Matrix metalloproteinase inhibitors: a structure-activity study.

Modifications around the dipeptide-mimetic core of a hydroxamic acid based matrix metalloproteinase inhibitor were studied. These variations incorporated a variety of natural, unnatural, and synthetic amino acids in addition to modifications of the P1' and P3' substituents. The results of this study indicate the following structural requirements: (1) Two key hydrogen bonds must be present between the enzyme and potent substrates. (2) Potent inhibitors must possess strong zinc-binding functionalities. (3) The potential importance of the hydrophobic group at position R3 as illustrated by its ability to impart greater relative potency against stromelysin when larger hydrophobic groups are used. (4) Requirements surrounding the nature of the amino acid appear to be more restrictive for stromelysin than for neutrophil collagenase, 72 kDa gelatinase, and 92 kDa gelatinase. These requirements may involve planar fused-ring aryl systems and possibly hydrogen-bonding capabilities.

Inhibition of angiogenesis by the matrix metalloprotease inhibitor N-[2R-2-(hydroxamidocarbonymethyl)-4-methylpentanoyl)]-L-tryptophan methylamide.

The inhibitor N-[2R-2-(hydroxamidocarbonymethyl)-4-methylpentanoyl)]-L- tryptophan methylamide specifically blocks several matrix metalloproteases, enzymes which are thought to be involved in angiogenesis. An extract of Walker 256 carcinoma in Hydron pellets implanted in the corneas of Sprague-Dawley rats was used to stimulate angiogenesis from the vessels of the limbus. Angiogenesis was graded visually as the distance penetrated into the cornea and the number of vessels generated. The vessel area was also measured by image analysis using Image 1 software. Continuous i.v. administration of N-[2-(hydroxamidocarbonymethyl)-4-methylpentanoyl)]- L-tryptophan methylamide at 32 mg/kg/day (n = 17) via syringe pump reduced vessel number [25.06 +/- 5.9 (SEM) compared to 65.33 +/- 9.0] and vessel area (26.14 +/- 3.2 mm2 compared with 40.96 +/- 4.6 mm2), but not distance penetrated, compared to vehicle-treated control eyes after 6 days. These results confirm the suspected role for matrix metalloproteases in angiogenesis and suggest that inhibitors of these enzymes may be angiostatic agents.

Inhibition of collagenolytic activity relates to quantitative reduction of invasion in vitro in a c-Ha-ras transfected glial cell line.

The function of proteases in brain tumor invasion is currently not well established. For tumors of epithelial and fibromatous origin collagenase production can enhance the invasive capacity of cells to penetrate basement membranes. We showed previously that a c-Ha-ras transformed glial cell line (CxT24neo3) invaded hamster brain tissue in vivo. These cells were also capable of invading reconstituted basement membrane and embryonic chick hearts in vitro. Since the histopathology of CxT24neo3 tumors mimics that of glioblastoma multiforme in humans, CxT24neo3 was used as the model in vitro for this type of brain tumor. Presently, we detected by zymogram analysis a gelatinase that was secreted by CxT24neo3 and that had an apparent molecular mass of 62 kD. To verify whether gelatinase affected invasion in vitro of these glial cells we determined the efficacy of a substrate specific collagenase inhibitor on invasion in vitro. GM6001 is a synthetic polypeptide that specifically occupies the substrate binding sites of metalloprotease. Since this drug did not show cytotoxicity, its specificity for metalloprotease is a valuable tool to evaluate the physiological function of these enzymes on invasion. We found that treatment of CxT24neo3 with GM6001 reduced the fraction of invading CxT24neo3 cells through reconstituted basement membrane. These data suggest that metalloproteases can stimulate brain tumor invasion.

Treatment of alkali-injured rabbit corneas with a synthetic inhibitor of matrix metalloproteinases.

