Chemical Synthesis of 19F-labeled HIV-1 Protease using Fmoc-Chemistry and ChemMatrix Resin

HIV-1 protease (PR) has been extensively studied due to its importance as a target in AIDS therapy. The enzyme can be obtained via expression of its cloned gene in an appropriate system, or via chemical synthesis. We required a reliable source of fluorine-labeled HIV-1 protease for NMR studies. As our attempts to incorporate a labeling step in overexpression experiments in E. coli failed, we turned to chemical synthesis. Herein is described the first chemical synthesis of an active, 99 amino acid residue HIV-1 encoded protease using Fmoc-chemistry on a total PEG-based resin (CM resin), and labeled with ¹⁹F at the Phe residue. Also reported here are NMR studies of the labeled synthetic protein with a synthetic dimerization inhibitor.     

Chemical replacement of P1' serine residue at the second reactive site of soybean protease inhibitor C-II

The P1' Ser(50) at the second reactive site of soybean protease inhibitor C-II was replaced with arginine to confirm the contribution of this residue to the inhibition. The Arg derivative had less trypsin inhibitory activity (Ki = 1 X 10(-7) M) than the Ser derivative (Ki = 2 X 10(-8) M), in contrast to the results obtained from studies on peanut protease inhibitor B-III reported in the previous paper (J. Biochem. 101, 723-728 (1987)). These results suggest that each Bowman-Birk type inhibitor has an amino acid at the P1' position inherently best suited to maintaining its inhibitory activity, and that serine is not unique for the P1' amino acid in Bowman-Birk type inhibitors.     

Crystal structure of the anticarcinogenic Bowman-Birk inhibitor from snail medic (Medicago scutellata) seeds complexed with bovine trypsin

The structure of the ternary complex of the anticarcinogenic Bowman-Birk protease inhibitor purified from snail medic (Medicago scutellata) seeds (MSTI) and two molecules of bovine trypsin has been solved by X-ray diffraction analysis of single crystals to a resolution of 2.0 A. This is the highest resolution model of a ternary complex of this type currently available. The two binding loops of the MSTI differ in only one amino acid and have in both cases an arginine in position P1. The distances between the residues of the inhibitor at the binding interface and the trypsin side chains that recognize them are almost identical in the two sites. When compared to the NMR model of the uncomplexed MSTI, the inhibitor in the functional assembly with trypsin shows the largest differences in the two P2' residues. Compared with the similar ternary complex of the soybean trypsin inhibitor, this model shows very small differences in the polypeptide chain of the trypsin binding sites and its largest difference in the area between Asp 26 and His 32 of the MSTI which in the soybean inhibitor has an extra Leu inserted in position 29.     

Complexation of two proteic insect inhibitors to the active site of chymotrypsin suggests decoupled roles for binding and selectivity

The crystal structures of two homologous inhibitors (PMP-C and PMP-D2v) from the insect Locusta migratoria have been determined in complex with bovine alpha-chymotrypsin at 2.1- and 3.0-A resolution, respectively. PMP-C is a potent bovine alpha-chymotrypsin inhibitor whereas native PMP-D2 is a weak inhibitor of bovine trypsin. One unique mutation at the P1 position converts PMP-D2 into a potent bovine alpha-chymotrypsin inhibitor. The two peptides have a similar overall conformation, which consists of a triple-stranded antiparallel beta-sheet connected by three disulfide bridges, thus defining a novel family of serine protease inhibitors. They have in common the protease interaction site, which is composed of the classical protease binding loop (position P5 to P'4, corresponding to residues 26-34) and of an internal segment (residues 15-18), held together by two disulfide bridges. Structural divergences between the two inhibitors result in an additional interaction site between PMP-D2v (position P10 to P6, residues 21-25) and the residues 172-175 of alpha-chymotrypsin. This unusual interaction may be responsible for species selectivity. A careful comparison of data on bound and free inhibitors (from this study and previous NMR studies, respectively) suggests that complexation to the protease stabilizes the flexible binding loop (from P5 to P'4).     

