or more substitutions mediated enhanced rates of cleavage, with an enhancement greater than 60-fold in the case of NC/p1. For the other two sites (p2/NC, p1/p6), the wild-type amino acid conferred optimal cleavage. The order of the relative rates of cleavage with the P1 amino acids Tyr, Met, and Leu suggests that processing sites can be placed into two groups and that the two groups are defined by the size of the P1 amino acid. These results point to a trans effect between the P1 and P1 amino acids that is likely to be a major determinant of the rate of cleavage at the individual sites and therefore also a determinant of the ordered cleavage of the Gag precursor.

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Peptide substrates and inhibitors of the HIV-1 protease

Cited by 160 publications

References 17 publications

Transition states of native and drug-resistant HIV-1 protease are the same

Transition states of native and drug-resistant HIV-1 protease are the same

In vitroSelection of Human Immunodeficiency Virus Type 1 Resistant to Ro 31-8959 Proteinase Inhibitor

Replacement of the P1 Amino Acid of Human Immunodeficiency Virus Type 1 Gag Processing Sites Can Inhibit or Enhance the Rate of Cleavage by the Viral Protease

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