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P Strop

Publications and source records attributed to P Strop.

At least 19 recordsLinked to original sources
PubMed1997

Configurations of diastereomeric hydroxyethylene isosteres strongly affect biological activities of a series of specific inhibitors of human-immunodeficiency-virus proteinase.

Human immunodeficiency virus (HIV) proteinase (PR) represents an important target for antiviral chemotherapy. We present an analysis of inhibitory activities of a series of pseudopeptide inhibitors of HIV-1 PR. All inhibitors were N-protected tetrapeptides with the scissile bond replaced by a nonhydrolysable hydroxyethylene or hydroxyethylamine isostere. To elucidate subtle structural requirements of the PR binding cleft, we synthesised inhibitors with four combinations of configurations at the asymmetric carbons of the isostere. Compounds were tested in vitro using purified recombinant enzyme and a chromogenic peptide substrate. The differences in inhibition constants between individual diastereoisomers reached three orders of magnitude. The most active hydroxyethylene-containing inhibitor possessed the 2R,4S,5S configuration at the isostere. Inhibitor activity was also tested in mammalian cell culture by analysing reduction of viral polyprotein processing and virus infectivity. The results obtained in tissue culture were generally in agreement with the in vitro data, giving a similar order of potency for the individual diastereoisomers. The most active compounds completely blocked production of infectious virus. A simulation method for interaction was employed to build a model of the inhibitors in the PR active site, to identify the interactions responsible for the differences in activities of individual stereoisomers, and to estimate the relative contribution of individual structural features to the overall inhibitory activity.

Animals↗
PubMed1997

Potency comparison of peptidomimetic inhibitors against HIV-1 and HIV-2 proteinases: design of equipotent lead compounds.

HIV-1 and HIV-2 proteinases (PR) are responsible for the processing of viral polyproteins, a step that is crucial for the formation of infectious virus particles. PR represents one of the most important targets for antiviral chemotherapy. Inhibitors of HIV-1 PR usually exhibit a 10- to 100-fold weaker affinity for HIV-2 PR. In order to design subnanomolar inhibitors for both HIV-1 and HIV-2 PRs, we prepared a series of compounds varying in the type of scissile bond replacement as well as in the P1, P1', and P2' side chains. While inhibitors containing reduced amide, hydroxyethylamine and statine isosteres had Ki values in the range of 10(-10)-10(-9) M against HIV-1 PR; their activities against HIV-2 PR were several orders of magnitude lower. Glutamic acid was identified to be the optimal P2' residue for both PRs. HIV-2 PR was shown to be more sensitive to P2' Glu-->Gln replacement. Using this data set we were able to design and prepare hydroxyethylene isostere containing inhibitors that were equipotent against both PRs.

Aspartic Acid Endopeptidases↗
PubMed1997

Crystal structures of a marginally active thymidylate synthase mutant, Arg 126-->Glu.

Thymidylate synthase (TS) is a long-standing target for anticancer drugs and is of interest for its rich mechanistic features. The enzyme catalyzes the conversion of dUMP to dTMP using the co-enzyme methylenetetrahydrofolate, and is perhaps the best studied of enzymes that catalyze carbon-carbon bond formation. Arg 126 is found in all TSs but forms only 1 of 13 hydrogen bonds to dUMP during catalysis, and just one of seven to the phosphate group alone. Despite this, when Arg 126 of TS from Escherichia coli was changed to glutamate (R126E), the resulting protein had kcat reduced 2000-fold and Km reduced 600-fold. The crystal structure of R126E was determined under two conditions--in the absence of bound ligand (2.4 A resolution), and with dUMP and the antifolate CB3717 (2.2 A resolution). The first crystals, which did not contain dUMP despite its presence in the crystallization drop, displayed Glu 126 in a position to sterically and electrostatically interfere with binding of the dUMP phosphate. The second crystals contained both dUMP and CB3717 in the active site, but Glu 126 formed three hydrogen bonds to nearby residues (two through water) and was in a position that partially overlapped with the normal phosphate binding site, resulting in a approximately 1 A shift in the phosphate group. Interestingly, the protein displayed the typical ligand-induced conformational change, and the covalent bond to Cys 146 was present in one of the protein's two active sites.

Antineoplastic Agents↗
PubMed1997

Substrates and inhibitors of human T-cell leukemia virus type 1 (HTLV-1) proteinase.

