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PubMed1995

The primary elastase inhibitor (elastasin) and trypsin inhibitor (contrapsin) in the goat are serpins related to human alpha 1-anti-chymotrypsin.

Two primary serine proteinase inhibitors in goat plasma have been isolated and characterized. The N-terminal sequence analysis of the purified proteins revealed that they are closely related to each other and are highly homologous to human alpha 1-anti-chymotrypsin rather than alpha 1-proteinase inhibitor. However, despite structural similarities the inhibitory specificity of the goat inhibitors differed from each other and from that of anti-chymotrypsin. In contrast with human anti-chymotrypsin, one of the goat inhibitors was shown to be a strong and specific inhibitor of trypsin (k(ass.) = 1.9 x 10(6) M-1.s-1), whereas the other was an efficient inhibitor of neutrophil elastase (k(ass.) = 1.5 x 10(6) M-1.S-1). Differences in the inhibitory specificity of each protein could readily be attributed to the amino acid sequence within the reactive site region. The trypsin inhibitor with an assumed arginine residue at the P1 position of the reactive-site peptide bond is referred to as 'contrapsin', and indicates that the occurrence of contrapsins is not restricted to rodents. In contrast, the inhibitory specificity, resistance to oxidative and proteolytic inactivation and the presence of a P1 leucine residue in the elastase inhibitor is unique among inhibitory serpins that have been characterized to date. Because this serpin is apparently the major elastase inhibitor in goat plasma, it is likely to be involved in the control of goat neutrophil elastase. Therefore, we suggest the name 'elastasin', and extend it to any other anti-chymotrypsin related serpins possessing neutrophil-elastase- inhibitory activity.

Amino Acid Sequence↗
PubMed1986

Some protease inhibitors are also inhibitors of poly(ADP-ribose) polymerase.

The low-molecular-weight peptide protease inhibitors, tosyl-lysine-chloromethyl ketone, antipain and leupeptin, inhibited poly(ADP-ribose) [poly(ADP-Rib)] polymerase in permeable cells. The concentrations required for 50% inhibition were 3.6, 5 and 29 mM, respectively. Two peptides without protease inhibitor activity, fibrinopeptide A and phenylalanine-leucine-(glutamine)2-leucine, also inhibited poly (ADP-Rib) synthesis; doses required for 50% inhibition were 0.37 and 11.2 mM, respectively. These concentrations lie within a range bracketed by the 50% inhibition concentrations of the strong and weak poly(ADP-Rib) synthesis inhibitors, 3-amino-benzamide (0.15 mM) and caffeine (greater than 100 mM), respectively. N-Ethylmaleimide also inhibited poly(ADP-Rib) synthesis, at a 50% inhibitory dose of 0.3 mM, in the absence of exogenous thiol reagents. High-molecular-weight protease inhibitors, such as soybean (including Bowman-Birk reagent) and lima bean trypsin inhibitors and human alpha 1-protease inhibitor, had no effect on poly(ADP-Rib) synthesis up to 2 mg/ml. Interference with transformation and other cellular effects that have been reported in carcinogen-damaged cells treated with low-molecular-weight peptide protease inhibitors may therefore involve common mechanisms with poly(ADP-Rib) inhibitors. Similar effects of high-molecular-weight protease inhibitors presumably involve different mechanisms.

Antipain↗
PubMed1995

Trypsin and elastase inhibitors from bitter gourd (Momordica charantia LINN.) seeds: purification, amino acid sequences, and inhibitory activities of four new inhibitors.

