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D H Rich

Publications and source records attributed to D H Rich.

At least 55 records · Page 3Linked to original sources
PubMed1992

New intramolecularly quenched fluorogenic peptide substrates for the study of the kinetic specificity of papain.

A series of new substrates for determining the catalytic activity of cysteine proteinases is described. The rate of hydrolysis by papain was monitored by a fluorescence continuous assay based on internal resonance energy transfer using 5-[(2-aminoethyl)amino]naphtalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) as fluorescent donor and quenching acceptor, respectively, in peptides with the general structure: DABCYL-Lys-Phe-Gly-Xxx-Ala-Ala-EDANS. The substrates were used to evaluate the effect of amino acid structure in the S1' position on the kinetic parameters for papain catalyzed hydrolysis.

Amino Acid Sequence↗
PubMed1992

Synthesis of a novel polyurethane co-polymer containing covalently attached RGD peptide.

The synthesis of a novel polyurethane block co-polymer containing a covalently attached, well-oriented RGD (Arg-Gly-Asp) peptide was explored. A poly(tetramethylene oxide) (PTMO)-based polyurethane was synthesized, and a bimolecular nucleophilic substitution reaction was then employed to incorporate ethyl carboxylate groups onto the polymer backbone (i.e. carboxylated polyurethane). Elemental analysis was used to determine the extent of carboxylation. The hexapeptide H-Gly-Arg-Gly-Asp-Ser-Tyr-OH was coupled to the carboxylated polyurethane via the formation of an amide bond. The attachment of the peptide was controlled by a protection-deprotection scheme. Nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopies were used to monitor the reactions. Sakaguchi assay and amino acid analysis confirmed that the RGD-containing peptide was successfully grafted onto the carboxylated polyurethane. This reaction scheme provides a new route for grafting end-linked, bioactive peptides onto polyurethanes.

Amino Acid Sequence↗
PubMed1991

Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay.

The kinetic properties and substrate specificity of two well-characterized peptidyl prolyl cis-trans isomerases (PPIases), cyclophilin and the FK-506 binding protein (FKBP), have been previously examined [Fischer, G., Bang, H., Berger, E., & Schellenberger, A. (1984) Biochim. Biophys. Acta 791, 87-97; Harrison, R.K., & Stein, R.L. (1990) Biochemistry 29, 1684-1689; Albers, M.W., Walsh, C.T., & Schreiber, S. L. (1990) J. Org. Chem. 55, 4984-4986]. The chymotrypsin-coupled enzymatic assay employed in these studies suffers from two serious shortcomings. Due to the low equilibrium population of the X-cis-Pro-Phe-pNA isomer (the PPIase substrate), in conjunction with the low solubility of p-nitroaniline generated by chymotrypsin hydrolysis, substrate concentrations in the saturating region are not experimentally attainable. Secondly, the uncatalyzed cis-trans isomerization obscures the interpretation of the initial velocity. As a result of these limitations, the steady-state kinetic parameters (Km,Kcat) have not been determined. Here we introduce an improved version of the spectrophotometric assay and report for the first time the Michaelis constants and turnover numbers for both PPIases with established substrates. The improvements in the experimental conditions originate in a medium-induced increase in the equilibrium population of the cis X-Pro conformer and in conducting the assay at 0 degrees C to suppress the uncatalyzed thermal isomerization. In addition, we present a rigorous mathematical model of the spectrophotometric progress curves that accounts for the contributions of the residual background rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Isomerases↗
PubMed1991

Is cyclophilin involved in the immunosuppressive and nephrotoxic mechanism of action of cyclosporin A?

In this report we have approached two questions relating to the mechanism of action of cyclosporin A (CsA). First, we address whether the major cytosolic protein for CsA, cyclophilin, is directly involved in mediating the immunosuppressive activity of this drug, and, in particular, whether inhibition of this protein's peptidyl-prolyl cis-trans isomerase (PPIase) activity results in inhibition of murine T cell activation. Second, we ask whether the nephrotoxicity observed with CsA is related to inhibition of PPIase-dependent pathways in cells other than lymphocytes. Using a series of 61 cyclosporin analogues, we generally found a good correlation between cyclophilin binding and immunosuppressive activity for the majority of analogues analyzed. However, a number of compounds of distinct structural classes were found that could interact with cyclophilin but were much less immunosuppressive than expected. The inability of these analogues to inhibit lymphocyte activation could not be explained by their failure to enter the cell and bind to cyclophilin under the conditions used in the cellular assays. Surprisingly, a nonimmunosuppressive analogue, MeAla-6, which bound well to cyclophilin and was active as a PPIase inhibitor, did not induce renal pathology in vivo. Furthermore, another analogue, MeBm2t, which was immunosuppressive in vitro, possessed little or no activity as a PPIase inhibitor. These findings pose serious questions concerning a direct role of cyclosporin in mediating CsA's immunosuppressive and nephrotoxic activities. In addition, they raise doubts about whether PPIase has a direct function in lymphocyte signal transduction.