Healing of corneal alkali injuries remains a severe clinical challenge. The authors evaluated the effect of a new synthetic inhibitor of matrix metalloproteinases (GM6001 or N-[2(R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-L-tryptophane methylamide) on preventing ulceration of rabbit corneas after alkali injury. Topical treatment of corneas with severe alkali injuries with 400 micrograms/ml or 40 micrograms/ml GM6001 alone prevented ulceration for 28 days, although 8 of 10 corneas treated with vehicle perforated. Corneas treated with 4 micrograms/ml GM6001 had midstromal depth ulcers. Corneas treated with 400 micrograms/ml of GM6001 contained very few inflammatory cells and had significantly reduced vessel ingrowth compared with vehicle-treated corneas. Epithelial regeneration after moderate alkali injuries also was investigated. Persistent epithelial defects developed 4 days after moderate alkali injury in rabbit corneas treated with vehicle and progressively increased to an average of 20% of the original 6 mm diameter wound by 27 days after moderate alkali injury. By contrast, epithelial regeneration was complete and persisted for 21 days for corneas treated with a formulation containing GM6001 (400 micrograms/ml), epidermal growth factor (10 micrograms/ml), fibronectin (500 micrograms/ml), and aprotinin (400 micrograms/ml). Sporadic punctate staining developed in 20% of the corneas treated with the combination of agents between days 21-28 after moderate alkali injury. These results demonstrate that topical application of GM6001 prevented corneal ulceration after severe alkali injury and that a combination containing GM6001, epidermal growth factor, fibronectin, and aprotinin promoted stable regeneration of corneal epithelium after moderate alkali injury.

Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids.

The hydroxamic acid HONHCOCH2CH(i-Bu)CO-L-Trp-NHMe, isomer 6A (GM 6001), inhibits human skin fibroblast collagenase with Ki of 0.4 nM using the synthetic thiol ester substrate Ac-Pro-Leu-Gly-SCH(i-Bu)CO-Leu-Gly-OEt at pH 6.5. The other isomer, 6B, which has the opposite configuration at the CH2CH(i-Bu)CO alpha-carbon atom, has a Ki of 200 nM for this enzyme. GM 6001 is one of the most potent inhibitors of human skin fibroblast collagenase yet reported. GM 6001 has a Ki of 20 nM against thermolysin and Pseudomonas aeruginosa elastase. Isomer 6B has a Ki of 7 nM against thermolysin and 2 nM against the elastase. 6A and 6B are the most potent hydroxamate inhibitors reported for these bacterial enzymes. The pattern of inhibition for all three enzymes suggests that isomer 6A is the (R,S) compound, stereochemically analogous to the L,L-dipeptide, and isomer 6B is the (S,S) compound, analogous to the DL-dipeptide. The tolerance of the D configuration by thermolysin and the elastase allows these inhibitors to discriminate between the human and bacterial enzymes simply by inversion of configuration at the CH2CH(i-Bu)CO alpha-carbon atom. Substitution of the potential metal liganding groups carboxylate and hydrazide for the hydroxamate group yields much weaker inhibitors for all three enzymes.

Inhibition of human skin fibroblast collagenase by phosphorus-containing peptides.

Substitution of the phosphonamidate linkage (PO2-NH) for the peptide bond (CO-NH) in substrate-like sequences produces inhibitors of human skin fibroblast collagenase with Ki's far below Km for the native collagen substrate. Using a thiol ester substrate at pH 6.5, phthaloyl-GlyP-Ile-Trp-(S)NHCH-(Me)Ph, the phosphonamidate analog of phthaloyl-Gly-Ile-Trp-(S)NHCH(Me)Ph, has a Ki of 20 nM. Peptide phosphonamidates with amino acid sequences extended further to the right or the left of the Gly-Ile-Trp sequence had higher Ki's. Substitution of the phosphinate linkage (PO2-CH2) for the peptide bond also gives potent inhibitors such as napthoyl-GlyP-C-Leu-Trp-NHBzl, the phosphinate analog of naphtholyl-Gly-Leu-Trp-NHBzl, which has a Ki of 10 nM. Some of the phosphonamidates and phosphinates are also excellent inhibitors of the bacterial zinc metalloproteases thermolysin and Pseudomonas aeruginosa elastase.