Serine proteinase inhibitors of the squash family were isolated from bitter gourd (Momordica charantia LINN.) seeds by the conventional purification method. Heat treatment of the extract of the seeds allowed removal of large amounts of protein without loss of trypsin and elastase inhibitory activities. From the supernatants thus obtained, the inhibitors were isolated to homogeneity by ion-exchange chromatography, gel filtration, and reversed phase chromatography. One trypsin inhibitor (Momordica charantia trypsin inhibitor-III; MCTI-III) and three elastase inhibitors (Momordica charantia elastase inhibitor-II, -III, and -IV; MCEI-II, -III, and -IV) were newly isolated in addition to trypsin inhibitors MCTI-I and -II and elastase inhibitor MCEI-I previously reported [Hara, S. et. al. (1989). J. Biochem. 105, 88-92]. The primary structures of the four new inhibitors were determined as follows. [sequence: see text] The dissociation constants, Ki, of MCTI-III complex with bovine beta-trypsin, and of MCEI-II, -III, -IV with porcine elastase were determined to be 1.9 x 10(-7) M, 9.4 x 10(-9) M, 4.0 x 10(-9) M, and 4.7 x 10(-9) M, respectively. Although MCTI-III differed from MCTI-I in only two amino acids, having Gly(3) and Gln(13) in place of Arg(3) and Arg(13), the Ki value of MCTI-III was 20-fold larger than that of MCTI-I. Addition of an amino terminal Glu residue, a dipeptide (Glu-Glu-), and a tripeptide (Glu-Glu-Glu-) to MCEI-I strengthened its elastase inhibitory activity by 200-fold.

Amino Acid Sequence↗
PubMed1997

Importance of beta-lactamase inhibitor pharmacokinetics in the pharmacodynamics of inhibitor-drug combinations: studies with piperacillin-tazobactam and piperacillin-sulbactam.

An in vitro pharmacokinetic model was used to study the pharmacodynamics of piperacillin-tazobactam and piperacillin-sulbactam against gram-negative bacilli producing plasmid-encoded beta-lactamases. Logarithmic-phase cultures were exposed to peak antibiotic concentrations observed in human serum after the administration of intravenous doses of 3 g of piperacillin and 0.375 g of tazobactam or 0.5 g of sulbactam. Piperacillin and inhibitor were either dosed simultaneously or piperacillin was dosed sequentially 0.5 h after dosing with the inhibitor. In studies with all four test strains, the pharmacodynamics observed after simultaneous dosing were similar to those observed with the sequential regimen. Since the ratio between piperacillin and tazobactam was in constant fluctuation after sequential dosing, these data suggest that the pharmacodynamics of the piperacillin-inhibitor combinations were not dependent upon maintenance of a critical ratio between the components. Furthermore, when regrowth was observed, the time at which bacterial counts began to increase was similar between the simultaneous and sequential dosing regimens. Since the pharmacokinetics of the inhibitors were the same for all regimens, these data suggest that the length of time that the antibacterial activity was maintained over the dosing interval with these combinations was dictated by the pharmacokinetics of the beta-lactamase inhibitor in the combination. The antibacterial activity of the combination appeared to be lost when the amount of inhibitor available fell below some critical concentration. This critical concentration varied depending upon the type and amount of enzyme produced, as well as the specific inhibitor used. These results indicate that the antibacterial activity of drug-inhibitor combinations, when dosed at their currently recommended ratios, is more dependent on the pharmacokinetics of the inhibitor than on those of the beta-lactam drug.

Bacteria↗
PubMed2004

Clinical efficacy of antiretroviral combination therapy based on protease inhibitors or non-nucleoside analogue reverse transcriptase inhibitors: indirect comparison of controlled trials.