Functional and structural similarities between protease nexin I and C1 inhibitor

Protease nexin I is a proteinase inhibitor that is secreted by human fibroblasts and forms stable complexes with certain serine proteinases; the complexes then bind to the fibroblasts and are rapidly internalized and degraded. In this report, we show that this inhibitor, which is present in very low concentrations in plasma, has functional and structural similarities to C1 inhibitor, an abundant proteinase inhibitor in plasma. Both inhibitors complex and inactivate certain proteinases that previously were known to rapidly react only with C1 inhibitor. Kinetic inhibition studies show that protease nexin I inhibits Factor XIIa and plasma kallikrein with second-order rate constants of 2.3 x 10(3) and 2.5 x 10(5) M-1 s-1, respectively, which are similar to the rate constants for inhibition of these proteinases by C1 inhibitor. Protease nexin I inhibits C1s about one-tenth as rapidly as does C1 inhibitor. Alignment of the amino acid sequences of protease nexin I and C1 inhibitor shows that these proteins have similarity at their reactive centers (from sites P7 to P1). The remaining regions of the two proteins share much less similarity. In contrast to protease nexin I, C1 inhibitor is not secreted by human fibroblasts. Although 125I-C1s-protease nexin I complexes readily bind to human fibroblasts, binding of 125I-C1s-C1 inhibitor complexes or other 125I-proteinase-C1-inhibitor complexes to these cells is not detectable. Thus, protease nexin I and C1 inhibitor may control some common regulatory proteinases in the extravascular and vascular compartments, respectively.     

Evaluation of a diverse set of potential P1 carboxylic acid bioisosteres in hepatitis C virus NS3 protease inhibitors

There is an urgent need for more efficient therapies for people infected with hepatitis C virus (HCV). HCV NS3 protease inhibitors have shown proof-of-concept in clinical trials, which make the virally encoded NS3 protease an attractive drug target. Product-based NS3 protease inhibitors comprising a P1 C-terminal carboxylic acid have shown to be effective and we were interested in finding alternatives to this crucial carboxylic acid group. Thus, a series of diverse P1 functional groups with different acidity and with possibilities to form a similar, or an even more powerful, hydrogen bond network as compared to the carboxylic acid were synthesized and incorporated into potential inhibitors of the NS3 protease. Biochemical evaluation of the inhibitors was performed in both enzyme and cell-based assays. Several non-acidic C-terminal groups, such as amides and hydrazides, were evaluated but failed to produce inhibitors more potent than the corresponding carboxylic acid inhibitor. The tetrazole moiety, although of similar acidity to a carboxylic acid, provided an inhibitor with mediocre potencies in both assays. However, the acyl cyanamide and the acyl sulfinamide groups rendered compounds with low nanomolar inhibitory potencies and were more potent than the corresponding carboxylic acid inhibitor in the enzymatic assay. Additionally, results from a pH-study suggest that the P(1) C-terminal of the inhibitors comprising a carboxylic acid, an acyl sulfonamide or an acyl cyanamide group binds in a similar mode in the active site of the NS3 protease.     

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.     

Identification of serine and histidine adducts in complexes of trypsin and trypsinogen with peptide and nonpeptide boronic acid inhibitors by 1H NMR spectroscopy.

We have previously shown, in 15N NMR studies of the enzyme's active site histidine residue, that boronic acid inhibitors can form two distinct types of complexes with alpha-lytic protease. Inhibitors that are structural analogs of good alpha-lytic protease substrates form transition-state-like tetrahedral complexes with the active site serine whereas those that are not form complexes in which N epsilon 2 of the active site histidine is covalently bonded to the boron of the inhibitor. This study also demonstrated that the serine and histidine adduct complexes exhibit quite distinctive and characteristic low-field 1H NMR spectra [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. A. (1988) Biochemistry 27, 7689-7697]. Here we have used low-field 1H NMR diagnostically for a series of boronic acid inhibitor complexes of trypsin and trypsinogen. The results show that H-D-Val-Leu-boroArg and Ac-Gly-boroArg, analogs of good trypsin substrates, form transition-state-like serine adducts with trypsin, whereas the nonsubstrate analog inhibitors boric acid, methane boronic acid, butane boronic acid, and triethanolamine borate all form histidine adducts, thereby paralleling the previous results obtained with alpha-lytic protease. However, with trypsinogen, Ac-Gly-boroArg forms predominantly a histidine adduct while H-D-Val-Leu-boroArg forms both histidine and serine adducts, with the histidine adduct predominating below pH 8.0 and the serine adduct predominating above pH 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of subtilisin BPN' by reaction site P1 mutants of Streptomyces subtilisin inhibitor