An inhibitor of the matrix metalloproteinase synthesized by rabbit corneal epithelium.

Normal and abnormal processes of cellular invasion often are initiated by degradation of basement membranes. The process of corneal ulceration might operate via similar mechanisms; degradation of the corneal stroma is not seen until after the basement membrane underlying the corneal epithelium in the preulcerative lesion is lost. Recent data implicate a member of the matrix metalloproteinase (MMP) family of enzymes, 92 kD gelatinase/type IV collagenase (MMP-9) in both cellular invasion processes and degradation of epithelial basement membrane before corneal ulceration. This suggests that use of nontoxic substances that block activity of MMP-9 might be useful in preventing or inhibiting pathologic invasion processes in vivo. An agent that fits these criteria is N-[D,L-2-isobutyl-3(N'-hydroxycarbonylamido)-propanoyl]-O- methyl-L-tyrosine methylamide, which previously has been characterized as an inhibitor of tumor cell collagenases. In this study, the authors show that the inhibitor can efficiently block activity of MMP-9 purified from cultures of rabbit corneal epithelial cells. Results suggest that the recently reported efficacy of a closely related inhibitor in blocking progression of alkali burns to ulceration might be attributable to its action against MMP-9.

92-kD type IV collagenase mediates invasion of human cytotrophoblasts.

The specialized interaction between embryonic and maternal tissues is unique to mammalian development. This interaction begins with invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. The transient tumor-like behavior of cytotrophoblasts, which peaks early in pregnancy, is developmentally regulated. Likewise, in culture only early-gestation human cytotrophoblasts invade a basement membrane-like substrate. These invasive cells synthesize both metalloproteinases and urokinase-type plasminogen activator. Metalloproteinase inhibitors and a function-perturbing antibody specific for the 92-kD type IV collagen-degrading metalloproteinase completely inhibited cytotrophoblast invasion, whereas inhibitors of the plasminogen activator system had only a partial (20-40%) inhibitory effect. We conclude that the 92-kD type IV collagenase is critical for cytotrophoblast invasion.

Selective phosphinate transition-state analogue inhibitors of the protease of human immunodeficiency virus.

The phosphinic acid isosteres of di-, tetra- and hexapeptides containing a hydrophobic amino acid side chains at the P1-P'1 positions are powerful inhibitors of Human Immunodeficiency Virus protease. Ki's ranged from 0.4 nM to 26 microM at pH 6.5 and were lower at pH 4.5. The compounds showed no activity against trypsin, weak activity against renin at pH 6.5, moderate activity against pepsin at pH 2.0 (Ki values in the microM range) and substantial activity against cathepsin D at pH 3.5 (Ki values from 9 to 300 nM).

Binding energetics of phosphorus-containing inhibitors of thermolysin.

The importance of a specific hydrogen bond between thermolysin and a phosphonamidate inhibitor, Z-NHCH2-PO(O-)-Leu-Leu (1) [Bartlett, P. A., & Marlowe, C. K. (1987) Science (Washington D.C.) 235, 569-571], has been reevaluated. We have determined the inhibition constants (binding free energies) for thermolysin of phosphonamidate n-hexyl-P(O)(O-)-Leu-Trp-NHMe (4), phosphonate n-hexyl-P-(O)(O-)OCH(iBu)CO-Trp-NHMe (5), and phosphinates n-hexyl-P(O)(O-)CH2CH(iBu)CO-Trp-NHMe (6) and Z-NHCH2PO(O-)CH2CH(iBu)CO-Leu (3). Replacement of the P-NH group by P-CH2 (1----3 and 4----6) weakens the overall binding free energy by about 1.5 kcal/mol. A negligible difference in solvation energy has been measured for these pairs, and the basicity of the P-O- ligand for zinc in each pair remains nearly unchanged as determined by pH titration of their 31P NMR resonances. Therefore, this value of 1.5 kcal/mol can be assigned to the specific hydrogen bond known to exist between the P-NH of 1 and thermolysin [Tronrud, D. E., Holden, H. M., & Matthews, B. W. (1987) Science (Washington, D.C.) 235, 871-574] and inferred to exist between 4 and the enzyme. Substitution of P-O for P-NH (1----2 [Bartlett, P. A., & Marlowe, C. K. (1987) Science (Washington, D.C.) 235, 569-571] and 4----5) weakens the overall binding free energy by 4.1 kcal/mol for each pair as the basicity of the P-O- ligand decreases by about 1.6 pH units. The measured solvation energy difference between 4 and 5 (and by inference between 1 and 2) is negligible.(ABSTRACT TRUNCATED AT 250 WORDS)