Amino Acid Isomerases↗
PubMed1990

Isotope-edited NMR of cyclosporin A bound to cyclophilin: evidence for a trans 9,10 amide bond.

The binding of a 13C-labeled cyclosporin A (CsA) analog to cyclophilin (peptidyl prolyl isomerase) was examined by means of isotope-edited nuclear magnetic resonance (NMR) techniques. A trans 9,10 peptide bond was adopted when CsA was bound to cyclophilin, in contrast to the cis 9,10 peptide bond found in the crystalline and solution conformations of CsA. Furthermore, nuclear Overhauser effects (NOEs) were observed between the zeta 3 and epsilon 3 protons of the methylleucine (MeLeu) residue at position 9 of CsA and tryptophan121 (Trp121) and phenylalanine (Phe) protons of cyclophilin, suggesting that the MeLeu9 residue of CsA interacts with cyclophilin. These results illustrate the power of isotope-edited NMR techniques for rapidly providing useful information about the conformations and active site environment of inhibitors bound to their target enzymes.

Amides↗
PubMed1990

Synthesis, conformation, and immunosuppressive activities of three analogues of cyclosporin A modified in the 1-position.

The syntheses of three new cyclosporin A (CsA) analogues that contain novel MeBmt derivatives in the 1-position are described. The MeBmt analogue that contains an additional methyl group on C4, (2S,3R,6E)-4,4-dimethyl-3-hydroxy-2-(N-methylamino)-6-octenoic acid (MeBm2t), was synthesized in four steps beginning with the reaction of Pmz-Sar-OtBu with (4E)-2,2-dimethyl-4-hexenal. The C4 desmethyl analogue of MeBmt, (2S,3R,6E)-3-hydroxy-2-(N-methylamino)-6-octenoic acid (MeBth), was synthesized in nine steps by a route based on the Sharpless chiral epoxidation procedure. The alkynyl derivative of MeBmt, (2S,3R,4R)-4-methyl-3-hydroxy-2-(N-methylamino)-6-octynoic acid (MeByt), was synthesized by a modification of the procedure described by Tung et al. for the synthesis of MeBmt. Each MeBmt analogue was protected as the N,O-acetonide and coupled with the hexapeptide Abu-Sar-MeLeu-Val-MeLeu-Ala-OBzl. The resulting heptapeptide was deprotected and coupled with Fmoc-D-Ala-MeLeu-MeLeu-MeVal-OH. The resulting undecapeptides were deprotected and cyclized to give the corresponding CsA analogues. Conformational analysis by 1D and 2D NMR methods was carried out for each analogue in chloroform, and the results are compared with the corresponding solution conformations of CsA and dihydrocyclosporin. The immunosuppressive activities of each analogue, determined in concanavalin A stimulated thymocytes, are lower than obtained for CsA. The results establish the important effect the methyl group and the double bond in MeBmt have on the solution conformation of the 1-position residue in CsA and on immunosuppressive activity.

Cyclosporins↗
PubMed1990

Synthesis, conformation, and immunosuppressive activity of cyclosporines that contain epsilon-oxygen (4R)-4-[(E)-butenyl]-4,N-dimethyl-L-threonine analogues in the 1-position.

