Two polymorphs of a covalent complex between papain and a diazomethylketone inhibitor.

The three-dimensional structure of two polymorphs of a ZLFG-CH2-papain covalent complex has been determined by X-ray crystallography. The structures indicate that: (i) the methylene carbon atom of the inhibitor is covalently bound to the Sgamma atom of Cys25 of papain; (ii) the hydrophobic S2 pocket formed by Pro68, Val133, Val157, and Asp158 is occupied by the inhibitor's phenylalanyl P2 side chain; (iii) extensive hydrogen bonding and hydrophobic interactions are responsible for the interaction of the inhibitor with the enzyme. Comparison with similar structures suggests that in covalent complexes preservation of main chain-main chain interactions between the enzyme and the inhibitor may have higher priority than the P-S interactions.

Structural studies of cysteine proteases and their inhibitors.

Cysteine proteases (CPs) are responsible for many biochemical processes occurring in living organisms and they have been implicated in the development and progression of several diseases that involve abnormal protein turnover. The activity of CPs is regulated among others by their specific inhibitors: cystatins. The main aim of this review is to discuss the structure-activity relationships of cysteine proteases and cystatins, as well as of some synthetic inhibitors of cysteine proteases structurally based on the binding fragments of cystatins.

Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping.

The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The dimerization occurs through three-dimensional domain swapping, a mechanism for forming oligomeric proteins. The reconstituted monomer-like domains are similar to chicken cystatin except for one inhibitory loop that unfolds to form the 'open interface' of the dimer. The structure explains the tendency of human cystatin C to dimerize and suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe 'conformational disease' is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage and death in young adults. The structure of the three-dimensional domain-swapped dimers shows how the L68Q mutation destabilizes the monomers and makes the partially unfolded intermediate less unstable. Higher aggregates may arise through the three-dimensional domain-swapping mechanism occurring in an open-ended fashion in which partially unfolded molecules are linked into infinite chains.

A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro.

Human cystatin C is a cysteine proteinase inhibitor belonging to the cystatin superfamily, which previously has been shown to inhibit bone resorption in bone organ culture. The aminoterminal segment, Arg(8)-Leu(9)-Val(10)-Gly(11) (RLVG), of the single polypeptide chain of cystatin C constitutes an essential part of its inhibitory center. In the present study, the effect of benzyloxycarbonyl-Arg(8)-Leu(9)-Val(10)-Gly(11)-diazomethane (Z-RLVG-CHN(2)) on bone resorption in vitro was compared with the effects of cystatin C and calcitonin. Bone resorption was assessed by the release of (45)Ca and (3)H from mouse calvarial bones prelabeled with [(45)Ca]CaCl(2) and [(3)H]-proline, respectively. Z-RLVG-CHN(2) concentration-dependently inhibited the release of (45)Ca and (3)H in bones stimulated by parathyroid hormone (PTH), with half-maximal inhibition obtained at 1 micromol/L. The inhibitory actions of Z-RLVG-CHN(2) and cystatin C were persistent, whereas action induced initially by calcitonin was lost with time. The inhibition caused by Z-RLVG-CHN(2) and cystatin C on PTH-stimulated (45)Ca release was observed after 6 h, whereas inhibition by calcitonin was seen already after 2 h. In contrast, the inhibitory effects of Z-RLVG-CHN(2) and cystatin C, as well as that of calcitonin, on (3)H release was seen already after 2 h. Z-RLVG-CHN(2), in which the reactive carboxyterminal diazomethane was substituted by nonreactive groups [-OH, -NH(2), or -N(CH(3))(2)], resulted in peptidyl derivatives, which, in contrast to Z-RLVG-CHN(2) and cystatin C, inhibited neither cysteine proteinases nor bone resorption. In contrast to wild-type cystatin C, recombinant human cystatin C with Gly substitutions for residues Arg(8), Leu(9), Val(10), and Trp(106), and with low or nonexistent affinity for cysteine proteinases, did not display any inhibitory effect on bone resorption. These data strongly indicate that Z-RLVG-CHN(2) inhibits bone resorption in vitro by a mechanism that seems primarily to be due to an inhibition of bone matrix degradation via cysteine proteinases. The data also corroborate the hypothesis that cystatin C inhibits bone resorption by virtue of its cysteine proteinase inhibitory capacity.

Expression of a selenomethionyl derivative and preliminary crystallographic studies of human cystatin C.

