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D H Rich

Publications and source records attributed to D H Rich.

At least 73 records · Page 4Linked to original sources
PubMed1988

Synthesis of sulfur-containing analogues of bestatin. Inhibition of aminopeptidases by alpha-thiolbestatin analogues.

Sulfur-containing amino acid and peptide analogues of bestatin [((2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl)-L-leucine] (1) have been synthesized and evaluated as inhibitors of aminopeptidase M (AP-M), leucine aminopeptidase (LAP), and aminopeptidase B (AP-B). The 2-thiolbestatin analogue (6) was found to be a potent inhibitor of all three aminopeptidases (AP-M, Ki = 4.4 microM; LAP, Ki = 0.55 microM; AP-B, Ki = 4.6 nM) but only a slightly better inhibitor of these aminopeptidases than the parent hydroxy-containing compound 1. Synthetic analogues of L-leucinethiol(4), a strong inhibitor of aminopeptidases, were prepared in which the carbon alpha to the thiol groups was substituted with methyl, methyl carboxylate, and carboxamide derivatives and found to be much weaker inhibitors of all aminopeptidases. A thioamide analogue of bestatin (49) is a modest inhibitor of AP-M (Ki = 40 microM), LAP (Ki = 0.33 microM), and AP-B (Ki = 2.4 microM). These results suggest that the sulfur atoms in 2-thiolbestatin and bestatin thioamide do not interact strongly with the active-site zinc atom of these aminopeptidases when the inhibitors are bound to the enzyme. These results are not consistent with proposed models for the inhibition of aminopeptidases by bestatin and related analogues.

Amino Acids↗
PubMed1988

Inhibition of porcine pepsin by two substrate analogues containing statine. The effect of histidine at the P2 subsite on the inhibition of aspartic proteinases.

Two new inhibitors, 4 and 5, of the aspartic proteinase porcine pepsin were synthesized. These compounds, which span the P4-P'3 binding subsites of the enzyme, were derived by replacing the Nph-Phe dipeptidyl unit of a good pepsin substrate, H2N-Phe-Gly-His-Nph-Phe-Ala-Phe-OMe (3), with statine [(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid, Sta]. Hexapeptide 5, H2N-Phe-Gly-Val-(S,S)-Sta-Ala-Phe-OMe, is an extremely potent inhibitor of pepsin with a Ki value less than 1 nM. This result is consistent with the proposal that statine functions as a bioisosteric replacement for a substrate dipeptidyl unit. Compound 4, which contains His at P2, is 2 orders of magnitude less active than the valine analogue 5 (Ki = 150 nM). The factor for the decrease in binding to pepsin effected by replacement of Val by His at P2 parallels the ratio of protonated vs unprotonated imidazole group in peptide 4 at pH 4, according to the Henderson-Hasselbach equation. This result suggests that a positively charged side chain at P2 is undesirable for maximum pepsin inhibition. Kinetic constants for several known inhibitors of pepsin and renin are presented that demonstrate that the effect of His incorporation at P2 on pepsin inhibition depends upon the peptide sequence and that the effect is considerably different for renin inhibitors. We further suggest that the high selectivity of potent renin inhibitors known to be only weak pepsin and cathepsin D inhibitors is due in part to the extent of histidine protonation at P2 arising from pH differences in the inhibition kinetics assay of renin (neutral conditions) compared to other aspartic proteinases (acid pH 2-4).

Algorithms↗
PubMed1987

L-lysinethiol: a subnanomolar inhibitor of aminopeptidase B.

L-Lysinethiol was found to be an extremely potent inhibitor of aminopeptidase B (AP-B) with a Ki = 9.1 X 10(-10) M. L-leucinethiol was also a potent inhibitor of AP-B (kj = 1.3 X 10(-7) M), while the D-isomer was much less effective (Ki = 9.8 X 10(-5) M). A thiol-zinc interaction at the active site is postulated for AP-B.

Amino Acids↗
PubMed1987

Analogues of the cytostatic and antimitogenic agents chlamydocin and HC-toxin: synthesis and biological activity of chloromethyl ketone and diazomethyl ketone functionalized cyclic tetrapeptides.