OBJECTIVE: To compare the clinical efficacy of triple antiretroviral regimens based on protease inhibitors and non-nucleoside analogue reverse transcriptase inhibitors (NNRTIs) in adults positive for antibodies to HIV-1. DESIGN: Systematic review and meta-analysis using indirect comparisons of clinical trials comparing three drug regimens based on two nucleoside reverse transcriptase inhibitors (NRTIs) and either a protease inhibitor or an NNRTI with two drug regimens (two NRTIs). Participants had no previous exposure to protease inhibitors or NNRTIs. DATA SOURCES: Medline, the Cochrane controlled trials register, Aidstrials, Aidsdrugs, conference proceedings, and trial registers. MAIN OUTCOME MEASURE: Progression to AIDS or death. RESULTS: 14 trials, totalling 6785 patients, were identified. Most patients had been exposed to an NRTI and had advanced immunodeficiency at baseline; 1096 progressed to AIDS or died. Seven trials assessed protease inhibitors based triple regimens and seven assessed NNRTI based triple regimens (nevirapine or delavirdine). Triple therapy was more effective than dual therapy. The effect was pronounced for protease inhibitor based regimens (odds ratio 0.49, 95% confidence interval 0.41 to 0.58) but non-significant for NNRTI based regimens (0.90, 0.71 to 1.15). Indirect comparison of the two regimens gave an odds ratio of 0.54 (0.49 to 0.73) in favour of protease inhibitor based treatments. Increases in CD4 cell counts were smaller and suppression of viral replication less with NNRTI based regimens. CONCLUSIONS: Indirect evidence shows that protease inhibitor based triple regimens are superior to regimens based on the NNRTIs nevirapine and delavirdine in patients with advanced immunodeficiency who have been exposed to NRTIs. Large trials with clinical end points are required.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Effects of angiotensinase inhibitors on plasma protein binding and IC50 determinations of renin inhibitors.

To establish whether the use of proteinase inhibitors in the routine determination of in vitro plasma renin activity overestimates the potency of renin inhibitors in vivo, we examined the effects of phenylmethylsulfonyl fluoride and 8-hydroxyquinoline sulfate on the binding to plasma proteins and the respective IC50 values (50% inhibiting concentrations) of three renin inhibitors. All three renin inhibitors, A-64662, A-65317, and A-74273, bound (> 60%) to plasma proteins at both pH 6.0 and 7.4, with slightly greater binding at pH 7.4. Phenylmethylsulfonyl fluoride (1.45 mmol/L) had no significant effect on the protein binding at either pH 6.0 or 7.4; 8-hydroxyquinoline sulfate (3.4 mmol/L) caused a modest dissociation (10-30%) of the renin inhibitors from plasma proteins at both pH values; and the effects of both proteinase inhibitors together were similar to those of 8-hydroxyquinoline alone. At pH 7.4, phenylmethylsulfonyl fluoride increased the potencies of the three renin inhibitors slightly (< or = 43%), whereas IC50 values determined in the presence of 8-hydroxyquinoline decreased by 1.5- to 3.7-fold. The greatest increase in potency occurred with the most hydrophilic compound, and with both angiotensinase inhibitors the effect was no greater than that of 8-hydroxyquinoline alone. The results show that any dissociation of the hypotensive activity measured in vivo from the plasma renin activity measured in vitro is not simply an artifact in the plasma renin activity assay stemming from the use of these angiotensinase inhibitors, especially if only phenylmethylsulfonyl fluoride is used.

Amides↗
PubMed1985

Wound-induced proteinase inhibitors from tomato leaves. II. The cDNA-deduced primary structure of pre-inhibitor II.

A cDNA containing the complete amino acid-coding region of wound-induced tomato Inhibitor II was constructed in the plasmid pUC9. The open reading frame codes for 148 amino acids including a 25-amino acid signal sequence preceding the N-terminal lysine of the mature Inhibitor II. The Inhibitor II sequence exhibits two domains, one domain having a trypsin inhibitory site and the other a chymotrypsin inhibitory site, apparently evolved from a smaller gene by a process of gene duplication and elongation. The amino acid sequence of tomato leaf Inhibitor II exhibits homology with two small proteinase inhibitors isolated from potato tuber and an inhibitor from eggplant. The small potato tuber inhibitors are homologous with 33 amino acids of the N-terminal domain and 19 amino acids from the C-terminal domain. Two identical nucleotide sequences of Inhibitor II cDNA in the 3' noncoding region were present that were also found in an Inhibitor I cDNA. These include an atypical polyadenylation signal, AATAAG, and a 10-base palindromic sequence, CATTATAATG, for which no function is yet known.

