Phe Psi Research Articles

Bradykinin decreases T-kininogen synthesis in a rat hepatoma cell line: evidence of bradykinin B2-type receptors.

Using the rat H4-II-E-C3 hepatoma cell line, we investigated the presence of [125I][Tyr8]BK binding sites and the direct modulation of T-kininogen synthesis, an acute phase protein of inflammation, by bradykinin (BK) analogues. H4-II-E-C3 membrane preparations exhibited [125I][Tyr8]BK binding sites with a Kd of 4 nM and a Bmax of 120 fmol/mg of protein. Des-Arg9-BK showed no affinity (Ki > 10(-4) M) for these sites. The B2 metabolism-resistant and selective agonist [Phe8 psi (CH2-NH)Arg9]BK decreased the T-kininogen concentration in H4-II-E-C3 medium by 23% (p < 0.05). This effect was reversed by coincubation with the B2 antagonist HOE140. The B1 agonist Sar[D-Phe8]des-Arg9-BK and the B1 antagonist Lys[Leu8]des-Arg9-BK did not modify T-kininogen concentrations. The interaction between cytokines and kinins in the modulation of T-kininogen synthesis was also studied. Preincubation of hepatoma cells for 1 h with interleukin-1 alpha (IL-1 alpha) alone reduced T-kininogen concentrations by 37%, and this effect was blocked by co-addition of HOE140. Preincubation with interleukin-6 (IL-6) increased T-kininogen levels by threefold. Coincubation in the presence of the B2 agonist decreased this augmentation by 24%. The latter effect was reversed by co-addition of HOE140. None of the cytokines tested induced a response to the B1 agonist or antagonist under the experimental conditions studied. Overall, these results support the presence of a functional B2 receptor on H4-II-E-C3 cells that modulates T-kininogen synthesis. We suggest that the receptor is involved in vivo in a retroaction loop between kinins and T-kininogen production during inflammation. We speculate that BK could be a mediator in the modulation of acute phase protein synthesis by the cytokines IL-1 alpha and IL-6.

Development of Highly Potent and Selective Phosphinic Peptide Inhibitors of Zinc Endopeptidase 24-15 Using Combinatorial Chemistry

Several hundred phosphinic peptides having the general formula Z-(L,D)Phe psi (PO2CH2)(L,D)Xaa'-Yaa'-Zaa', where Xaa' = Gly or Ala and Yaa' and Zaa' represent 20 different amino acids, have been synthesized by the combinatorial chemistry approach. Peptide mixtures or individual peptides were evaluated for their ability to inhibit the rat brain zinc endopeptidases 24-15 and 24-16. Numerous phosphinic peptides of this series act as potent (Ki in the nanomolar range) mixed inhibitors of these two peptidases. However, our systematic and comparative strategy led us to delineate the residues located in P2' and P3' positions of the inhibitors that are preferred by these two peptidases. Thus, endopeptidase 24-15 exhibits a marked preference for inhibitors containing a basic residue (Arg or Lys) in the P2' position, while 24-16 prefers a proline in this position. The P3' position has less influence on the inhibitory potency and selectivity, both peptidases preferring a hydrophobic residue at this position. On the basis of these observations, we have prepared highly potent and selective inhibitors of endopeptidase 24-15. The Z-(L,D)Phe psi-(PO2CH2)(L,D)Ala-Arg-Met compound (mixture of the four diastereoisomers) displays a Ki value of 70 pM for endopeptidase 24-15. The most selective inhibitor of endopeptidase 24-15 in this series, Z-(L,D)Phe psi (PO2-CH2)(L,D)Ala-Arg-Phe, exhibits a Ki value of 0.160 nM and is more than 3 orders of magnitude less potent toward endopeptidase 24-16 (Ki = 530 nM). Furthermore, at 1 microM this selective inhibitor is unable to affect the activity of several other zinc peptidases, namely endopeptidase 24-11, angiotensin-converting enzyme, aminopeptidase M, leucine aminopeptidase, and carboxypeptidases A and B. Therefore, Z-(L,D)Phe psi (PO2CH2)(L,D)Ala-Arg-Phe can be considered as the most potent and specific inhibitor of endopeptidase 24-15 developed to date. This new inhibitor should be useful in assessing the contribution of this proteolytic activity in the physiological inactivation of neuropeptides known to be hydrolyzed, at least in vitro, by endopeptidase 24-15. Our study also demonstrates that the combinatorial chemistry approach leading to the development of phosphinic peptide libraries is a powerful strategy for discovering highly potent and selective inhibitors of zinc metalloproteases and should find a broader application in studies of this important class of enzymes.