The synthesis and biological activity of four novel analogues of the cytostatic and antimitogenic agents chlamydocin and HC-toxin are reported in which the natural products' reactive epoxy ketone side-chain moiety is replaced by a chloromethyl or a diazomethyl ketone functionality, but the respective 12-membered cyclic tetrapeptide ring systems are retained. Syntheses of the linear tetrapeptide sequences were, in each case, achieved by conventional methodology and designed such that cyclization would be onto proline. The use of suitably protected L-2-aminosuberic acid (Asu) enabled the ready assimilation of the desired chloromethyl and diazomethyl ketone functionalities after cyclization. Cyclization was accomplished by using bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl). Yields of cyclic product were comparable to or, in the case of the HC-toxin ring system, better than those previously reported. Liberation of the Asu-side-chain acid and manipulation to the required functionalities via mixed anhydride to the diazomethyl ketone and quenching with HCl to yield the chloromethyl ketone was achieved in excellent yield for the HC-toxin analogues but in only moderate yield for the chlamydocin analogue. The antimitogenic activities of HC-toxin chloromethyl ketone (IC50 = 30-40 ng/mL) and chlamydocin chloromethyl ketone (IC50 = 3-10 ng/mL) were found to be 3-4-fold lower than those of the natural products themselves. The diazomethyl ketone analogue of HC-toxin was found to be inactive (IC50 greater than 2000 ng/mL). A modification of the HC-toxin peptide ring system, [L-Phe]3-HC-toxin chloromethyl ketone was found not to be a more active analogue (IC50 = 40-100 ng/mL). The nature of the putative target molecule, the binding interactions of the various analogues and the contribution of rate of inhibition toward activity are briefly discussed. The chloromethyl ketones herein reported constitute the most potent synthetic antimitogenic cyclic tetrapeptide analogues yet designed.

Animals↗
PubMed1987

Inhibition of aspartic proteinases by peptides containing lysine and ornithine side-chain analogues of statine.

The synthesis of two new analogues of statine are reported corresponding to analogues with the lysine side chain and the ornithine side chain. These analogues were designed on the basis of substrate specificity and molecular modeling of three-dimensional structures of the penicillopepsin: Iva-Val-Sta-OEt crystal structure. 4,8-Diamino-3-hydroxyoctanoic acid [LySta] and 4,7-diamino-3-hydroxyheptanoic acid [OrnSta] were synthesized respectively from Boc-Lys(Z)-al and Boc-Orn(Bzl,Z)-al by addition of lithio ethyl acetate to the aldehyde group. The [LySta] derivative was converted to the trichloroethoxycarbonyl derivative and separated into the corresponding 3S,4S and 3R,4R diastereomers. The [OrnSta] derivative was used as a mixture of 3-position diastereomers. These new amino acids were used to prepare the following inhibitors: Iva-Val-Val-[LySta]-OEt and Iva-Val-Val-[OrnSta]-OEt as well as the corresponding synthetic intermediates. Inhibition constants (Ki values) were measured for inhibition of porcine pepsin and penicillopepsin. Both compounds were potent inhibitors of penicillopepsin with Ki values 10-100 times smaller (2.1 and 1.1 nM, respectively) than the Ki of Iva-Val-Val-Sta-OEt (47 nM). In contrast both inhibitors are exceptionally weak inhibitors of porcine pepsin with Ki values greater than 1 microM. These results are correlated with the ability of the basic group in the new inhibitors to bind to aspartic acid-77 in penicillopepsin.

Amino Acids↗
PubMed1987

Synthetic and enzyme inhibition studies of pepstatin analogues containing hydroxyethylene and ketomethylene dipeptide isosteres.

Synthetic details for the preparation of a series of hydroxyethylene and ketomethylene dipeptide isosteres with control of stereochemistry at C(2) are described. Incorporation of the isosteres into peptide sequences derived from pepstatin afforded potent inhibitors of the aspartic protease porcine pepsin. When Leu-OH-Ala or Leu-OH-Phe was substituted for statine [3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid), inhibitors equipotent to the parent compound were obtained, whereas Leu-OH-Gly was a much less effective replacement for statine. A similar trend was evident in the corresponding ketones. The finding that structural features for good substrates do not closely parallel those for good inhibitors is discussed.