Human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of papain-like mammalian proteases, has been produced in its full-length form by recombinant techniques and crystallized in two polymorphic forms: cubic and tetragonal. A selenomethionyl derivative of the protein, obtained by Escherichia coli expression and with complete Met-->Se-Met substitution confirmed by mass spectrometry, amino-acid analysis and X-ray absorption spectra, was crystallized in the cubic form. A truncated variant of the protein, lacking ten N-terminal residues, has also been crystallized. The crystals of this variant are tetragonal and, like the two polymorphs of the full-length protein, contain multiple copies of the molecule in the asymmetric unit, suggesting oligomerization of the protein.

Binding modes of a new epoxysuccinyl-peptide inhibitor of cysteine proteases. Where and how do cysteine proteases express their selectivity?

Papain from Carica papaya, an easily available cysteine protease, is the best-studied representative of this family of enzymes. The three dimensional structure of papain is very similar to that of other cysteine proteases of either plant (actinidin, caricain, papaya protease IV) or animal (cathepsins B, K, L, H) origin. As abnormalities in the activities of mammalian cysteine proteases accompany a variety of diseases, there has been a long-lasting interest in the development of potent and selective inhibitors for these enzymes. A covalent inhibitor of cysteine proteases, designed as a combination of epoxysuccinyl and peptide moieties, has been modeled in the catalytic pocket of papain. A number of its configurations have been generated and relaxed by constrained simulated annealing-molecular dynamics in water. A clear conformational variability of this inhibitor is discussed in the context of a conspicuous conformational diversity observed earlier in several solid-state structures of other complexes between cysteine proteases and covalent inhibitors. The catalytic pockets S2 and even more so S3, as defined by the pioneering studies on the papain-ZPACK, papain-E64c and papain-leupeptin complexes, appear elusive in view of the evident flexibility of the present inhibitor and in confrontation with the obvious conformational scatter seen in other examples. This predicts limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit the minute differences in the catalytic pockets of various members of this family. A simultaneous comparison of the three published proenzyme structures suggests the enzyme's prosegment binding loop-prosegment interface as a new potential target for selective inhibitors of papain-related thiol proteases.

Identification of binding domains of pituitary adenylate cyclase activating polypeptide (PACAP) for its type 1 receptor by photoaffinity labeling.

Structure-function studies and photoaffinity labeling experiments were performed to identify residues and domains of PACAP involved in the interaction with PACAP receptors. For this purpose, a series of photoreactive analogues of PACAP(1-27) containing a photoreactive benzophenone (BP) residue in different peptide structural domains were utilized to analyze the interaction of PACAP(1-27) with pig PACAP type 1 receptors. Five PACAP derivatives were created with a photoreactive amino acid in the following peptide domains: either the disordered N-terminal or the helical C-terminal domain or a short loop region within the C-terminal helical domain of the peptide. Their receptor binding properties and efficiencies were tested on pig brain PACAP receptors. The results indicate the importance of the helical C-terminal domain of PACAP(1-27) for receptor binding affinity. Monoiodination of the photoreactive analogues did not change their binding affinities. Experiments with pig brain membranes demonstrated that the 125I-labeled photoreactive analogues specifically label a protein band of M(r) 66,000. The efficiency of photoreactive labeling differed for the various analogues. These findings suggest that Tyr22 and Lys15 in PACAP (1-27) are located in or close to the hormone binding site of the PACAP type 1 receptor. The results provide evidence that the alpha-helical C-terminal region of PACAP is directly involved in receptor binding.

Fluorescence study of neurohypophyseal hormones and their analogues. Distance distributions in a series of arginine-vasopressin analogues.

Analogues of arginine-vasopressin (AVP) in which substitution of the proline residue in position 7 (by either sarcosine or N-methylalanine) combined with replacement of the cysteine residue in position 1 were the subject of a fluorescence and molecular mechanics study. We obtained two groups of analogues: selective antidiuretic agonists (cysteine or beta-mercaptopropionic acid in position 1) and pressor and uterotonic antagonists (deaminopenicillamine or beta-mercapto-beta, beta-cyclopentamethylenepropionic acid in position 1). Using frequency-domain measurements of fluorescence resonance energy transfer (FRET) we estimated the distance distribution between the phenolic ring of Tyr2 and the disulphide bridge Cys1-Cys6. We also analyzed acrylamide quenching of tyrosyl fluorescence to determine the exposure of the tyrosyl ring to the solvent. Results from fluorescence experiments were compared with those from Monte Carlo simulation (ECEPP/3 force-field).

Crystallization and preliminary crystallographic studies of a new crystal form of papain from Carica papaya.