Stereoselective synthesis of Xaaψ[CH2CH(OH)]Yaa dipeptidomimetics and their inclusion in HIV‐1 protease inhibitors

Two stereoselective syntheses of a new pseudodipeptide isostere, the right-hand hydroxyethylene dipeptidomimetic (Xaa psi[CH2CH(OH)]Yaa), are presented. In one method readily available amino acids are used as starting materials for Evans chiral aldol condensation chemistry. The second method relies on the synthesis of an anti-aldol product for the hydroxyethylene isostere via an E-selective ethyl hydrocinnamate enolization, and thus allows for the synthesis of isosteres having side chains other than those available from amino acids. Both methods are illustrated by the chiral synthesis of Boc-Phe psi[CH2CH(OH)]Phe. Two diastereomers, (S,S,R) and (S,R,R), are incorporated into an HIV-1 protease inhibitor template which yields potent inhibitors of HIV-1 protease when the pseudodipeptide isostere is Phe psi[CH(OH)CH2]Phe or Phe psi[CH(OH)CH(OH)]Phe. The resulting Phe psi[CH2CH(OH)]Phe-containing inhibitors possess modest potency.

MDL-101,562 blocks the stimulatory effects of bombesin and gastrin-releasing peptide on hypothalamic dopaminergic neurons.

The effects of central administration of bombesin, gastrin-releasing peptide (GRP) and a putative bombesin/GRP receptor antagonist [Phe8 psi[CH2S]Leu9]litorin (MDL-101,562) were determined on the activities of hypothalamic tuberoinfundibular and periventricular-hypophysial dopaminergic neurons in male rats. Dopaminergic neuronal activity was estimated by measuring concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence and the intermediate lobe of the posterior pituitary which contain terminals of these neurons. Intracerebroventricular injections of bombesin and GRP increased DOPAC concentrations in both the median eminence and intermediate lobe. MDL-101,562 had no effect on median eminence or intermediate lobe DOPAC concentrations per se, but blocked the stimulatory actions of bombesin and GRP on DOPAC concentrations in these regions. These results suggest that MDL-101,562 may be a useful pharmacologic tool in the study of the central actions of bombesin and related peptides.

Potent pseudopeptide bombesin‐like agonists and antagonists

Bombesin-like pseudopeptides have been synthesized, and certain physicochemical properties and biological activities have been examined. Bombesin and the related peptide litorin were modified at positions 13-14 and 8-9, respectively, with psi[CH2S] and psi[CH2N(CH3)]. [Phe13 psi[CH2S]Leu14]bombesin and [Phe8 psi[CH2S]-Leu9]litorin bound to the murine pancreatic bombesin/gastrin releasing peptide receptor with similar dissociation constants (Kd = 3.9 and 3.4 nM, respectively). Increased potency was achieved by oxidation of the thiomethylene ether to two diastereomeric sulfoxides (isomer I, Kd = 1.6 nM and isomer II, Kd = 0.89 nM. Further oxidation to the sulfone decreased potency ([Phe8 psi[CH2SO2]Leu9]litorin, Kd = 9.9 nM). All five analogs were receptor antagonists as determined by phosphatidylinositol turnover in murine pancreas. In contrast to these peptide backbone substitutions, a psi[CH2N(CH3)] at the 8-9 amide bond position resulted in an agonist. The analogs were compared with those of litorin (Kd = 0.1 nM) and [Leu9]litorin (Kd = 0.17 nM) by CD and fluorescence spectroscopy. The CD spectra demonstrated ordered conformation for all the peptides in TFE. Different conformations corresponding to agonist and antagonist peptides were suggested by CD. Based on the pH-dependence of the fluorescence spectra of the peptides in a zwitterionic detergent, two titratable groups were identified (pKa = 6.3 and 8.5). The lower pKa is found in the agonist analogs but not in the psi [CH2S]-containing antagonist.