A series of CsA analogues that contain novel epsilon-oxygen isosteres of (4R)-4-[(E)-butenyl]-4,N-dimethyl-L-threonine (MeBmt) in the 1-position were synthesized. The key steps for the syntheses of enantiomerically pure epsilon-oxygen MeBmt analogues 4-7 were based on the stereoselective epoxidation of cis-allylic alcohol derivative 12 with a peracid, followed by the application of a base-catalyzed intramolecular rearrangement of epoxyurethane 15, which was derived from the reaction of epoxy alcohol 14 and methyl isocyanate. All epsilon-oxygen MeBmt analogues have the same stereochemistry and the same functional groups as those on the alpha,beta,gamma-carbons of MeBmt except for the double bond of MeBmt, which is replaced by the -OCH2-group. The syntheses of the peptide portion of CsA analogues followed the strategy we reported previously. The immunosuppressive activities of CsA analogues 28a-e, determined by inhibition of concanavalin A stimulated thymocytes, showed that 28b, which has the closest structural resemblance to MeBmt, retains about 7-10% of activity of CsA, whereas the analogues 28a, 28c, and 28e retain about 2-5% activity. It is interesting to note that 28d, which has the larger benzyl group on the end of the side chain, is about 20-25% as active as CsA. Extensive conformational analyses by 1D and 2D NMR indicated that the conformation of the 33-membered peptide ring system for all CsA analogues was very similar to that of CsA. However, the NMR analyses revealed that the 1-position side chain of all these CsA analogues adopted a novel conformation in chloroform by forming a different intramolecular hydrogen bond between the beta-OH and the epsilon-oxygen of the same residue. The NMR data also suggest that the chloroform conformation of these CsA analogues is similar to the conformation observed in the crystal structure of CsA in that the 1-position side chain is folded across the cyclic peptide ring system.

Chemical Phenomena↗
PubMed1990

X-ray crystallographic structure of a complex between a synthetic protease of human immunodeficiency virus 1 and a substrate-based hydroxyethylamine inhibitor.

The structure of a crystal complex of the chemically synthesized protease of human immunodeficiency virus 1 with a heptapeptide-derived inhibitor bound in the active site has been determined. The sequence of the inhibitor JG-365 is Ac-Ser-Leu-Asn-Phe-psi[CH(OH)CH2N]-Pro-Ile-Val-OMe; the Ki is 0.24 nM. The hydroxyethylamine moiety, in place of the normal scissile bond of the substrate, is believed to mimic a tetrahedral reaction intermediate. The structure of the complex has been refined to an R factor of 0.146 at 2.4-A resolution by using restrained least squares with rms deviations in bond lengths of 0.02 A and bond angles of 4. The bound inhibitor diastereomer has the S configuration at the hydroxyethylamine chiral carbon, and the hydroxyl group is positioned between the active site aspartate carboxyl groups within hydrogen bonding distance. Comparison of this structure with a reduced peptide bond inhibitor-protease complex indicates that these contacts confer the exceptional binding strength of JG-365.

Amino Acid Sequence↗
PubMed1990

Synthesis and immunosuppressive activities of conformationally restricted cyclosporin lactam analogues.

Cyclosporin A (CsA, 1), an immunosuppressive cyclic undecapeptide, is known to adopt predominantly a single conformation in chloroform solution, characterized in part by a type II' beta-turn encompassing Abu-Sar-MeLeu-Val (residues 2-5). In order to evaluate whether this beta-turn is bound by the receptor, we previously had prepared a conformationally restricted beta-turn analogue, (delta-lactam) CsA (2), which was found to retain only weak immunosuppressive activity, a result that could indicate that steric hindrance between receptor and the lactam atoms in 2 diminished activity or that the type II' beta-turn is not a feature in the bioactive conformation of CsA. In an attempt to distinguish between these two possibilities, we have synthesized two new CsA analogues, (gamma-lactam) CsA (3) and (des-N-methyl-lactam) CsA (4), which contain less sterically demanding conformational restrictions. The immunosupressive activity of each analogue (4-13% and 7-17%, respectively, relative to CsA), measured in an assay that determined the inhibition of concanavalin A stimulated thymocytes, is essentially equipotent with that of the delta-lactam. The chemical shifts and temperature dependencies of the protons in analogues 3 and 4 are very similar to the corresponding protons in CsA and in 2, which suggest that the solution conformations of the small lactam analogues are very similar to that of the delta-lactam 2. The synthesis of the lactam components and the corresponding CsA derivatives is described. Reduction in the size of the lactam ring does not lead to enhanced immunosuppressive activity.

Amino Acid Sequence↗
PubMed1989

Affinity purification of the novel cysteine proteinase papaya proteinase IV, and papain from papaya latex.