A new crystal form of papain from the latex of Carica papaya, complexed with an inhibitor (Z-Arg-Leu-Val-Gly-CHN2) was obtained by the vapor-diffusion method using a methanol/ethanol mixture as a precipitant. The slat-like crystals are monoclinic, space group P2(1), with unit cell parameters a = 42.6 A, b = 49.8 A, c = 50.5 A, A = 111.9 degrees, and contain one molecule in the asymmetric unit. The crystals are stable in the X-ray beam and diffract beyond 1.8 A. A molecular model has been placed in the unit cell by molecular replacement.

1H-NMR and 13C-NMR investigation of complexes of Mn2+ with ocytocin analogues in (2H6)dimethylsulfoxide.

Several ocytocin analogues were synthesised by substitution of the Pro residue with sarcosine or N-methylalanine, the glutamine residue with threonine and one of the cysteines with 2-mercaptopropionic acid. All the derivatives were investigated by NMR in dimethylsulfoxide solutions and evidence was obtained for similar preferred conformations in the solution free state. All peptides were shown to form complexes with Mn2+ in solution by the strong paramagnetic effects experienced by several proton resonances. Two structures could be determined, one formed by peptides containing threonine and the other by the remaining peptides. The two structures were delineated by molecular modelling using the Mn(2+)-proton distances obtained by NMR as restraints.

Exploration of the conformational space of oxytocin and arginine-vasopressin using the electrostatically driven Monte Carlo and molecular dynamics methods.

Conformational analysis of the neurohypophyseal hormones oxytocin (OT) and arginine-vasopressin (AVP) has been carried out using two different computational approaches and three force fields, namely by the Electrostatically Driven Monte Carlo (EDMC) method, with the Empirical Conformational Energy Program for Peptides (ECEPP/3) force field or with the ECEPP/3 force field plus a hydration-shell model, and by simulated-annealing molecular dynamics with the Consistent Valence Force Field (CVFF). The low-energy conformations obtained for both hormones were classified using the minimal-tree clustering algorithm and characterized according to the locations of beta-turns in the cyclic moieties. Calculations with the CVFF force field located conformations with a beta-turn at residues 3 and 4 as the lowest energy ones both for OT and for AVP. In the ECEPP/3 force field the lowest energy conformation of OT contained a beta-turn at residues 2 and 3, conformations with this location of the turn being higher in energy for AVP. The latter difference can be attributed to the difference in the size of the side chain in position 3 of the sequences: the bulkier phenylalanine residue of AVP in combination with the bulky Tyr2 residue hinders the formation of a turn at residues 2 and 3. Conformations of OT and AVP with a turn at residues 3,4 were in the best agreement with the x-ray structures of deaminooxytocin and pressinoic acid (the cyclic moiety of vasopressin), respectively, and with the nmr-derived distance constraints. Generally, the low-energy conformations obtained with the hydration-shell model were in a better agreement with the experimental data than the conformations calculated in vacuo. It was found, however, that the obtained low-energy conformations do not satisfy all of the nmr-derived distance constraints and the nuclear Overhauser effect pattern observed in nmr studies can be fully explained only by assuming a dynamic equilibrium between conformations with beta-turns at residues 2,3, 3,4, and 4,5. The low-energy structures of OT with a beta-turn at residues 2,3 have the disulfide ring conformations close to the model proposed recently for a potent bicyclic antagonist of OT [M. D. Shenderovich et al. (1994) Polish Journal of Chemistry, Vol. 25, pp. 921-927], although the native hormone differs from the bicyclic analogue by the conformation of the C-terminal tripeptide. This finding confirms the hypothesis of different receptor-bound conformations of agonists and antagonists of OT.

Conformational aspects of differences in requirements for oxytocin and vasopressin receptors.

Conformational energy calculations were carried out on three non-peptide antagonists of oxytocin and vasopressin: penicilide (compound 1; selective for oxytocin receptors), 1-¿1-[4-(3-acetylaminopropoxy(benzoyl]-4-piperidyl¿-3,4-dihydro-2( 1H)-quinoline (compound 2; selective for vasopressin V1 receptors) and 5-dimethylamino-1-¿(2-methylbenzylamino)-benzoyl¿-2,3,4,5-tetrahyd ro-1H-benzapine (compound 3; selective for vasopressin V2 receptors). The obtained low-energy conformations of compound 1 were compared with low-energy conformations of oxytocin (OT) and low-energy conformations of compounds 2 and 3 were compared with low-energy conformations of arginine vasopressin (AVP). It was found that the affinity of the non-peptide antagonists and their selectivity for vasopressin and oxytocin receptors is probably connected with mimicking the aromatic rings of the Tyr2 and the Phe3 residues of AVP in the case of compounds 2 and 3 and with mimicking the Tyr2 residue and the Ile3 or Leu8 residues of OT by the outer benzene ring and the isobutyl group of compound 1. Application of the results in the design of more potent non-peptide antagonists of OT and VP is also discussed.