The ketone cinnamoyl-(1-13C-Phe)-CGly-Pro-Pro is a tetrahedral transition state analog inhibitor of C. histolyticum collagenase.

The ketone cinnamoyl-(1-13C-Phe)-CGly-Pro-Pro [(4-13C-5-cinnamido-4-oxo-6-phenylhexanoyl)-Pro-Pro 2] competitively inhibits a mixture of collagenases from Clostridium histolyticum with a Ki of 40 +/- 6 nM. 13C-nmr spectroscopy of the ketone in the presence of this collagenase shows a bound 13C resonance at 102.6 ppm and the resonance of the free ketone at 212 ppm. Ketone alone shows no trace (less than 0.5%) of a resonance in the region around 100 ppm. The bound resonance is displaceable by another competitive inhibitor. This ketone is thus a transition state analog which is rehybridized from trigonal planar to tetrahedral upon binding to collagenase.

Ionization states of the complex formed between 2-benzyl-3-phosphonopropionic acid and carboxypeptidase A.

The binding to carboxypeptidase A of two phosphonic acid analogues of 2-benzylsuccinate, 2-DL-2-benzyl-3-phosphonopropionic acid (inhibitor I) and 2-DL-2-benzyl-3-(-O-ethylphosphono)propionic acid (inhibitor II) was studied by observing their 31P resonances when free and bound to the enzyme in the range of pH from 5 to 10. The binding of I by co-ordination to the active-site Zn(II) lowered the highest pKa of I from a value of 7.66(+/- 0.10) to a value of 6.71(+/- 0.17). No titration of any protons on II occurred over the pH range studied. The enzyme-bound inhibitor II also did not titrate over the pH range 6.17-7.60. The pH-dependencies of the apparent inhibition constants for I and II were also investigated by using N-(-2-(furanacryloyl)-L-phenylalanyl-L-phenylalanine as substrate. Two enzymic functional groups with pKa values of 5.90(+/- 0.06) and 9.79(+/- 0.14) must be protonated for binding of inhibitor I, and two groups with pKa values of 6.29(+/- 0.10) and 9.19(+/- 0.15) for binding of inhibitor II. Over the pH range from 6.71 to 7.66, inhibitor I binds to the enzyme in a complex of the enzyme in a more protonated form, and the inhibitor in a less protonated form than the predominant unligated forms at this pH. Mock & Tsay [(1986) Biochemistry 25, 2920-2927] made a similar finding for the binding of L-2-(1-carboxy-2-phenylethyl)-4-phenylazophenol over a pH range of nearly 4 units. The true inhibition constant for the dianionic form of inhibitor I (racemic) was calculated to be 54.0(+/- 5.9) nM and that of the trianionic form to be 5.92(+/- 0.65) nM. The true inhibition constant of the fully ionized II (racemic) was calculated to be 79.8(+/- 6.4) nM.

19F nuclear magnetic resonance spectroscopy study of the complex of 2-benzyl-4-oxo-5,5,5-trifluoropentanoic acid and carboxypeptidase A.