Amino Acid Sequence↗
PubMed1987

Synthetic bovine prothrombin precursor 13-29 for studies of vitamin K dependent carboxylase.

The synthesis of the amino acid sequence found in bovine prothrombin precursor 13-29 (PTP 13-29) has been achieved by solid-phase synthesis of the bis(acetamidomethyl)-protected linear peptide followed by cyclization to the monomeric disulfide. Synthesis of the disulfide bond was achieved by deprotection with mercuric acetate in acetic acid followed by oxidation with potassium ferricyanide. Experimental conditions for closure of the disulfide bond were identified by obtaining the circular dichroism spectra of the linear precursor in a variety of solvent systems. Cyclization in organic solvent systems was not successful but led to the formation of insoluble polymers. Synthetic PTP 13-29 was tested as a substrate for the vitamin K dependent carboxylase. Neither the linear nor cyclic synthetic 17 amino acid peptides were carboxylated as well as the standard, Boc-Glu-Glu-Leu-OMe, at mM concentrations. The estimated Km of synthetic PTP 13-29 is greater than 1 mM. Thus, bovine prothrombin precursor 13-29 is not an unusually effective substrate for the carboxylase as reported by Soute et al.

Animals↗
PubMed1986

Purification of cathepsin B by a new form of affinity chromatography.

Human cathepsin B was purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal linked to Sepharose 4B, with elution by 2,2'-dipyridyl disulphide at pH 4.0. The product obtained in high yield by the single step from crude starting material was 80-100% active cathepsin B. The possibility that this new form of affinity chromatography may be of general usefulness in the purification of cysteine proteinases is discussed.

2,2'-Dipyridyl↗
PubMed1986

Pepsinogen activation: genesis of the binding site.

Previous experiments have suggested that a substrate binding site may appear relatively early in the sequence of transformations that pepsinogen undergoes during its unimolecular activation. To test this possibility, tert-Boc-L-valyl-[3(S)-hydroxy-4(S)-amino-5-(2-naphthyl) pentanoyl]-L-alanylisoamylamide, a fluorescent analogue of pepstatin, has been used to measure the rate of appearance of the binding site. This probe is a potent inhibitor of pepsin, and its naphthyl group is an environmentally sensitive fluorophore that permits binding to be detected by fluorescence spectroscopy. In stopped-flow experiments, the fluorescence change following acidification of pepsinogen due to binding of the probe was found to obey a two-term exponential decay law. Analysis of the data obtained at various pH values permits us to rule out a sequential model for the kinetics. Rather, we were able to demonstrate that a concurrent model, with two species undergoing simultaneous transformation at different rates, is consistent with the results. The two species are related to each other by the protonation of a single site with a pKa of approximately 2.2. We obtained essentially identical results in an analysis of the early events observed in the acidification of pepsinogen labeled with 6-(p-toluidinyl)naphthalene-2-sulfonyl chloride (Auer & Glick, 1984). The protonic equilibrium probably occurs early after exposure to acid and is proposed to lead to a conformational change that freezes the distribution between the two species. It may be that these two conformers persist as identifiable entities at later stages of activation as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Synthesis and antimitogenic activities of four analogues of cyclosporin A modified in the 1-position.

Cyclosporin A (CSA, 1), an immunosuppressive cyclic undecapeptide, contains a unique N-methylated amino acid, (2S,3R,4R,6E)-3-hydroxy-4-methyl-2-(N-methylamino)-6-octenoic++ + acid, called both C-9-ene and MeBmt [(4R)-N-methyl-4-butenyl-4-methylthreonine] that may be essential for the biological activity of CSA. In order to determine the minimal portion of MeBmt needed for antimitogenic activity, four analogues of CSA specifically modified in the 1-position have been synthesized. These are (MeThr1)CSA (4), (MeAbu1)CSA (5), (MeAbu1,Sar10)CSA (6), and [(MeLeu(3-OH)1)]CSA (7). The synthesis of analogues was carried out by forming a linear undecapeptide that was cyclized at the two non-N-methylated amino acids. The structure of cyclic analogues 4-7 and their corresponding precursors were established unequivocally by 1H NMR, FAB mass spectrometry, elemental analysis, and HPLC. The inhibition of Con A stimulated thymocytes by CSA (1), DH-CSA (2), 7, 4, 5, and 6 gave IC50's (nM) of 4, 10, 600, 8 X 10(3), 15 X 10(3), and 40 X 10(3), respectively. The increase in IC50 by modification of the side chain in MeBmt suggested the importance of this amino acid in the 1-position of CSA for full antimitogenic activity.