It has been shown that the P1 site (the center of the reactive site) of protease inhibitors corresponds to the specificity of the cognate protease, and consequently specificity of Streptomyces subtilisin inhibitor (SSI) can be altered by substitution of a single amino acid at the P1 site. In this paper, to investigate whether similar correlation between inhibitory activity of mutated SSI and substrate preference of protease is observed for subtilisin BPN', which has broad substrate specificity, a complete set of mutants of SSI at the reaction site P1 (position 73) was constructed by cassette and site-directed mutagenesis and their inhibitory activities toward subtilisin BPN' were measured. Mutated SSIs which have a polar (Ser, Thr, Gln, Asn), basic (Lys, Arg), or aromatic amino acid (Tyr, Phe, Trp, His), or Ala or Leu, at the P1 site showed almost the same strong inhibitory activity toward subtilisin as the wild type (Met) SSI. However, the inhibitory activity of SSI variants with an acidic (Glu, Asp), or a beta-branched aliphatic amino acid (Val, Ile), or Gly or Pro, at P1 was decreased. The values of the inhibitor constant (Ki) of mutated SSIs toward subtilisin BPN' were consistent with the substrate preference of subtilisin BPN'. A linear correlation was observed between log(1/Ki) of mutated SSIs and log(1/Km) of synthetic substrates. These results demonstrate that the inhibitory activities of P1 site mutants of SSI are linearly related to the substrate preference of subtilisin BPN', and indicate that the binding mode of the inhibitors with the protease may be similar to that of substrates, as in the case of trypsin and chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis, antiviral activity, and conformational studies of a P3 aza-peptide analog of a potent macrocyclic tripeptide HCV protease inhibitor

BILN 2061 is a macrocyclic tripeptide inhibitor of hepatitis C virus NS3-4A protease that has shown efficacy in the clinic for treating patients infected with HCV. We have synthesized a P3 aza-peptide analog of a potent macrocyclic tripeptide inhibitor closely related to BILN 2061. This aza-derivative was found to be >2 orders of magnitude less active than the parent macrocycle in both isolated enzyme (HCV NS3-4A) and HCV subgenomic replicon assays. NMR studies of P3 aza-peptides revealed these compounds adopt a beta-turn conformation stabilized by an intramolecular H-bonding interaction. Molecular models of these structures indicate a D-like configuration of the P3 aza-residue. Thus, the configurationally undefined nature at P3 in the aza-peptide allows the compound to adopt an H-bond stabilized conformation that is substantially different from that necessary for tight binding to the active site of HCV NS3 protease.     

Enzyme inhibition by allosteric capture of an inactive conformation

All members of the human herpesvirus protease (HHV Pr) family are active as weakly associating dimers but inactive as monomers. A small-molecule allosteric inhibitor of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) traps the enzyme in an inactive monomeric state where the C-terminal helices are unfolded and the hydrophobic dimer interface is exposed. NMR titration studies demonstrate that the inhibitor binds to KSHV Pr monomers with low micromolar affinity. A 2.0-Å-resolution X-ray crystal structure of a C-terminal truncated KSHV Pr-inhibitor complex locates the binding pocket at the dimer interface and displays significant conformational perturbations at the active site, 15 Å from the allosteric site. NMR and CD data suggest that the small molecule inhibits human cytomegalovirus protease via a similar mechanism. As all HHV Prs are functionally and structurally homologous, the inhibitor represents a class of compounds that may be developed into broad-spectrum therapeutics that allosterically regulate enzymatic activity by disrupting protein-protein interactions.     

Kazal型蛋白酶抑制剂结构与功能研究进展

蛋白酶抑制剂广泛存在于生物体内,在许多生命活动过程中发挥必不可少的作用,特别是对蛋白酶活性进行精确调控。其中Kazal型蛋白酶抑制剂是最重要的、研究最为广泛的酶抑制剂之一,该类抑制剂一般由一个或几个结构域组成,每一个结构域具有保守的序列和分子构象,同时发现该类抑制剂与蛋白酶作用的结合部位高度易变,它们大多数暴露于与溶剂接触的环上,其中P1部位是抑制作用的关键部位,抑制剂的专一性由P1部位氨基酸残基的性质决定,其它残基取代结合部位残基对抑制剂-酶的结合常数有显著的影响。Laskowski算法可直接从Kazal型丝氨酸蛋白酶抑制剂的序列推测其与6种丝氨酸蛋白酶之间的抑制常数(Ki)。目前在生物体内发现大量的Kazal型蛋白酶抑制剂,并证实其有重要的生物学功能。     