A procedure is described for the purification of a previously undetected cysteine proteinase, which we have called papaya proteinase IV, from spray-dried latex of the papaya (Carica papaya) plant. The purification involves affinity chromatography on Gly-Phe-aminoacetonitrile linked to CH-Sepharose 4B, with elution by 2-hydroxyethyl disulphide at pH 4.5. The product thus obtained is a mixture of almost fully active papain and papay proteinase IV, which are then separated by cation-exchange chromatography. A preliminary characterization of papaya proteinase IV showed it to be very similar to chymopapain in both molecular size and charge. However, the new enzyme is immunologically distinct from the previously characterized cysteine proteinases of papaya latex. It also differs in its lack of activity against the synthetic substrates of the other papaya proteinases, in its narrow specificity against protein substrates and its lack of inhibition by chicken cystatin. Papaya proteinase IV is abundant, contributing almost 30% of the protein in spray-dried papaya latex, and contamination of chymopapain preparations with this enzyme may account for some of the previously reported heterogeneity of chymopapain.

Catalysis↗
PubMed1989

Inhibition of aminopeptidases by peptides containing ketomethylene and hydroxyethylene amide bond replacements.

Inhibitors of aminopeptidase enzymes have been prepared by the synthesis of peptide substrate analogues in which the scissile amide bond has been replaced with the hydrolytically stable ketomethylene (-COCH2-) and hydroxyethylene [-CH(OH)CH2-] functionalities. Two synthetic strategies were used to prepare the inhibitors, and the advantages and disadvantages of each are discussed. The synthesis of peptides that contain the hydroxyethylene isostere was complicated by competing lactone and lactam formation, and attempts to prepare free N-terminal dipeptide hydroxyethylene isostere derivatives were unsuccessful. All ketomethylene isosteres examined were weak inhibitors of both leucine aminopeptidase and aminopeptidase M. However, the ketomethylene inhibitor LysK(RS)Phe (58) (Ki = 4 nM) is a potent inhibitor comparable to the natural product, arphamenine A (ArgKPhe; Ki = 2.5 nM). Normal Michaelis-Menten kinetics for inhibition of membrane leucine aminopeptidase are observed in the absence of magnesium ion, but nonlinear kinetics were obtained in the presence of Mg2+.

Alkenes↗
PubMed1989

Synthesis of the novel pi-(benzyloxymethyl)-protected histidine analogue of statine. Inhibition of penicillopepsin by pepstatin-derived peptides containing different statine side-chain derivatives.

The synthesis of aspartic proteinase inhibitors derived from a new histidine side-chain analogue of statine (Sta), (3S,4S)-4-amino-3-hydroxy-5-(imidazol-4-yl)pentanoic acid (HiSta, 20), is reported. Boc-HiSta(BOM)-OMe (7) was prepared in 16% overall yield from Boc-His(pi-BOM)-OH via formation of the tetramic acid derivative 11 and stereoselective cis reduction with NaBH4 to the 4-hydroxy lactam 12. Removal of the Boc group from ester 7 (enantiomeric purity ee = 88-90%) and coupling to the tripeptide segment Iva-Val-Val-OH (13) by the DCC/HOBt preactivation method followed by hydrogenolytic removal of the pi-BOM group over Pd(OH)2 on carbon gave Iva-Val-Val-HiSta-OMe (16). This new peptide 16 is a very potent inhibitor of the fungal aspartic proteinase penicillopepsin (Ki = 4.5 x 10(-9) M) that is 10 times more active than the comparable Sta-containing inhibitor 3 and 2-3 times more potent than the new (3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA) analogue 17 (Ki = 1.5 x 10(-8) M). However, compound 16, which has an imidazole residue at the P1 position, is a significantly weaker inhibitor of the enzyme than the corresponding analogues with the lysine (5) and ornithine (6) side chains at P1. Considerations that led to the synthesis of 16 and the results of the enzyme kinetics are discussed in detail.

Amino Acids↗
PubMed1989

Synthesis, biological activity, and conformational analysis of (2S,3R,4S)-MeBmt1-cyclosporin, a novel 1-position epimer of cyclosporin A.