(dl)-2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is a strong transition state analog inhibitor of the zinc protease carboxypeptidase A. 19F NMR spectroscopy of the aqueous solution of this inhibitor shows the hydrate of the ketone carbonyl to be the major species, with a shift of -9.95 ppm. As the pH is varied from 4.9 to 13.1, a 1.53 ppm downfield shift occurs, giving a pK alpha of 11.10. When excess inhibitor is added to the enzyme, a new, bound peak appears at -8.84 ppm, in addition to the free hydrate peak. Spectra taken at pH's from 4.90 to 9.15 show no change in the position of the bound resonance; from 9.15 to 12.15, a 0.26 ppm upfield shift occurs. The interpretation is that the monoanion of the hydrate is the form that binds to the enzyme.

Ketone-substrate analogues of Clostridium histolyticum collagenases: tight-binding transition-state analogue inhibitors.

A series of ketone-substrate analogues has been synthesized for the two classes of collagenases from Clostridium histolyticum and shown to be competitive inhibitors. These compounds have sequences that match those of specific peptide substrates for these enzymes. The best inhibitor is the ketone analogue of cinnamoyl-Leu-Gly-Pro-Pro, which has a KI value of 18 nM for epsilon-collagenase, a class II enzyme. This is the tightest binding inhibitor reported for any collagenase to date. Plots of log KI for the inhibitors vs log KM/kcat for the matched substrates for both collagenases are linear with slopes near unity, indicating that the ketones are transition-state analogues. This strongly implies that the ketone carbon atoms of these inhibitors are tetrahedral when bound to the enzymes.

Effects of some cyclic elements containing amphiphilic compounds on stability and transport properties of model lecithin membranes.

The paper presents results of studies on changes in model membrane properties induced by some single-chain amphiphilic quaternary ammonium salts with fungicidal activity, N-dodecyl-N-methylmorpholinium chloride (IVC), N-dodecyloxymethylene-N-methylmorpholinium chloride (IVD), N-dodecyl-N-methylpiperidinium chloride (VIC), N-dodecyloxymethylene-N-methylpiperidinium chloride (VID) and N-dodecyl-N,N-dimethyl-N-benzy-lammonium chloride (IB). Two different lipid systems were used, namely, unilamellar liposomes and black planar membranes (BLM). All the compounds studied interfered with sulphate ion transport across liposomal membranes as well as with calcium ion desorption from the membranes. The compounds were found also to change the stability of black lecithin membranes. Generally the sequence of effectiveness of the salts studied on both models used was: IB greater than VID greater than IVD greater than VIC greater than IVC, although there were some differences for particular processes. The results obtained are discussed in view of a possible mechanism of the interaction between the salts studied and the lipid model including the role the salt head group structure and charge distribution can play in this interaction.

Inhibition of angiotensin converting enzyme by aldehyde and ketone substrate analogues.

Three classes of carbonyl-containing substrate analogues and partial substrate analogues have been tested for their ability to inhibit angiotensin converting enzyme. (4-Oxobutanoyl)-L-proline is proposed to occupy the S1' and S2' subsites on the enzyme, thus locating its aldehyde carbonyl group at the position of the active site zinc atom. This aldehyde is 70% hydrated in aqueous solution and could mimic a tetrahedral intermediate occurring during enzyme-catalyzed substrate hydrolysis, but its Ki is only 760 microM. Carbobenzoxy-L-isoleucyl-L-histidyl-L-prolyl-L-phenylalaninal is proposed to occupy the S1 through S4 subsites on the other side of the zinc atom. Its weak Ki of 60 microM is nearly equipotent to its parent peptide terminating in phenylalanine. However, ketoace, (5RS)-(5-benzamido-4-oxo-6-phenylhexanoyl)-L-proline [Almquist, R.G., Chao, W.R., Ellis, M.E., & Johnson, H.L. (1980) J. Med. Chem. 23, 1392-1398], one of the third class of inhibitors proposed to occupy subsites S1 through S2' on both sides of the zinc atom, has a Ki of 0.0006 microM under our assay conditions, orders of magnitude more potent than its parent peptide. The carbonyl carbon of ketoace is less than 3% hydrated in aqueous solution as determined by carbon-13 nuclear magnetic resonance spectroscopy. If the hydrate is the species bound to converting enzyme, its Ki must be less than 18 pM. Ketoace is a slow-binding inhibitor of converting enzyme, but its overall Ki is dependent on its concentration and therefore prevents calculation of kinetic constants for slow binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of carboxypeptidase A by ketones and alcohols that are isosteric with peptide substrates.