Human T-cell leukemia virus type 1 (HTLV-1) proteinase, a 125 residue polypeptide, was chemically synthesized using the solid phase method. The crude product was purified, renaturated and proteolytic activity was tested using oligopeptide substrates derived from processing sites of various retroviral polyproteins. Cleavage of the oligopeptide substrates together with an initial study using a series of HIV-1 and MAV (myeloblastosis associated virus) proteinase inhibitors suggest that the substrate specificity of HTLV-1 proteinase is very close to that of BLV (bovine leukemia virus) proteinase and distinct from that of both HIV-1 and MAV proteinases.

Amino Acid Sequence↗
PubMed1997

Peptide inhibitors of HIV-1 and HIV-2 proteases: a comparative study.

HIV-1 and HIV-2 proteases (PR) which play the key role in the formation of infectious viral particles offer a target for inhibitors that could block the maturation step. Inhibitors o HIV-1 PR exhibit mostly 1-2 orders of magnitude weaker affinity for HIV-2 PR. The subsite specificity study of the HIV-1 and HIV-2 proteases performed with inhibitors varying in the type of nonhydrolysable bonds and amino acid residues in the P1, P1'and P2'positions has led us to the design of inhibitors with 2S,4S and 2R,4S stereomeres of the hydroxyethylene isostere and Glu or Gln in the P2'positions. These compounds inhibit HIV-1 and HIV-2 proteases in vitro in subnanomolar concentrations and exhibit the activity in tissue culture.

Animals↗
PubMed1996

Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

Amino Acid Sequence↗
PubMed1995

Cloning, bacterial expression, and characterization of the Mason-Pfizer monkey virus proteinase.

We have cloned and expressed the 3' region of the Mason-Pfizer monkey virus pro gene in Escherichia coli. The recombinant 26-kDa precursor undergoes rapid self-processing both in E. coli and in vitro at the NH2 terminus, yielding a proteolytically active 17-kDa protein, p17. This initial cleavage is followed in vitro by a much slower self-processing that leads to emergence of proteolytically active p12 and a COOH-terminal cleavage product p5. We have found the NH2-terminal processing site of both the p17 and p12 to be identical and similar to the amino terminus of the mouse mammary tumor virus proteinase. We have also identified the COOH-terminal processing site of the p12 form. Using purified recombinant proteins and synthetic oligopeptide substrates based on naturally occurring retroviral processing sites, we have determined the enzymatic activity and specificity of the Mason-Pfizer monkey virus proteinase to be more closely related to that of myeloblastosis-associated virus proteinase rather than that of the Human immunodeficiency virus type 1 proteinase. Inhibition studies using peptide inhibitors support these results.

Amino Acid Sequence↗
PubMed1995

Comparative analysis of the X-ray structures of HIV-1 and HIV-2 proteases in complex with CGP 53820, a novel pseudosymmetric inhibitor.

BACKGROUND: The human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). Two subtypes of the virus, HIV-1 and HIV-2, have been characterized. The protease enzymes from these two subtypes, which are aspartic acid proteases and have been found to be essential for maturation of the infectious particle, share about 50% sequence identity. Differences in substrate and inhibitor binding between these enzymes have been previously reported. RESULTS: We report the X-ray crystal structures of both HIV-1 and HIV-2 proteases each in complex with the pseudosymmetric inhibitor, CGP 53820, to 2.2 A and 2.3 A, respectively. In both structures, the entire enzyme and inhibitor could be located. The structures confirmed earlier modeling studies. Differences between the CGP 53820 inhibitory binding constants for the two enzymes could be correlated with structural differences. CONCLUSIONS: Minor sequence changes in subsites at the active site can explain some of the observed differences in substrate and inhibitor binding between the two enzymes. The information gained from this investigation may help in the design of equipotent HIV-1/HIV-2 protease inhibitors.

Amino Acid Sequence↗
PubMed1995

Proteolytic processing of particle-associated retroviral polyproteins by homologous and heterologous viral proteinases.

Retroviral proteinase(PR)-catalyzed cleavage of the viral Gag and Gag-Pol polyproteins within the nascent virus particle is required for productive viral infection. Kinetic characterization and specificity analyses have been reported for several retroviral PR using oligopeptide substrates. In this study, we performed a comparative analysis of PR from avian, bovine, simian and human retroviruses using polyproteins of human immunodeficiency virus (HIV) type 1 or avian leukosis virus as substrates. Polyproteins were derived from immature virus-like particles purified from culture medium of transfected or recombinant baculovirus-infected cells. Specific cleavage to the correct size intermediate and end products occurred in the presence of detergent and homologous PR. HIV-1 PR cleaved its Gag precursor to completion at a concentration of approximately 25 nM but cleaved the Gag-Pol precursor incompletely even at fourfold higher PR concentration. In contrast to the requirement for high ionic strength for peptide cleavage reported previously, we found that Gag protein cleavage by HIV-1 PR proceeded best at low ionic strength, for both of the protein substrates tested. HIV-2 PR was approximately sixfold less active than HIV-1 PR. PR from avian myeloblastosis-associated virus (MAV) yielded efficient cleavage of the HIV-1 polyprotein only at concentrations above 1 microM. Both enzymes were stimulated by high salt and their cleavage products were identical or very similar to those of HIV-1 PR. A mutant of MAV PR engineered to cleave HIV-1 peptide substrates did not cleave the HIV-1 polyprotein at a concentration of 0.4 microM. The PR of Mason Pfizer monkey virus cleaved this polyprotein very poorly, whereas PR of bovine leukemia virus cleaved it, albeit at different sites.