Amino Acid Sequence↗
PubMed1996

Protein inhibitor of mitochondrial ATP synthase: relationship of inhibitor structure to pH-dependent regulation.

In the absence of an electrochemical proton gradient, the F1 moiety of the mitochondrial ATP synthase catalyzes the hydrolysis of ATP. This reaction is inhibited by a natural protein inhibitor, in a process characterized by an increase in ATPase inhibition as pH is decreased from 8.0 to 6.0. In order to gain greater insight into the molecular and chemical events underlying this regulatory process, the relationships among pH, helicity of the inhibitor protein, and its capacity to inhibit F1-ATPase activity were examined. First, peptides corresponding to four regions of the 82-amino-acid inhibitor protein were chemically synthesized and assessed for both retention of secondary structure, and capacity to inhibit F1-ATPase activity. These studies showed that a region of only 24-amino-acid residues, from Phe 22 through Len 45, accounts for the inhibitory capacity of the inhibitor protein, and that retention of native helical structure in this region is not essential for inhibition. Second, three mutants (33P34, 39P40, and 43P44) of the intact inhibitor protein were prepared in which a proline residue was inserted within the inhibitory region to disrupt native helical structure. The secondary structures and inhibitory capacities of these mutants were analyzed as a function of pH. These studies revealed that, despite the initial loss of helical structure within the inhibitory region due to proline insertion, a further loss of helical structure is required to modulate inhibitory activity. These results suggest that a loss of helical structure outside the inhibitory region correlates with an increase in inhibitory capacity. Finally, two separate mutants (H48A and H55A) were prepared in which a conserved histidine residue in the wild-type inhibitor protein was replaced with an alanine. The secondary structures and inhibitory capacities of these mutants were also investigated as a function of pH. Results indicated that, although histidine residues do not directly affect the inhibitory capacity of the protein, they are important for maintaining the inhibitor protein in an inactive form at high pH. Furthermore, these results show that loss in helical structure, although correlated with an increase in inhibitory capacity, is not essential for this function. These novel experiments are consistent with a model in which the inhibitor protein is envisioned as consisting of two regions, an inhibitory region and a regulatory region. It is suggested that reduction of pH allows for the protonation of a histidine residue blocking the interaction between the two regions, thus activating the inhibitory response. The pH reduction also correlates with a partial unfolding of the protein that may either cause or result from the loss of interaction between the two helices. This unfolding may be necessary for further optimization of inhibitor function.

Amino Acid Sequence↗
PubMed1998

Structures of Tyr188Leu mutant and wild-type HIV-1 reverse transcriptase complexed with the non-nucleoside inhibitor HBY 097: inhibitor flexibility is a useful design feature for reducing drug resistance.

The second generation Hoechst-Bayer non-nucleoside inhibitor, HBY 097 (S-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3, 4-dihydroqui noxalin-2(1H)-thione), is an extremely potent inhibitor of HIV-1 reverse transcriptase (RT) and of HIV-1 infection in cell culture. HBY 097 selects for unusual drug-resistance mutations in HIV-1 RT (e.g. Gly190Glu) when compared with other non-nucleoside RT inhibitors (NNRTIs), such as nevirapine, alpha-APA and TIBO. We have determined the structure of HBY 097 complexed with wild-type HIV-1 RT at 3.1 A resolution. The HIV-1 RT/HBY 097 structure reveals an overall inhibitor geometry and binding mode differing significantly from RT/NNRTI structures reported earlier, in that HBY 097 does not adopt the usual butterfly-like shape. We have determined the structure of the Tyr188Leu HIV-1 RT drug-resistant mutant in complex with HBY 097 at 3.3 A resolution. HBY 097 binds to the mutant RT in a manner similar to that seen in the wild-type RT/HBY 097 complex, although there are some repositioning and conformational alterations of the inhibitor. Conformational changes of the structural elements forming the inhibitor-binding pocket, including the orientation of some side-chains, are observed. Reduction in the size of the 188 side-chain and repositioning of the Phe227 side-chain increases the volume of the binding cavity in the Tyr188Leu HIV-1 RT/HBY 097 complex. Loss of important protein-inhibitor interactions may account for the reduced potency of HBY 097 against the Tyr188Leu HIV-1 RT mutant. The loss of binding energy may be partially offset by additional contacts resulting from conformational changes of the inhibitor and nearby amino acid residues. This would suggest that inhibitor flexibility can help to minimize drug resistance.