Two-step binding mechanism for HIV protease inhibitors.

Rate constants for binding of five inhibitors of human immunodeficiency virus (HIV) protease were determined by stopped-flow spectrofluorometry. The two isomers of quinoline-2-carbonyl-Asn-Phe psi-[CH(OH)CH2N]Pro-O-t-Bu (R diastereomer = 1R; S diastereomer = 1S) quenched the protein fluorescence of HIV protease and thus provided a spectrofluorometric method to determine their binding rate constants. The dissociation rate constants for acetyl-Thr-Ile-Leu psi(CH2NH)Leu-Gln-Arg-NH2 (2), (carbobenzyloxy)-Phe psi[CH(OH)CH2N]Pro-O-t-Bu (3), and pepstatin were determined by trapping free enzyme with 1R as 2, 3, and pepstatin dissociated from the respective enzyme.inhibitor complex. Association rate constants of 1R, 2, and pepstatin were calculated from the time-dependent inhibition of protease-catalyzed hydrolysis of the fluorescent substrate (2-aminobenzoyl)-Thr-Ile-Nle-Phe(NO2)-Gln-Arg-NH2 (4). The kinetic data for binding of 1S to the protease fit a two-step mechanism. Kd values for these inhibitors were calculated from the rate constants for binding and were similar to the respective steady-state Ki values.

Synthesis and inhibitory activities against aminopeptidase B and enkephalin-degrading enzymes of ketomethylene dipeptide analogues of arphamenines.

Three ketomethylene pseudodideptide analogues [(S)Lys psi(COCH2)(R and S)Phe (14 or 15 and 15 or 14) and (S)Lys psi(COCH2)(xi Trp (19)] of natural arphamenine A [(S)Arg psi(COCH2(R,S)Phe (1)] were easily prepared by a route involving two successive main reactions: a malonic ester alkylation with Z-protected lysine iodomethyl ketone and the introduction of a benzyl or (indol-3-yl)methyl moiety in position 2 of the resulting 4-ketodiester. The isomer of 1 with reversed sequence, (S)Phe psi(COCH2)(R,S)Arg (22) was synthesized by guanidylation and subsequent deprotection of Z-(S)Phe psi(COCH2)(R,S)Orn. The inhibitory effects of compounds 14, 15, 19, and 22, and the related ketomethylene dipeptides (S)Ala psi(COCH2)(R,S)Phe (3), (S)Phe psi(COCH2)(R,S)X [X = Ala (4), Orn (5)] and (S)Trp psi(COCH2)(R,S)Y [Y = Orn (6), Lys (7), Arg (8)] on aminopeptidase B (AP-B), and enkephalin-degrading enzymes [aminopeptidase N (APN) and neutral endopeptidase (NEP)] were compared with that of the model compound 1.

Pseudopeptide analogs of substance P and leucine enkephalinamide containing the .psi.(methyleneoxy) modification: synthesis and biological activity