Alpharetrovirus↗
PubMed1995

One-bead-one-structure combinatorial libraries.

Combinatorial libraries employing the one-bead-one-compound technique are reviewed. Two distinguishing features characterize this technique. First, each compound is identified with a unique solid support, enabling facile segregation of active compounds. Second, the identity of a compound on a positively reacting bead is elucidated only after its biological relevance is established. Direct methods of structure identification (Edman degradation and mass spectroscopy) as well as indirect "coding" methods facilitating the synthesis and screening of nonpeptide libraries are discussed. Nonpeptide and "scaffold" libraries, together with a new approach for the discovery of a peptide binding motif using a "library of libraries," are also discussed. In addition, the ability to use combinatorial libraries to optimize initially discovered leads is illustrated with examples using peptide libraries.

Amino Acid Sequence↗
PubMed1993

Structural and functional studies in vitro on the p6 protein from the HIV-1 gag open reading frame.

Protein p6 from HIV-1 gag open reading frame is reported to affect both the final phase of assembly of the viral particle and the early stage of the gag polyprotein maturation in vitro. Two separate hypotheses have been proposed, on only one of these reported effects. We think that both observations may be eventually explained if p6 protein strongly inhibits the HIV-1 proteinase. Protein p6 was synthesised by solid-phase peptide synthesis. Several methods of folding the p6 protein were tested, each resulting in the random structure according to both CD and 1D proton NMR spectra. A uniformly high exposure of NH protons to the solution was confirmed by temperature-dependent NMR spectra and isotope exchange experiments. Thus the p6 protein does not have any rigid conformation in solution. A rigid structure is not formed after further cleavage by HIV-1 proteinase as neither the protein nor its fragments are cleaved by this proteinase. In addition, the p6 protein itself does not act as inhibitor of HIV-1 proteinase. This excludes a direct role of p6 protein and supports the hypothesis that p6 is involved in forming the appropriate structure of gag polyprotein precursor. The role of slowly cleaved tight gag-proteinase in the final stage of maturation may be to slow down maturation of the precursor polyproteins prior to their transport to final location in the membrane.

Amino Acid Sequence↗
PubMed1993

Specificity mapping of HIV-1 protease by reduced bond inhibitors.

A series of 47 N-truncated reduced bond inhibitors, systematically modified at individual positions (P1, P'1, P'2, P'3, and P'4), were synthesized and used to map the subsite preferences of HIV-1 protease. The tight binding inhibitor of HIV-1 protease t-butoxycarbonyl-Phe-[CH2NH]Phe-Glu-Phe-NH2 (Ki = 0.2 nM) was chosen as the parent structure for further modifications. The P'2 glutamic acid was found to fit well into the S'2 subsite of the protease. The conformational restriction of any phenylalanine residue or saturation of more than one phenylalanine side chain in P'1 or P'3 lead is to a large Ki increase. Introduction of tyrosine in the P1 position improves the binding by an order of magnitude. The S'4 subsite of the protease was shown to accommodate large structural changes in the inhibitor at this position. Therefore P'4 may serve as an ideal region for further modification in order to improve bioavailability of this type of compound. An improved method of direct comparison of tight binding inhibitors with subnanomolar Ki values has been described.

Binding Sites↗
PubMed1993

Myeloblastosis associated virus (MAV) proteinase site-mutated to be HIV-like has a higher activity and allows production of infectious but morphologically altered virus.