An iodinated vasopressin (V1) antagonist blocks flank marking and selectively labels neural binding sites in golden hamsters.

An arginine-vasopressin (AVP) derivative, [d(CH2)5,Sar7]AVP (SAVP), has been characterized as an antagonist to vasopressin V1 receptors. Using AVP-dependent flank-marking behavior as a bioassay, it was possible to verify that iodinated SAVP (I-SAVP) retains biological activity within the central nervous system, as the antagonist blocked the behavior. Furthermore, 125I-SAVP was used to localize specific V1 binding sites in the brain. The resulting binding was localized to discrete anatomical sites, and highly specific to V1-like receptors. While we confirmed previous findings using 3H-AVP in golden hamsters, we also identified binding in many areas previously unreported (e.g., arcuate and paraventricular nuclei of the hypothalamus, tenia tecta, posteromedial cortical nucleus of the amygdala, and zona incerta), suggesting that 125I-SAVP provides a greater level of resolution. In addition, specific binding was observed in the lateral septum, anterior hypothalamus, and midbrain central gray, areas that have previously been shown to trigger flank marking in response to AVP microinjection. The presence of AVP binding sites in limbic and mesencephalic areas involved in the regulation of flank marking suggests that this neuropeptide may play an important role as a neurotransmitter at multiple levels in the neural circuits controlling this behavior.

Potent V2/V1a vasopressin antagonists with C-terminal ethylenediamine-linked retro-amino acids.

We report the solid-phase synthesis and antagonistic potencies of 25 analogues (1-25) of [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-O-ethyl-D-tyrosine,4-valine]arginine-vasopressin (d(CH2)5D-Tyr(Et)2-VAVP) (A) and of the related Ile4 (D) and [D-Phe2,Ile4] (E) analogues, potent antagonists of the antidiuretic (V2-receptor) and of the vasopressor (V1a-receptor) responses to arginine-vasopressin (AVP). Six of these peptides (1, 13, 17, 19, 21, and 23) have the Pro-Arg-Gly-NH2 tripeptide side chain fully or partially replaced or extended by ethylenediamine (Eda). The remaining 19 peptides have L- or D-amino acids retrolinked to these six C-terminal Eda peptides. Peptides 1, 13, 17, and 19 all have the ring structure of (A). Their side-chain structures are as follows: 1, Eda; 13, Pro-Eda; 17, Pro-Arg-Eda; 19, Arg-Gly-Eda. Peptide 21 is the Pro-Arg-Eda analogue of D; peptide 23 is the Pro-Arg-Gly-Eda analogue of E. Peptide 2 is the retro-Arg analogue of 1. Its side-chain structure is Eda<--Arg. Peptides 3-6 are analogues of 2 which have the D-Tyr-(Et)2 residue replaced by L-Tyr(Et)2 (3), D-Phe2 (4), D-Ile2 (5), or D-Leu2 (6), respectively. Peptides 7-12 are analogues of 2 which have the C-terminal retro-Arg replaced in retrofashion by D-Arg (7), Gly (8), Orn (9), D-Orn (10), D-Lys (11), or Arg-Arg (12). Peptides 14-16 have D-Orn (14), D-Lys (15), and D-Arg (16) retrosubstituted to peptide 13. Peptides 18, 20, and 22 are the retro-Arg-substituted analogues of 17, 19, and 21, respectively. Peptides 24 and 25 have Val and D-Val in retrolinkage with 23, respectively. All 25 peptides were examined for agonistic and antagonistic potencies in AVP V2/V1a assays. With the exception of peptides 5 and 6, all exhibit potent anti-V1a antagonism, with anti-V1a pA2 values in the range 7.64-8.33.(ABSTRACT TRUNCATED AT 400 WORDS)

Vascular action of natural vasopressin-like peptides in isolated rat tail artery.

The literature data regarding the vasoconstriction potency of natural vasopressin-like peptides are contradictory. The cumulative concentration-response curve for arginine-vasopressin (AVP), lysine-vasopressin (LVP), arginine-vasotocin (AVT), lysine-vasotocin (LVT) and phenypressin (PHP) on the isolated rat tail artery was determined. The potency rank of these peptides on the vascular smooth muscle of the rat tail artery was the following: AVP greater than LVP greater than AVT = LVT greater than PHP. The results are discussed in comparison to the data in the literature.

MNDO study of the mechanism of the inhibition of cysteine proteinases by diazomethyl ketones.