Functional tolerance of Streptomyces subtilisin inhibitor toward conformational and stability changes caused by single-point mutations in the hydrophobic core

Single amino acid mutations of Met103 in the hydrophobic core of a serine protease inhibitor, Streptomyces subtilisin inhibitor, caused little change in the inhibitory activity, as measured by the inhibitor constant, although some altered the thermodynamic stability of the protein considerably. (1)H NMR investigations showed that the conformational stress caused by the replacement of Met103 with Gly, Ala, Val, and Ile, namely, the effects of the cavities generated by replacements with smaller side-chains and of the steric distortions generated by beta-branched side-chains, caused considerable changes in the structural arrangement of the side-chains within the core. However, these structural changes were absorbed within the hydrophobic core, without distorting the structure of the reactive site essential for the protein function. These results provide an excellent example of the conformational flexibility of a protein core and the degree of its tolerance of an amino acid replacement. The results also reveal the crucially designed structural relationship between the core of the inhibitor and the enzyme-binding segment with the reactive site in a serine protease inhibitor.     

Substrate properties of C1 inhibitor Ma (alanine 434----glutamic acid). Genetic and structural evidence suggesting that the P12-region contains critical determinants of serine protease inhibitor/substrate status

The serine protease inhibitor (serpin) C1 inhibitor inactivates enzymes involved in the regulation of vascular permeability. A patient from the Ma family with the genetic disorder hereditary angioedema inherited a dysfunctional C1 inhibitor allele. Relative to normal plasma, the patients's plasma contained an additional C1 inhibitor immunoreactive band, which comigrated with normal C1 inhibitor cleaved by plasma kallikrein, C1s, or factor XIIa. C1 inhibitor Ma did not react with a monoclonal antibody to a neoepitope that is present in complexed and cleaved normal C1 inhibitor, suggesting conformational differences between cleaved normal C1- inhibitor and cleaved C1 inhibitor Ma. Molecular cloning and sequencing of exon 8 of the C1 inhibitor Ma allele revealed a single C to A mutation, changing alanine 434 to glutamic acid. Ala 434 of C1 inhibitor aligns with the P12 residue of the prototypical serpin alpha 1-antitrypsin. The P12 amino acid of all inhibitory serpins is alanine, and it is present in a highly conserved region on the amino-terminal side of the serpin-reactive center loop. Whereas normal C1 inhibitor expressed by transfected COS-1 cells formed complexes with and was cleaved by kallikrein, fXIIa, and C1s, COS-1-expressed Ala434---Glu C1 inhibitor was cleaved by these enzymes but did not form complexes with them. These results, together with evidence from other studies, suggest that serpin protease inhibitor activity is the result of protein conformational change that occurs when the P12 region of a serpin moves from a surface location, on the reactive site loop of the native molecule, to an internal location within sheet A of the complexed inhibitor.     

A conserved cis peptide bond is necessary for the activity of Bowman-Birk inhibitor protein

The Bowman-Birk inhibitor (BBI) family of protease inhibitors has an inhibitory region comprising a disulfide-linked nine-residue loop that adopts the characteristic canonical motif found in many serine protease inhibitors. A unique feature of the BBI loop is the presence of a cis peptide bond at the edge of the inhibitory loop. BBI-related protein fragments that encapsulate this loop retain the structure and inhibitory activity of the parent protein. The most common BBI loop sequence has a proline-proline element with a cis-trans geometry at P3'-P4'. We have examined this element by analysis of the inhibitory activity and structure for a series of synthetic fragments where each of these proline residues has been systematically replaced with alanine. The results show that only when a proline is present at P3' are potent inhibition and a cis peptide bond at that position in the solution structure observed, suggesting that this conformation is required for biological activity. Though a P4' proline is not essential for activity, it effectively stabilizes the cis conformation at P3' by suppressing alternative conformations. This is most evident from the Pro-Ala variant, which comprises a 1:1 mixture of slowly exchanging and structurally different cis and trans isomers. Monitoring the action of trypsin on this mixture by NMR shows that this protease interacts selectively with the cis P3' structure, providing direct evidence for the link between activity and the nativelike structure of the cis isomer. This is, to the best of our knowledge, the first example where cis isomer selectivity can be demonstrated for a proteinase.     