Animals↗
PubMed1986

Structure and solution conformation of the cytostatic cyclic tetrapeptide WF-3161, cyclo[L-leucyl-L-pipecolyl-L-(2-amino-8-oxo-9, 10-epoxydecanoyl)-D-phenylalanyl].

The sequence and configuration of amino acids in the cytostatic cyclic tetrapeptide WF-3161 are established as cyclo(L-Leu-L-Pip-L-Aoe-D-Phe) where Pip = pipecolic acid and Aoe = 2-amino-8-oxo-9,10-epoxydecanoic acid. In chloroform, WF-3161 adopts a conformation with a possible gamma-turn between Leu NH and Aoe C = O and a cis amide bond between Leu and Pip. The torsion angles for this conformation are L-Aoe, phi, -95 degrees, psi, +85 degrees, omega, -155 degrees; D-Phe, phi, +120 degrees, psi, -80 degrees, omega, -175 degrees; L-Leu, phi, -145 degrees, psi, +35 degrees, omega, -10 degrees; L-Pip, phi, +20 degrees, psi, -135 degrees, omega, -170 degrees. The cis,trans,trans,trans amide bond sequence is related to the dimethyl sulfoxide conformation of chlamydocin, another cytostatic cyclic tetrapeptide.

Amino Acid Sequence↗
PubMed1986

Inhibition of cathepsin D by substrate analogues containing statine and by analogues of pepstatin.

Five new cathepsin D inhibitors were synthesized and tested as inhibitors of bovine cathepsin D. The compounds were derived by replacing a Phe-Phe dipeptidyl unit of a good cathepsin D substrate, Boc-Phe-Leu-Ala-Phe-Phe-Val-Leu-OR, with statine [3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid, Sta) or with Sta-Phe. The best inhibitor, Boc-Phe-Leu-Ala-(S,S)-Sta-Val-Leu-OMe, inhibited cathepsin D with a Ki value of 1.1 nM. In general, the more effective inhibitors were consistent with the proposal that statine functions as a replacement for a dipeptidyl unit. Thirty-five known pepstatin analogues also were evaluated as cathepsin D inhibitors. Substituents in the P4 and P3' positions are important for maximal inhibition of this aspartic proteinase, and the P4 substituent appears more important for inhibition of cathepsin D than for inhibition of other aspartic proteinases.

Amino Acid Sequence↗
PubMed1985

Inhibition of aspartic proteases by pepstatin and 3-methylstatine derivatives of pepstatin. Evidence for collected-substrate enzyme inhibition.

The synthesis of 10 analogues of pepstatin modified so that statine is replaced by 4-amino-3-hydroxy-3,6-dimethylheptanoic acid (Me3Sta) or 4-amino-3-hydroxy-3-methyl-5-phenylpentanoic acid (Me3AHPPA) residues is reported. Both the 3S,4S and 3R,4S diastereomers of each analogue were tested as inhibitors of the aspartic proteases, porcine pepsin, cathepsin D, and penicillopepsin. In all cases the 3R,4S diastereomer (rather than the 3S,4S diastereomer) of the Me3Sta and Me3AHPPA derivatives was found to be the more potent inhibitor of the aspartic protease (Ki = 1.5-10 nM for the best inhibitors), in contrast to the results obtained with statine (Sta) or AHPPA derivatives, where the 3S,4S diastereomer is the more potent inhibitor for each diastereomeric pair of analogues. The Me3Sta- and Me3AHPPA-containing analogues are only about 10-fold less potent than the corresponding statine and AHPPA analogues and 100-1000-fold more potent than the corresponding inhibitors lacking the C-3 hydroxyl group. Difference NMR spectroscopy indicates that the (3R,4S)-Me3Sta derivative induces conformational changes in porcine pepsin comparable to those induced by the binding of pepstatin and that the (3S,4S)-Me3Sta derivatives do not induce the difference NMR spectrum. These results require that the C-3 methylated analogues of statine-containing peptides must inhibit enzymes by a different mechanism than the corresponding statine peptides. It is proposed that pepstatin and (3S)-statine-containing peptides inhibit aspartic proteases by a collected-substrate inhibition mechanism. The enzyme-inhibitor complex is stabilized, relative to pepstatin analogues lacking the C-3 hydroxyl groups, by the favorable entropy derived when enzyme-bound water is returned to bulk solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartic Acid Endopeptidases↗
PubMed1985