The Ki's of three peptide ketone and three peptide alcohol inhibitors of carboxypeptidase A are compared with Ki's of their respective isosteric peptide substrates, N alpha-benzoyl-L-phenylalanine, N alpha-benzoylglycyl-L-phenylalanine, and N alpha-carbobenzoxyglycylglycyl-L-phenylalanine. For the isosteric ketone analogues of these substrates, the respective Ki's are as follows: (2RS)-2-benzyl-4-(3-methoxyphenyl)-4-oxobutanoic acid, 180 +/- 40 microM; (2RS)-5-benzamido-2-benzyl-4-oxopentanoic acid (V), 48 +/- 7 microM; (2RS)-2-benzyl-5-(carbobenzoxyglycinamido)-4-oxopentanoic acid (IX), 9 +/- 0.1 microM. For the alcohols derived by reduction of each of these ketones, Ki's are as follows: (2RS,4RS)-2-benzyl-4-(3-methoxyphenyl)-4-hydroxybutanoic acid, 190 +/- 10 microM; (2RS,4RS)-5-benzamido-2-benzyl-4-hydroxybutanoic acid (IV), 160 +/- 62 microM; (2RS,4RS)-2-benzyl-5-(carbobenzoxyglycinamido)-4-hy droxypentanoic acid (XI), 600 +/- 100 microM. Ki values for the competitive peptide ketone inhibitors decrease with increasing peptide chain length. This is consistent with the possibility of increased binding interaction between inhibitor and enzyme by simple occupation of additional binding subsites by adding more amino acid residues to the inhibitor. In contrast, the Ki values of the alcohols (competitive or mixed inhibition) increased or remain essentially unchanged with increasing chain length. Increasing the chain length of ketone inhibitor V to give IX decreases Ki by one-fifth. The Ki of ketone IX is also less than 1/30th the Ki of its isosteric peptide and almost 1/70th that of its isosteric alcohol, XI.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Phosphonopropionic acids inhibit carboxypeptidase A as multisubstrate analogues or transition-state analogues.

A series of phosphonic acid analogues of 2-benzylsuccinate were tested as inhibitors of carboxypeptidase A. The most potent of these, (2RS)-2-benzyl-3-phosphonopropionic acid, had a Ki of 0.22 +/- 0.05 microM, equipotent to (2RS)-2-benzylsuccinate and thus one of the most potent reversible inhibitors known for this enzyme. Lengthening by one methylene group to (2RS)-2-benzyl-4-phosphonobutyric acid increased the Ki to 370 +/- 60 microM. The monoethyl ester (2RS)-2-benzyl-3-(O-ethylphosphono)propionic acid was nearly as potent as (2RS)-2-benzyl-3-phosphonopropionic acid, with a Ki of 0.72 +/- 0.3 microM. The sulphur analogue of the monoethyl ester, 2-ambo-P-ambo-2-benzyl-3-(O-ethylthiophosphono)propionic acid, had a Ki of 2.1 +/- 0.6 microM, nearly as active as (2RS)-2-benzyl-3-(O-ethylphosphono)propionic acid. These phosphonic acids and esters could be considered to be multisubstrate inhibitors of carboxypeptidase A by virtue of their structural analogy with 2-benzylsuccinate. Alternatively, the tetrahedral hybridization at the phosphorus atom suggests that they could be mimicking a tetrahedral transition state for the enzyme-catalysed hydrolysis of substrate.