Antiviral Agents↗
PubMed1982

Effect of cyclooxygenase inhibitors and protease inhibitors on phorbol-induced stimulation of oxygen consumption and superoxide production by rat pulmonary macrophages.

Oxygen consumption and superoxide anion production by pulmonary macrophages are both increased by phorbol myristate acetate (PMA) but the two processes have been separated using protease inhibitors and cyclooxygenase inhibitors. Pretreatment with the protease inhibitors (L-1-tosylamido-2-phenylethylchloromethyl ketone (TPCK) and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), as well as with the cyclooxygenase inhibitors acetylsalicylic acid (ASA) and ibuprofen (IBU), inhibited the stimulation of superoxide production and oxygen consumption by phorbol myristate acetate. However, whereas the order of potency for inhibition of stimulation of superoxide production was TPCK greater than TLCK greater than IBU greater than ASA, the order of potency for inhibition of stimulation of oxygen consumption was ASA greater than IBU greater than TPCK = TLCK. Although all four agents were effective inhibitors of PMA-stimulated superoxide production and oxygen consumption when added before PMA, in contrast to the cyclooxygenase inhibitors. TPCK was unable to inhibit oxygen consumption by more than 70-80% regardless of the concentration used, although superoxide generation could be inhibited completely. When added after PMA, ASA did not suppress either oxygen consumption or superoxide production and ibuprofen was only one-half as effective as an inhibitor. TPCK and TLCK, when added after PMA, accelerated the return to basal rates of both oxygen consumption and superoxide production. None of the four agents had any effect on basal superoxide production or oxygen consumption at the concentrations used. The data support the interpretation that both prostaglandin biosynthesis and protease activity may be associated with the activation of the superoxide-generating system of pulmonary macrophages. The consumption of molecular oxygen following stimulation of the cells with phorbol myristate acetate is not due solely to the generation of superoxide, however, since each process is inhibited with different potency by the same group of inhibitors. There appears to be a component of oxygen consumption which results from the activation of cyclooxygenase and, unlike superoxide production, cannot be completely inhibited by treatment with protease inhibitors.

Animals↗
PubMed1985

Pre-alpha 2-elastase inhibitor of the horse: a hybrid molecule between alpha 1-proteinase inhibitor and alpha 2-beta 1-glycoprotein.

Pre-alpha 2-elastase inhibitor of horse plasma has recently been isolated in our laboratory. In this article we demonstrate that the inhibitor is a composite structure built of alpha 1-proteinase inhibitor and alpha 1-beta 1-glycoprotein. The compound inhibitor is biologically active, although it has previously been shown that its enzyme specificity is different from that of free alpha 1-proteinase inhibitor. Our observations are based on immunochemical cross-reactions between pre-alpha 1-elastase inhibitor and antibodies to alpha 2-beta 1-glycoprotein as well as antibodies to alpha 1-proteinase inhibitor. Pre-alpha 2-elastase inhibitor could be dissociated into its parent components by dithiothreitol. After dissociation, the alpha 1-proteinase inhibitor portion retained its biological activity.

Animals↗
PubMed1987

Purification of a proteinase inhibitor from bovine serum with C1-inhibitor activity.