Identification of oxygen nucleophiles in tetrahedral intermediates: 2H and 18O induced isotope shifts in 13C NMR spectra of pepsin-bound peptide ketone pseudosubstrates.

The synthetic ketone peptide analogue of pepstatin, isovaleryl-L-valyl-[3-13C]-(3-oxo-4S)-amino-6-methylheptanoyl-L-al anyl-isoamylamide is a strong inhibitor of aspartyl proteases. When the peptide is added to porcine pepsin in H2O at pH 5.1, the 13C NMR chemical shift of the ketone carbon moves from 208 ppm for the inhibitor in solution to 99.07 ppm when bound to the enzyme active site. In 2H2O the bound shift is 98.71 ppm, 0.36 ppm upfield. For the analogous experiment contrasting H216O and H218O, the 13C chemical shift was 0.05 ppm to higher field for the heavier isotope. These data show that water, and not an enzyme nucleophile, adds to the peptide carbonyl to yield a tetrahedral diol adduct in the enzyme-catalyzed reaction, and provide a method for differentiating between covalent and non-covalent mechanisms.

Animals↗
PubMed1984

Vitamin K-dependent carboxylase. Carboxylation of aspartyl residues to beta-carboxyaspartyl residues in synthetic substrates.

The rat liver microsomal vitamin K-dependent carboxylase has been shown to carboxylate the aspartyl residue in N alpha-t-butyloxycarbonylaspartic acid alpha-benzyl ester to a beta-carboxyaspartyl residue. The enzyme also carboxylates aspartyl residues in low molecular weight peptides to beta-carboxyaspartyl residues, but much less effectively than Glu residues in homologous peptides are carboxylated to gamma-carboxyglutamyl residues. The carboxylated product was identified by comparison to authentic beta-carboxyaspartate, by loss of 50% radioactivity incorporated from 14CO2 upon acid hydrolysis, and by identification of [14C]aspartic acid following acid hydrolysis. When peptides containing both aspartyl and glutamyl residues were used as substrates for the enzyme, the only carboxylated product detected was gamma-carboxyglutamic acid.

Animals↗
PubMed1984

Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes.

Amastatin [(2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl-L-valyl-L-valyl-L- aspartic acid] and bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine] are slow-binding, competitive inhibitors of aminopeptidase M (AP-M) with net inhibition constants (Ki) of 1.9 X 10(-8) and 4.1 X 10(-6) M, respectively. The effect of inhibitor structure on net Ki and on slow-binding inhibition was evaluated for analogues of both inhibitors on AP-M and leucine aminopeptidase (LAP). The (2S)-hydroxyl group contributes to the stabilization of a collision complex [EI], which is formed rapidly. In contrast, increasing the peptide chain length of the inhibitor produces more potent inhibitors as a consequence of a slower binding process. A statine analogue of amastatin [(3S,4S)-Sta-Val-Val-Asp] stimulated rather than inhibited LAP. AP-M binds tri- and tetrapeptide inhibitors more strongly than dipeptide inhibitors, whereas LAP binds dipeptide inhibitors more strongly. The difference in binding can be used to distinguish cytosolic from membrane-bound aminopeptidases.

Aminopeptidases↗