The isosteric methyleneoxy psi (CH2O) function was employed as a novel peptide-bond surrogate and incorporated into sequences of two neuropeptides, substance P (SP) and enkephalin. A pseudopeptide analogue [pGlu6,Phe8 psi(CH2O)Gly9]SP6-11 (7) of SP related C-terminal hexapeptide [pGlu6]SP6-11 and two pseudopeptide analogues of [Leu5]enkephalinamide, [Tyr1 psi (CH2O)Gly2, Leu5] enkephalinamide (11) and [Gly2 psi (CH2O)-Gly3, Leu5]enkephalinamide (17), were synthesized. The N alpha-protected pseudodipeptidic units were incorporated in the appropriate peptide sequences by using conventional coupling methods in solution. Compound 7 was a potent agonist (EC50 = 4.8 nM) of substance P as compared to the parent peptide [pGlu6]SP6-11 (EC50 = 1.2 nM), in stimulating contraction of the isolated guinea pig ileum (GPI). Analogue 7 was more potent on the neuronal (NK-3) than on the muscular (NK-1) tachykinin receptors in the GPI as shown by the ratio of activities, EC50 (NK-1)/EC50 (NK-3) = 3.16, thus displaying an improved selectivity for the NK-3 tachykinin receptor subtype as compared to that of [pGlu6]SP6-11, EC50 (NK-1)/EC50 (NK-3) = 0.44. In the rat vas deferens (RVD) assay, a typical NK-2 system, the pseudopeptide analogue 7 was (EC50 = 2 microM) 10-fold more potent than the parent peptide and 20-fold less potent than eledoisin, an NK-2 selective tachykinin. The pseudopeptide enkephalin analogue 17 had low biological activity when tested in the electrically induced GPI (EC50 = 2.3 microM) and was inactive in the mouse vas deferens (MVD) assay. In the rat brain membrane (RBM) binding assay analogue 17 had low affinity (in the micromolar range) for both the mu and delta binding sites. In contrast, analogue 11 was a potent enkephalin agonist (EC50 = 30 nM), being equipotent to [D-Ala2, Leu5]enkephalinamide (DALE) in the GPI assay. In the MVD, analogue 11 showed a substantially reduced activity (EC50 = 92 nM), being about 10-fold less potent than DALE. In the RBM binding assay analogue 11 showed high affinity (in the nanomolar range) for both mu and delta binding sites with increased selectivity for the delta sites as shown by the ratio of the apparent affinities for both receptors, Ki (delta)/Ki (mu) = 2.1. The contribution of the modified peptide bonds in the mode of interaction of SP and enkephalin at their corresponding receptors is discussed.

Specificity and inhibition of proteases from human immunodeficiency viruses 1 and 2.

Highly purified, recombinant preparations of the virally encoded proteases from human immunodeficiency viruses (HIV) 1 and 2 have been compared relative to 1) their specificities toward non-viral protein and synthetic peptide substrates, and 2) their inhibition by several P1-P1' pseudodipeptidyl-modified substrate analogs. Hydrolysis of the Leu-Leu and Leu-Ala bonds in the Pseudomonas exotoxin derivative, Lys-PE40, is qualitatively the same for HIV-2 protease as published earlier for the HIV-1 enzyme (Tomasselli, A. G., Hui, J. O., Sawyer, T. K., Staples, D. J., FitzGerald, D. J., Chaudhary, V. K., Pastan, I., and Heinrikson, R. L. (1990) J. Biol. Chem. 265, 408-413). However, the rates of cleavage at these two sites are reversed for the HIV-2 protease which prefers the Leu-Ala bond. The kinetics of hydrolysis of this protein substrate by both enzymes are mirrored by those obtained from cleavage of model peptides. Hydrolysis by the two proteases of other synthetic peptides modeled after processing sites in HIV-1 and HIV-2 gag polyproteins and selected analogs thereof demonstrated differences, as well as similarities, in selectivity. For example, while the two proteases were nearly identical in their rates of cleavage of the Tyr-Pro bond in the HIV-1 gag fragment, Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val, the HIV-1 protease showed a 64-fold enhancement over the HIV-2 enzyme in hydrolysis of a Tyr-Val bond in the same template. Accordingly, the HIV-2 protease appears to have a different specificity than the HIV-1 enzyme; it is better able to hydrolyze substrates with small amino acids in P1 and P1', but is variable in its rate of hydrolysis of peptides with bulky substituents in these positions. In addition to these comparisons of the two proteases with respect to substrate specificity, we present inhibitor structure-activity data for the HIV-2 protease. Relative to P1-P1' statine or Phe psi [CH2N]Pro-modified pseudopeptidyl inhibitors, compounds having Xaa psi[CH(OH)CH2]Yaa inserts were found to show significantly higher affinities to both enzymes, generally binding from 10 to 100 times stronger to HIV-1 protease than to the HIV-2 enzyme. Molecular modeling comparisons based upon the sequence homology of the two enzymes and x-ray crystal structures of HIV-1 protease suggest that most of the nonconservative amino acid replacements occur in regions well outside the catalytic cleft, while only subtle structural differences exist within the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Substrate analog inhibition and active site titration of purified recombinant HIV-1 protease