N alpha-(diphenoxyphosphoryl)-L-alanyl-L-proline, N alpha-[bis (4-nitrophenoxy)phosphoryl]-L-alanyl-L-proline, and N alpha-[ (2-phenylethyl)phenoxyphosphoryl]-L-alanyl-L-proline: releasers of potent inhibitors of angiotensin converting enzyme at physiological pH and temperature.

The rate of loss of phenol or 4-nitrophenol from N alpha-(diphenoxyphosphoryl)-L-alanyl-L-proline (2), N alpha-[bis(4-nitrophenoxy)phosphoryl]-L-alanyl-L-proline (5), and N alpha-[(2-phenylethyl)phenoxyphosphoryl]-L-alanyl-L-proline (12) was determined spectrophotometrically at pH 7.5 and 37 degrees C in both Tris and phosphate buffers. These moderately potent inhibitors of angiotensin converting enzyme (Ki greater than 0.8 microM) all hydrolyze, losing 1 mol of phenol to yield highly potent inhibitors (Ki = 0.5-18 nM). The half-times for loss of 1 mol of phenol in Tris buffer are 22 days (2), 3.4 h (5), and 21 days (12). The half-times in phosphate buffer were not significantly different. The mono(4-nitrophenoxy) ester 6 (Ki = 18 nM) loses its 1 mol of nitrophenol with a half-time of 35 h to yield N alpha-phosphoryl-L-alanyl-L-proline 16 (Ki = 1.4 nM), which hydrolyzes at the P-N bond with a half-time of 2.2 h. Hydrolysis of the P-N bond in 2 and 12 was not observed during the time course of the kinetic experiments. The two phosphoramidate diesters 2 and 5 and the phosphonamidate monoester 12 thus release powerful inhibitors of angiotensin converting enzyme with a known time course at physiological pH and temperature in vitro. A time-dependent increase in inhibitory potency against converting enzyme that paralleled the kinetics of phenyl ester hydrolysis was confirmed in vitro.

Aldehyde and ketone substrate analogues inhibit the collagenase of Clostridium histolyticum.

The collagenase from Clostridium histolyticum is a mixture of several collagenases, all of which are zinc metalloproteases. This enzyme catalyzes the cleavage of the X-Gly peptide bond in the repeating sequence of collagen: -Gly-Pro-X-Gly-Pro-X-. Thus the S3, S2, and S1 subsites on the enzyme appear to be occupied by the sequence -Gly-Pro-X- and the S1', S2', and S3' subsites also by -Gly-Pro-X-. Short peptides up to and including N alpha-acyltetrapeptides containing the repeat sequence do not detectably inhibit the enzyme (IC50 greater than 10 mM). However, peptide aldehydes of the form aminoacyl-X-glycinal, presumably occupying the S1, S2, ..., Sn subsites, are inhibitors. The most potent of these was Pro6-Gly-Pro-glycinal, with an IC50 of 340 +/- 70 microM. The single peptide aldehyde investigated, which could occupy the S1' and S2' subsites, 4-oxobutanoyl-L-proline, did not inhibit collagenase (IC50 greater than 20 mM). The peptide ketone 5-benzamido-4-oxo-6-phenylhexanoyl-Pro-Ala (XXV), which could occupy the S1-S3' subsites, inhibits collagenase with an IC50 of 120 +/- 50 microM, over 80-fold more potently than its parent peptide analogue benzoyl-Phe-Gly-Pro-Ala (XXIII). The alcohol analogue of XXV, 5-benzamido-4-hydroxy-6-phenylhexanoyl-Pro-Ala (XXVI), is over 60-fold less potent with an IC50 of 8 +/- 2mM. Extending the peptide ketone XXV to occupy the S2-S3' subsites gave 5-(N alpha-carbobenzoxy-L-prolinamido)-4-oxo-6-phenylhexanoyl-Pro -Ala (XXVII). Surprisingly, XXVII had an IC50 of only 5.2 +/- 2 mM.(ABSTRACT TRUNCATED AT 250 WORDS)