This report describes the purification of a novel proteinase inhibitor from bovine serum. This protein was purified to apparent homogeneity employing affinity binding to sulfated dextran and precipitation by ammonium sulfate, followed by sequential chromatography on DEAE-cellulose, heparin-Sepharose and Sephacryl S-200. Quantitative enzyme-linked immunosorbent assays revealed that the concentration of this inhibitor is approximately 3 microM in bovine serum. The inhibitor is a single polypeptide chain with an estimated Mr of 83,000 as determined by SDS-polyacrylamide gel electrophoresis. An aspartic acid was found at the amino terminus of the protein; N-terminal amino acid sequence data indicated that there was no significant homology with other reported amino acid sequences. This bovine inhibitor covalently complexed the human proteinases C1-r, C1-s, factor XIIa and plasma kallikrein, which are also complexed and inactivated by human C1-inhibitor. In addition, the bovine inhibitor complexed and inactivated bovine chymotrypsin, a feature which functionally distinguishes it from human C1-inhibitor. Although the bovine inhibitor appears functionally very similar to C1-inhibitor, we found no evidence for structural homology with the human counterpart.

Amino Acid Sequence↗
PubMed1987

Antibodies against mammary derived growth inhibitor (MDGI) react with a fibroblast growth inhibitor and with heart fatty acid binding protein.

A polypeptide growth inhibitor, designated as mammary derived growth inhibitor, has previously been purified from lactating bovine mammary glands. Polyclonal rabbit antiserum raised against mammary derived growth inhibitor cross-reacts with bovine heart fatty acid binding protein and bovine peripheral myelin P2 protein. These results are consistent with the observation that the amino acid sequence of mammary derived growth inhibitor showed homology to the sequences of these proteins. In a parallel series of immunoblotting experiments, rabbit anti-mammary derived growth inhibitor also reacted specifically with a fibroblast growth inhibitor purified from the conditioned medium of cultured mouse 3T3 fibroblasts. These data suggest that bovine mammary derived growth inhibitor and mouse fibroblast growth inhibitor may share common structural features and raise the possibility that these growth inhibitors may together define a new family of growth regulatory molecules.

Animals↗
PubMed1998

The cysteine protease activity of Colorado potato beetle (Leptinotarsa decemlineata Say) guts, which is insensitive to potato protease inhibitors, is inhibited by thyroglobulin type-1 domain inhibitors.

High levels of protease inhibitors are induced in potato leaves by wounding. These inhibitors, when ingested by Colorado potato beetle (Leptinotarsa decemlineata Say) larvae, induce expression of specific proteolytic activities in the gut. Induced protease activities cannot be inhibited by potato inhibitors and thus enable the insects to overcome this defence mechanism of potato plants. The induced aminopeptidase and endoproteolytic activities both have the characteristics of cysteine proteases. Twenty-one protein inhibitors of different structural types have been examined for their ability to inhibit these activities in vitro. Members of the cystatin superfamily were found to be poor inhibitors of the induced endoproteolytic activities, except for the third domain of human kininogen, which was a fairly strong inhibitor (75% inhibition). The strongest inhibition (85%) of induced endoproteolytic activity was obtained using structurally different thyroglobulin type-1 domain-like inhibitors--equistatin and MHC class II-associated p41 invariant fragment. Experiments performed using three synthetic substrates for endoproteases gave similar results and indicate the existence of at least different endoproteolytic enzymes resistant to potato inhibitors. The induced aminopeptidase activity can be inhibited only by stefin family of inhibitors in cystatin superfamily. In in vivo experiments, Colorado potato beetle larvae fed on equistatin-coated potato leaves were strongly retarded in their growth and almost 50% died after 4 days. This demonstrated the potential of equistatin to protect crops from insect attack.

Animals↗
PubMed2002

Evidence for a global inhibitor-induced conformation change on the Ca(2+)-ATPase of sarcoplasmic reticulum from paired inhibitor studies.