The aspartyl protease of human immunodeficiency virus 1 (HIV-1) has been expressed in Escherichia coli at high levels, resulting in the formation of inclusion bodies which contain denatured insoluble aggregates of the protease. After solubilization of these inclusion bodies in guanidinium chloride, the protease was purified to apparent homogeneity by a single-step reverse-phase HPLC procedure. The purified, but inactive, protein was denatured in 8 M urea and refolded to produce the active protease. Enzyme activity was demonstrated against the substrate H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-OH, modeled after the cleavage region between residues 128 and 135 in the HIV gag polyprotein. With this substrate, a Vmax of 1.3 +/- 0.2 mumol/(min.mg) and KM of 2.0 +/- 0.3 mM were determined at pH 5.5. Pepstatin (Iva-Val-Val-Sta-Ala-Sta-OH) and substrate analogues with the Tyr-Pro residues substituted by Sta, by Phe psi [CH2N]Pro, and by Leu psi [CH(OH)CH2]Val inhibited the protease with KI values of 360 nM, 3690 nM, 3520 nM, and less than 10 nM, respectively. All were competitive inhibitors, and the tightest binding compound provided an active site titrant for the quantitative determination of enzymatically active HIV-1 protease.

Dehydro ketomethylene and ketomethylene analogs of substance P: synthesis and biological activity

The synthesis of dehydro keto methylene and keto methylene analogues of substance P using classical peptide synthesis is described. The following analogues were prepared: [pGlu6,Gly9psi(COCH2)(RS)Leu10]SP6-11 (4) and [pGlu6,-(RS)Phe7 psi(COCH2)(RS)Phe8]SP6-11 (8). The use of an improved deprotection scheme employing Meerwein's reagent (Et3OBF4) made possible the syntheses of the novel dehydro keto methylene analogue [pGlu6,(RS)Phe7 psi-(COCH2)delta(E)Phe8]SP6-11 (26) adn the tetrapeptide analogue [pGlu6,(RS)Phe8 psi(COCH2)Gly9]SP6-9(-OMe) (23). Compound 4 was a weak agonist in provoking contractions of the guinea pig ileum. Compound 26 was a potent inhibitor of SP degradation in rat hypothalamus preparations, with an IC50 value of 1.8 microM.

ChemInform Abstract: Ketomethylene Pseudopeptide Analogues of Substance P: Synthesis and Biological Activity.

Two pseudopeptide analogues [Bz-(RS)Phe8 psi (COCH2)Gly9]SP8-11 (I) and [pGlu6,(RS)Phe8 psi (COCH2)Gly9]SP6-11 (II) of the substance P related C-terminal hexapeptide [pGlu6]SP6-11 were prepared as follows. The pseudodipeptidic unit H(RS)Phe psi (COCH2)GlyOH was synthesized by using a modified Dakin-West reaction between Bz-Phe-OH and monomethyl succinoyl chloride. The N alpha-protected pseudopeptidic unit was then incorporated into the appropriate peptide by using various coupling methods. The two pseudopeptide analogues were purified, characterized, and tested for their biological activity and inhibitory effect on SP degrading enzymes. Analogue II was a full agonist contracting the isolated guinea pig ileum with a potency of 70% compared to the parent hexapeptide [pGlu6]SP6-11. It was also a potent inhibitor of SP degrading activity in rat diencephalon membranes with a Ki of 20 microM whereas analogue I was a weak inhibitor.

Ketomethylene pseudopeptide analogs of substance P: synthesis and biological activity

Two pseudopeptide analogues [Bz-(RS)Phe8 psi (COCH2)Gly9]SP8-11 (I) and [pGlu6,(RS)Phe8 psi (COCH2)Gly9]SP6-11 (II) of the substance P related C-terminal hexapeptide [pGlu6]SP6-11 were prepared as follows. The pseudodipeptidic unit H(RS)Phe psi (COCH2)GlyOH was synthesized by using a modified Dakin-West reaction between Bz-Phe-OH and monomethyl succinoyl chloride. The N alpha-protected pseudopeptidic unit was then incorporated into the appropriate peptide by using various coupling methods. The two pseudopeptide analogues were purified, characterized, and tested for their biological activity and inhibitory effect on SP degrading enzymes. Analogue II was a full agonist contracting the isolated guinea pig ileum with a potency of 70% compared to the parent hexapeptide [pGlu6]SP6-11. It was also a potent inhibitor of SP degrading activity in rat diencephalon membranes with a Ki of 20 microM whereas analogue I was a weak inhibitor.