Cyclosporin A (CsA, 1), an immunosuppressive cyclic undecapeptide, contains a unique amino acid, (4R)-4-[(E)-butenyl]-4,N-dimethyl-L-threonine (MeBmt), that appears to be critically involved in the biological activity of CsA. In order to further explore the effect that structural elements in MeBmt have on the conformation and biological activity of CsA, the 4-epimer of MeBmt [(4S)-MeBmt, 2] and the corresponding CsA analogue [(4S)-MeBmt1-CsA, 3] have been synthesized. Biological assay using concanavalin A stimulated thymocytes indicated that (4S)-MeBmt1-CsA (3) has only 2-4% immunosuppressive activity relative to CsA. The NMR analysis by 1D and 2D NMR methods establishes the conformation of 3, of which the 33-membered cyclic peptide ring system in chloroform is very similar to that of CsA. However, the NMR analysis also reveals that the 1-position side chain orientation in (4S)-MeBmt1-CsA (3) is very different from that of CsA. Specifically, the (4S)-MeBmt alpha,beta-torsion angle (chi 1) has been rotated approximately 120 degrees relative to that of CsA, and the orientation of the butenyl side chain relative to the 33-membered peptide backbond is different. The orientation of the (4S)-MeBmt side chain is consistent with the possible conformations calculated for (4S)-MeBmt1-CsA (3) by using molecular mechanics (in vacuo) calculations. The conformational analysis suggests that the loss of biological activity for 3 results from an altered conformation of the 1-position side chain relative to the peptide backbond due to the changed chirality at C4 of MeBmt.

Chemical Phenomena↗
PubMed1988

Inhibition of arginine aminopeptidase by bestatin and arphamenine analogues. Evidence for a new mode of binding to aminopeptidases.

The synthesis and inhibition kinetics of a new, potent inhibitor of arginine aminopeptidase (aminopeptidase B; EC 3.4.11.6) are reported. The inhibitor is a reduced isostere of bestatin in which the amide carbonyl is replaced by the methylene (-CH2-) moiety. Analysis of the inhibition of arginine aminopeptidase by this inhibitor according to the method of Lineweaver and Burk yields an unusual noncompetitive double-reciprocal plot. The replot of the slopes versus [inhibitor] is linear (Kis = 66 nM), but the replot of the y intercepts (1/V) versus [inhibitor] is hyperbolic (Kii = 10 nM, Kid = 17 nM). These results provide evidence for a kinetic mechanism in which the inhibitor binds to the S1' and S2' subsites on the enzyme, not the S1 and S1' subsites occupied by dipeptide substrates. Furthermore, structure-activity data for a series of ketomethylene dipeptide isosteres in which the amide (-CONH-) of a dipeptide is replaced with the ketomethylene (-COCH2-) moiety show that the S1 and S1' subsites preferentially bind basic and aromatic side chains, respectively. These results are in agreement with the known substrate specificity of arginine aminopeptidase. The structure-activity data for several bestatin analogues, however, show that these compounds do not bind to the S1 and S1' sites of arginine aminopeptidase. A comparison of the data provides evidence that bestatin inhibits arginine aminopeptidase and possibly other aminopeptidases by binding to the S1' and S2' sites of the enzyme.

Aminopeptidases↗
PubMed1988

Synthesis, conformation, and immunosuppressive activity of a conformationally restricted cyclosporine lactam analogue.

Cyclosporine A (CsA, 1), an immunosuppressive cyclic undecapeptide, in apolar solvents adopts a II' beta-turn at the Sar3-MeLeu4 residues. [D-Proline3]Cs has been reported to be a nonimmunosuppressive analogue in which the II' beta-turn is retained. In order to determine if this loss of activity is caused by steric hindrance between the Cs analogue and its receptor or is caused by a change in the peptide conformation, an analogue that stabilizes a II' beta-turn has been synthesized, [lactam3,4]Cs. We also have studied the solution conformation of two other analogues, [D-MeAla3]Cs and [L-MeAla3]Cs. The conformations have been established by 1D difference NOE and 2D (NOESY or ROESY) NMR. The conformations of [lactam3,4]Cs and [D-MeAla3]Cs are indistinguishable from that of CsA in solution. [L-MeAla3]Cs was found to adopt a conformation with a cis amide bond between Sar3 and MeLeu4. The inhibition of concanavalin A stimulated thymocytes by CsA, [D-MeAla3]Cs, [L-MeAla3]Cs, and [lactam3,4]Cs gave IC50 values (nM) of 5, 6, 100, and 100, respectively. The weak immunosuppressive activity of [lactam3,4]Cs possessing the II' beta-turn suggests that the loss of activity for 4 is due to steric hindrance with the Cs receptor.

Animals↗