We have characterized the structure and infectivity of an avian retrovirus, myeloblastosis associated virus (MAV), containing a genetically altered proteinase (PR). A site-directed mutant of MAV-PR that shows an increased proteolytic activity in vitro (about 20 times higher kcat/Km) as a consequence of substituting five amino acids from the substrate-binding pocket with those corresponding to the HIV-1 PR was cloned into a full-sized MAV plasmid. In particular, the wild-type MAV-PR gene was replaced with the mutant one. Despite encoding for an enzyme with increased PR activity, mutant plasmid-transfected turkey fibroblasts displayed an unimpaired virus production in cell cultures. Further, the mutant progeny virus was infectious and its pattern of gag processing products appeared identical to that of wild-type virus. However, by electron microscopy we found that the predominant morphology of mutant viral particles was altered. Instead of a centrally collapsed avian retroviral core, a more diffuse core was visualized for wild-type mutant virions, similar to that observed in mammalian C-type retroviruses.

Alpharetrovirus↗
PubMed1992

15gag proteinase of myeloblastosis-associated virus: specificity studies with substrate-based inhibitors.

The specificity of the proteinase of myeloblastosis-associated virus (MAV) was studied with (a) 21 substrate-based inhibitors, (b) 9 inhibitors with pseudopalindrome sequences, (c) 8 chimeric inhibitors, and (d) 3 compounds designed as human immunodeficiency virus 1 (HIV-1) proteinase inhibitors. The central inhibitory unit (transition state or cleaved bond analog) and the role of the inhibitor side chains from P4 to P4' were investigated. MAV proteinase prefers an aromatic side chain in P1 and a small aliphatic nonpolar chain in P2 and P2'. Residues in P5 and P4 positions are outside of the short catalytic cleft of the enzyme, but still influence binding considerably. The data obtained provide evidence that the MAV proteinase has generally lower specificity and poorer binding than the HIV proteinase.

Amino Acid Sequence↗
PubMed1992

Different requirements for productive interaction between the active site of HIV-1 proteinase and substrates containing -hydrophobic*hydrophobic- or -aromatic*pro- cleavage sites.

The sequence requirements for HIV-1 proteinase catalyzed cleavage of oligopeptides containing two distinct types of junctions (-hydrophobic*hydrophobic- or -aromatic*Pro-) has been investigated. For the first type of junction (-hydrophobic*hydrophobic-) the optimal residues in the P2 and P2' positions were found to be Val and Glu, respectively, in accord with recent statistical analysis of natural cleavage sites [Poorman, R. A., Tomasselli, A. G., Heinrikson, R. L., & Kézdy, F. J. (1991) J. Biol. Chem. 266, 14554-14561]. For the -aromatic*Pro- type of junction, in the specific sequence context studied here, the value of Glu in the P2' position was again observed. An explanation for the inefficient cleavage observed for peptides with the sequence -Val-Tyr*Pro- has been provided from molecular modeling of the putative enzyme-substrate complex. A significant effect upon cleavage rates due to the amino acid in the P5 position has also been documented. While lysine in the P5 position in one sequence of the -hydrophobic*hydrophobic- type produces a peptide cleaved very efficiently (kcat greater than 15 s-1 for Lys-Ala-Arg-Val-Nle*p-nitrophenylalanine-P2'-Ala-Nle-NH2, for P2' = Glu, Gln, Ile, Val, or Ala), for substrates of the -aromatic*Pro- type, the P5 residue can exert either a positive or negative effect on cleavage rates. These results have again been interpreted in light of molecular modeling. We suggest that interaction of the substrate sequence on the periphery of the active site cleft may influence the match of the enzyme-substrate pair and, hence, control the efficiency of catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Reduced-bond tight-binding inhibitors of HIV-1 protease. Fine tuning of the enzyme subsite specificity.

Truncation of a peptide substrate in the N-terminus and replacement of its scissile amide bond with a non-cleavable reduced bond results in a potent inhibitor of HIV-1 protease. A series of such inhibitors has been synthesized, and S2-S3' subsites of the protease binding cleft mapped. The S2 pocket requires bulky Boc or PIV groups, large aromatic Phe residues are preferred in P1 and P1' and Glu in P2'. The S3' pocket prefers Phe over small Ala or Val. Introduction of a Glu residue into the P2' position yields a tight-binding inhibitor of HIV-1 protease, Boc-Phe-[CH2-NH]-Phe-Glu-Phe-OMe, with a subnanomolar inhibition constant. The relevant peptide derived from the same amino acid sequence binds to the protease with a Ki of 110 nM, thus still demonstrating a good fit of the amino acid residues into the protease binding pockets and also the importance of the flexibility of P1-P1' linkage for proper binding. A new type of peptide bond mimetic, N-hydroxylamine -CH2-N(OH)-, has been synthesized. Binding of hydroxylamino inhibitor of HIV-1 protease is further improved with respect to reduced-bond inhibitor.

Amino Acid Sequence↗