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by a variety of hydrophobic, hydroxy-containing molecules. A kinetic method has been used to study competition between binding of pairs of inhibitors to the ATPase. The presence of 2,5-di-tert-butyl-1,4-dihydroxybenzene (BHQ) decreases the affinity of the ATPase for 2,5-dipropyl-1,4-dihydroxybenzene (PHQ), suggesting that PHQ and BHQ bind to the same site on the ATPase. In contrast, the presence of BHQ increases the affinity of the ATPase for curcumin and vice versa. This suggests that BHQ and curcumin bind to separate sites on the ATPase and that binding of the first inhibitor to the ATPase results in a change to a conformation with higher affinity for the second inhibitor. This is consistent with previous experiments with BHQ and thapsigargin suggesting a conformation change on inhibitor binding, E2 + I <--> 2; E2I <--> 2; E2(A)I, with E2(A)I having a higher affinity for the second inhibitor than E2. The affinity for BHQ is also increased by binding of diethylstilbesterol, ellagic acid, or nonylphenol, and the affinity for curcumin is also increased by ellagic acid. These results showing that binding of a variety of inhibitors of very different structures all result in a general increase in inhibitor affinity point to a global conformational change on the Ca(2+)-ATPase caused by inhibitor binding, as well as any local, inhibitor-specific changes in conformation.

Binding, Competitive↗
PubMed1996

Ureido-based peptidomimetic inhibitor of herpes simplex virus ribonucleotide reductase: an investigation of inhibitor bioactive conformation.

We have been investigating peptidomimetic inhibitors of herpes simplex virus (HSV) ribonucleotide reductase (RR). These inhibitors bind to the HSV RR large subunit and consequently prevent subunit association and subsequent enzymatic activity. This report introduces a new series of compounds that contain an extra nitrogen (a ureido function) at the inhibitor N-terminus. This nitrogen improves inhibitor binding potency 50-fold over our first published inhibitor series. Evidence supports that this improvement in potency results from a new hydrogen-bonding contact between the inhibitor and the RR large subunit. This report also provides evidence for the bioactive conformation around two important amino acid residues contained in our inhibitors. A tert-butyl group, which contributes 100-fold to inhibitor potency but does not directly bind to the large subunit, favors an extended beta-strand conformation that is prevalent in solution and in the bound state. More significantly, the bioactive conformation around a pyrrolidine-modified asparagine residue, which contributes over 30 000-fold to inhibitor potency, is elucidated through a series of conformationally restricted analogues.

Enzyme Inhibitors↗
PubMed1997

Design and synthesis of novel imidazole-substituted dipeptide amides as potent and selective inhibitors of Candida albicans myristoylCoA:protein N-myristoyltransferase and identification of related tripeptide inhibitors with mechanism-based antifungal activity.