NMR solution structure of Apis mellifera chymotrypsin/cathepsin G inhibitor-1 (AMCI-1): structural similarity with Ascaris protease inhibitors

The three-dimensional structure of the 56 residue polypeptide Apis mellifera chymotrypsin/cathepsin G inhibitor 1 (AMCI-1) isolated from honey bee hemolymph was calculated based on 730 experimental NMR restraints. It consists of two approximately perpendicular beta-sheets, several turns, and a long exposed loop that includes the protease binding site. The lack of extensive secondary structure features or hydrophobic core is compensated by the presence of five disulfide bridges that stabilize both the protein scaffold and the binding loop segment. A detailed analysis of the protease binding loop conformation reveals that it is similar to those found in other canonical serine protease inhibitors. The AMCI-1 structure exhibits a common fold with a novel family of inhibitors from the intestinal parasitic worm Ascaris suum. The pH-induced conformational changes in the binding loop region observed in the Ascaris inhibitor ATI are absent in AMCI-1. Similar binding site sequences and structures strongly suggest that the lack of the conformational change can be attributed to a Glu-->Gln substitution at the P1' position in AMCI-1, compared to ATI. Analysis of amide proton temperature coefficients shows very good correlation with the presence of hydrogen bond donors in the calculated AMCI-1 structure.     

NMR and ESR studies on human pregnancy zone protein. Comparison with human alpha 2-macroglobulin.

NMR and ESR spectroscopies have been used to examine the plasma protease inhibitor pregnancy zone protein (PZP) and its complex with chymotrypsin. The 1H NMR spectrum of PZP shows relatively few sharp resonances, which, by analogy with human alpha 2-macroglobulin, probably arise from the proteolytically sensitive bait region. Upon reaction with chymotrypsin to form a 1:1 protease.PZP tetramer complex, there is a large increase in the intensity of sharp resonances due to an increase in mobility of these residues. 35Cl NMR has been used to follow binding of zinc and manganese to apo-PZP. Zinc binding causes a linear broadening of the bulk Cl-, consistent with access of Cl- to PZP-bound zinc. Since zinc in the two highest affinity sites in human alpha 2-macroglobulin causes no broadening of Cl-, it is concluded that these zinc sites are absent from PZP. The mobility of chymotrypsin in the PZP.chymotrypsin complex was examined by covalently attaching a nitroxide spin label at the serine residue in the active site of the enzyme and examining the appearance of the ESR spectrum. The chymotrypsin is rigidly held by the PZP to which it is covalently bound. In an analogous experiment performed previously on alpha 2-macroglobulin, chymotrypsin, bound in the presence of methylamine and therefore largely noncovalently bound, was found to be free to rotate inside the cage formed by the protease inhibitor.     

Purification of extracellular acid protease and analysis of fermentation metabolites by Synergistes sp. utilizing proteinaceous solid waste from tanneries

The untanned proteinaceous tannery solid waste, animal fleshing (ANFL), was used as a substrate for acid protease production by Synergistes sp. The strain was isolated from an anaerobic digester used for the treatment of tannery solid waste and was selected for its enhanced protease production at activity 350-420 U/ml. The optimum pH was in the acidic range of 5.5-6.5 and optimum temperature was in mesophilic range of 25-35 degrees C. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the zymogram analyses of the purified protein indicated an estimated molecular mass of 60 kDa. This protease could be classified as aspartic protease based on its inhibition by aspartate type protease inhibitor pepstatin and on non-inhibition by 1,10-phenanthroline, EDTA, EGTA and phenylmethylsulfonyl fluoride. The degradation of ANFL was confirmed by Gas Chromatography-Mass Spectroscopy (GC-MS), Proton Nuclear Magnetic Resonance Spectroscopy (H1 NMR) and Scanning Electron Microscopy (SEM) analyses. In this study we found that the activity of acid protease depended on factors such as calcium concentration, pH and temperature. Based on these lines of evidence, we postulate that this protease is a highly catalytic novel protease of its type.     

3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.

Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size.