Diazomethyl ketones are one of the most effective irreversible inhibitors of cysteine proteinases and are therefore very important in drug design. In the present study a mechanism of inactivation is proposed based on the results of model MNDO calculations of the possible pathways. It was found that the mercaptide nucleophile, on approaching the carbonyl carbon as in the catalytic reaction path, binds to the inner diazo nitrogen. The intermediate thus formed can rearrange giving a stable product, beta-thioketone, and molecular nitrogen, with a considerable energy gain. The energy barrier to this process is equal to 36.9 kcal/mol, and corresponds to a pyramidal transition state with the vertex at the methylene carbon and the base formed by the carbonyl, thiol, and diazo groups. The energy barrier can be lowered on deprotonation of the intermediate. Based on the results obtained it was concluded that good irreversible inhibitors of cysteine proteases must fulfil two structural requirements: i) the dimensions and charge distribution must be similar to those of the peptide bond and ii) a second electrophilic center must be present in the neighbourhood of the carbonyl carbon. These are requirements which are satisfied by other strong cysteine proteinase inhibitors: beta-chloroketones and beta-ketooxiranes.

Investigation of the inhibition pathway of glucosamine synthase by N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid by semiempirical quantum mechanical and molecular mechanics methods.

Glucosamine synthase (E.C. 2.6.1.16) is a promising target in antifungal drug design. It has been reported that its potent inhibitor, N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP), inactivates the enzyme by the Michael addition of the S-H group to the FMDP molecule followed by cyclisation reactions. In this study we have investigated, by means of semiempirical MNDO, PM3 and molecular mechanics methods, the energetics and kinetic possibility of the formation of various stereoisomers of the products of cyclisation of the Michael addition products detected experimentally. It was found that the substituted 1,4-thiazin-3-one can be formed in one step under alkaline conditions; the stereoisomers of this compound predicted to be the most stable on the basis of theoretical calculations are also the dominant ones in reality.

Conformation of [8-arginine]vasopressin and V1 antagonists in dimethyl sulfoxide solution derived from two-dimensional NMR spectroscopy and molecular dynamics simulation.

Structural and dynamic properties of [8-arginine]vasopressin and a class of highly potent vasopressin V1 antagonists which contain 3-mercapto-3,3-cyclopentamethylene propionic acid (Mca) in position 1 of the vasopressin sequence have been determined. On the basis of two-dimensional NMR experiments in dimethyl sulfoxide solution, interproton distances were derived according to which model conformations were built and refined using molecular dynamics simulations. The conformation of vasopressin and the V1 antagonists differ mainly in the region of the mutated residue. The antagonistic property was found to be related to an inversed chirality of the disulfide bridge. In all investigated molecules, characteristic beta-turn structure elements were found for the backbone conformation of the endocyclic residues Tyr2-Asn5. For this portion of the peptide sequence, various conformational equilibria were detected which matched different time scales. For [Arg8]vasopressin, averaged NMR parameters were obtained which could be explained by rapid interconversion between different beta-turn geometries, whereas multiple slowly exchanging conformations were observed for the V1 antagonists. V1 antagonists containing sarcosine in position 7 exhibited multiple spectral patterns for the exocyclic part attributed to cis/trans isomerization. The X-ray structure of deamino-oxytocin [Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., Hruby, V. J., Buku, A., Fischman, A. J. & Wyssbrod, H. R. (1986) Science 232, 633-636] was found to represent one sample of the conformational space covered by the multiple conformations found for [Mca1, Arg8]vasopressin.

Effect of vasopressin, oxytocin and LHRH on the proliferation and metabolism of rat bone marrow stromal cells in culture.

Effect of vasopressin, oxytocin and LHRH (10 and 20 pg/ml medium) on the proliferation and metabolism of cultured rat bone marrow stromal cells was investigated by methyl-3H-thymidine incorporation, cytochemistry and estimation of enzyme activities. Vasopressin did not change of the activity of tetrahydrofolate dehydrogenase (4HFDH), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PD) and the level of reduced glutathione (GSH). However, the higher concentration of vasopressin significantly lowered the activity of acetylcholinesterase (AchE). As compared with the control cultures, stromal cells grown in the presence of oxytocin showed higher (at lower hormone concentration) and lower (at higher concentration) LDH activity as well as lower G6PD activity (only at higher concentration), while the activity of AchE and the level of GSH was not changed. LHRH significantly increased G6PD and AchE activity and decreased LDH activity in the cultured cells. As revealed by cytochemistry, LHRH specifically enhanced 4HFDH activity in reticular cells.