A new class of antifungal agents has been discovered which exert their activity by blockade of myristoylCoA: protein N-myristoyltransferase (NMT; EC 2.1.3.97). Genetic experiments have established that NMT is needed to maintain the viability of Candida albicans and Cryptococcus neoformans,the two principal causes of systemic fungal infections in immunocompromised humans. Beginning with a weak octapeptide inhibitor ALYASKLS-NH2 (2, Ki = 15.3 +/- 6.4 microM), a series of imidazole-substituted Ser-Lys dipeptide amides have been designed and synthesized as potent and selective inhibitors of Candida albicans NMT. The strategy that led to these inhibitors evolved from the identification of those functional groups in the high-affinity octapeptide substrate GLYASKLS-NH2 1a necessary for tight binding, truncation of the C-terminus, replacement of the four amino acids at the N-terminus by a spacer group, and substitution of the glycine amino group with an N-linked 2-methylimidazole moiety. Initial structure-activity studies led to the identification of 31 as a potent and selective peptidomimetic inhibitor with an IC50 of 56 nM and 250-fold selectivity versus human NMT. 2-Methylimidazole as the N-terminal amine replacement in combination with a 4-substituted phenacetyl moiety imparts remarkable potency and selectivity to this novel class of inhibitors. The (S,S) stereochemistry of serine and lysine residues is critical for the inhibitory activity, since the (R,R) enantiomer 40 is 10(3)-fold less active than the (S,S) isomer 31. The inhibitory profile exhibited by this new class of NMT ligands is a function of the pKa of the imidazole substituent as illustrated by the benzimidazole analog 35 which is about 10-fold less potent than 31. The measured pKa (7.1 +/- 0.5) of 2-methylimidazole in 31 is comparable with the estimated pKa (approximately 8.0) of the glycyl residue in the high-affinity substrate 1a. Groups bulkier than methyl, such as ethyl, isopropyl, or iodo, at the imidazole 2-position have a detrimental effect on potency. Further refinement of 31 by grafting an alpha-methyl group at the benzylic position adjacent to the serine residue led to 61 with an IC50 of 40 nM. Subsequent chiral chromatography of 61 culminated in the discovery of the most potent Candida NMT inhibitor 61a reported to date with an IC50 of 20 nM and 400-fold selectivity versus the human enzyme. Both 31 and 61a are competitive inhibitors of Candida NMT with respect to the octapeptide substrate GNAASARR-NH2 with Ki(app) = 30 and 27 nM, respectively. The potency and selectivity displayed by these inhibitors are dependent upon the size and orientation of the alpha-substituent. An alpha-methyl group with the R configuration corresponding to the (S)-methyl-4-alanine in 2 confers maximum potency and selectivity. Structural modification of 31 and 61 by appending an (S)-carboxyl group beta to the cyclohexyl moiety provided the less potent tripeptide inhibitors 73a and 73b with an IC50 of 1.45 +/- 0.08 and 0.38 +/- 0.03 microM, respectively. However, these tripeptides (73a and 73b) exhibited a pronounced selectivity of 560- and 2200-fold versus the human NMT. More importantly 73a displayed fungistatic activity against C albicans with an EC50 of 51 +/- 17 microM in cell culture. Compound 73b also exhibited a similar antifungal activity. An Arf protein gel mobility shift assay for monitoring intracellular myristoylation revealed that a single dose of 200 microM of 73a or 73b produced < 50% reduction in Arf N-myristoylation, after 24 and 48 h, consistent with their fungistatic rather than fungicidal activity. In contrast, the enantiomer 73d which had an IC50 > 1000 microM against C. albicans NMT did not exhibit antifungal activity and produced no detectable reduction in Arf N-myristoylation in cultures of C. albicans. These studies confirm that the observed antifungal activity of 73a and 73b is due to the attenuation of NMT activity and that NMT represents an attractive tar

Acyltransferases↗
PubMed1998

Use of papain as a model for the structure-based design of cathepsin K inhibitors: crystal structures of two papain-inhibitor complexes demonstrate binding to S'-subsites.

Papain has been used as a surrogate enzyme in a drug design effort to obtain potent and selective inhibitors of cathepsin K, a new member of the papain superfamily of cysteine proteases that is selectively and highly expressed in osteoclasts and is implicated in bone resorption. Here we report the crystal structures of two papain-inhibitor complexes and the rational design of novel cathepsin K inhibitors. Unlike previously known crystal structures of papain-inhibitor complexes, our papain structures show ligand binding extending deep within the S'-subsites. The two inhibitor complexes, carbobenzyloxyleucinyl-leucinyl-leucinal and carbobenzyloxy-L-leucinyl-L-leucinyl methoxymethyl ketone, were refined to 2.2- and 2.5-A resolution with R-factors of 0.190 and 0. 217, respectively. The S'-subsite interactions with the inhibitors are dominated by an aromatic-aromatic stacking and an oxygen-aromatic ring edge interaction. The knowledge of S'-subsite interactions led to a design strategy for an inhibitor spanning both subsites and yielded a novel, symmetric inhibitor selective for cathepsin K. Simultaneous exploitation of both S- and S'-sites provides a general strategy for the design of cysteine protease inhibitors having high specificity to their target enzymes.

Binding Sites↗