Gene_locus Report for: pig-ppce

We have investigated the effect of regiospecifically introducing substituents in the P2 part of the typical dipeptide derived basic structure of PREP inhibitors. This hitherto unexplored modification type can be used to improve target affinity, selectivity, and physicochemical parameters in drug discovery programs focusing on PREP inhibitors. Biochemical evaluation of the produced inhibitors identified several substituent types that significantly increase target affinity, thereby reducing the need for an electrophilic "warhead" functionality. Pronounced PREP specificity within the group of Clan SC proteases was generally observed. Omission of the P1 electrophilic function did not affect the overall binding mode of three representative compounds, as studied by X-ray crystallography, while the P2 substituents were demonstrated to be accommodated in a cavity of PREP that, to date, has not been probed by inhibitors. Finally, we report on results of selected inhibitors in a SH-SY5Y cellular model of synucleinopathy and demonstrate a significant antiaggregation effect on alpha-synuclein.

Prolyl oligopeptidase is a large cytosolic enzyme that belongs to a new class of serine peptidases. The enzyme is involved in the maturation and degradation of peptide hormones and neuropeptides, which relate to the induction of amnesia. The 1.4 A resolution crystal structure is presented here. The enzyme contains a peptidase domain with an alpha/beta hydrolase fold, and its catalytic triad (Ser554, His680, Asp641) is covered by the central tunnel of an unusual beta propeller. This domain makes prolyl oligopeptidase an oligopeptidase by excluding large structured peptides from the active site. In this way, the propeller protects larger peptides and proteins from proteolysis in the cytosol. The structure is also obtained with a transition state inhibitor, which may facilitate drug design to treat memory disorders.

Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80,751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. [3H]Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.

Prolyl specific oligopeptidase (POP), is one of the highly expressed enzymes in the brain and is a prime target to treat disorders related to the central nervous system. Here, we describe the structure-based design of the tacrine derivatives, selective, and brain-permeable POP inhibitors. These compounds inactivate POP in-vitro specifically and sustainably at very low concentrations (nano molar). Among this series, compound 6b (IC(50) = 0.81 +/- 0.04 microM) exhibited most potent inhibition. Furthermore, kinetic study revealed that these molecules target active site of POP which is further confirmed by in-silico molecular interaction analysis. The computational docking results indicates that the compounds are well fitted in the active site with high binding score (i.e., > -7 to > -4 kcal/mol) where Trp595, Arg643, Tyr473, and Ser554 plays important role in binding with the active compounds. The molecular dynamic simulation of most active compounds (6a, 6b, 6d, and 6f) displayed higher free energy binding, when compared to the standard drug in MM-PBSA based binding free energy calculation. In addition, the predicted pharmacokinetic profile suggests that these compounds can serve as excellent inhibitors upon additional optimization which makes them prime choice for further investigation.Communicated by Ramaswamy H. Sarma.

Altered prolyl oligopeptidase (PREP) activity is found in many common neurological and other genetic disorders, and in some cases PREP inhibition may be a promising treatment. The active site of PREP resides in an internal cavity; in addition to the direct interaction between active site and substrate or inhibitor, the pathway to reach the active site (the gating mechanism) must be understood for more rational inhibitor design and understanding PREP function. The gating mechanism of PREP has been investigated through molecular dynamics (MD) simulation combined with crystallographic and mutagenesis studies. The MD results indicate the inter-domain loop structure, comprised of 3 loops at residues, 189-209 (loop A), 577-608 (loop B), and 636-646 (loop C) (porcine PREP numbering), are important components of the gating mechanism. The results from enzyme kinetics of PREP variants also support this hypothesis: When loop A is (1) locked to loop B through a disulphide bridge, all enzyme activity is halted, (2) nicked, enzyme activity is increased, and (3) removed, enzyme activity is only reduced. Limited proteolysis study also supports the hypothesis of a loop A driven gating mechanism. The MD results show a stable network of H-bonds that hold the two protein domains together. Crystallographic study indicates that a set of known PREP inhibitors inhabit a common binding conformation, and this H-bond network is not significantly altered. Thus the domain separation, seen to occur in lower taxa, is not involved in the gating mechanism for mammalian PREP. In two of the MD simulations we observed a conformational change that involved the breaking of the H-bond network holding loops A and B together. We also found that this network was more stable when the active site was occupied, thus decreasing the likelihood of this transition.

Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189-209) and loop B (res. 577-608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.

We have investigated the effect of regiospecifically introducing substituents in the P2 part of the typical dipeptide derived basic structure of PREP inhibitors. This hitherto unexplored modification type can be used to improve target affinity, selectivity, and physicochemical parameters in drug discovery programs focusing on PREP inhibitors. Biochemical evaluation of the produced inhibitors identified several substituent types that significantly increase target affinity, thereby reducing the need for an electrophilic "warhead" functionality. Pronounced PREP specificity within the group of Clan SC proteases was generally observed. Omission of the P1 electrophilic function did not affect the overall binding mode of three representative compounds, as studied by X-ray crystallography, while the P2 substituents were demonstrated to be accommodated in a cavity of PREP that, to date, has not been probed by inhibitors. Finally, we report on results of selected inhibitors in a SH-SY5Y cellular model of synucleinopathy and demonstrate a significant antiaggregation effect on alpha-synuclein.

A new inhibitor, containing a linked proline-piperidine structure, for the enzyme prolyl oligopeptidase (POP) has been synthesised and demonstrated to bind covalently with the enzyme at the active site. This provides evidence that covalent inhibitors of POP do not have to be limited to structures containing five-membered N-containing heterocyclic rings.

The positive electrostatic environment of the active site of prolyl oligopeptidase was investigated by using substrates with glutamic acid at positions P2, P3, P4, and P5, respectively. The different substrates gave various pH rate profiles. The pKa values extracted from the curves are apparent parameters, presumably affected by the nearby charged residues, and do not reflect the ionization of a simple catalytic histidine as found in the classic serine peptidases like chymotrypsin and subtilisin. The temperature dependence of kcat/Km did not produce linear Arrhenius plots, indicating different changes in the individual rate constants with the increase in temperature. This rendered it possible to calculate these constants, i.e. the formation (k1) and decomposition (k-1) of the enzyme-substrate complex and the acylation constant (k2), as well as the corresponding activation energies. The results have revealed the relationship between the complex Michaelis parameters and the individual rate constants. Structure determination of the enzyme-substrate complexes has shown that the different substrates display a uniform binding mode. None of the glutamic acids interacts with a charged group. We conclude that the specific rate constant is controlled by k1 rather than k2 and that the charged residues from the substrate and the enzyme can markedly affect the formation but not the structure of the enzyme-substrate complexes.

Prolyl oligopeptidase, a member of a new family of serine peptidases, plays an important role in memory disorders. Earlier x-ray crystallographic investigations indicated that stabilization of the tetrahedral transition state of the reaction involved hydrogen bond formation between the oxyanion of the tetrahedral intermediate and the OH group of Tyr(473). The contribution of the OH group was tested with the Y473F variant using various substrates. The charged succinyl-Gly-Pro-4-nitroanilide was hydrolyzed with a much lower k(cat)/K(m) compared with the neutral benzyloxycarbonyl-G1y-Pro-2-naphthylamide, although the binding modes of the two substrates were similar, as shown by x-ray crystallography. This suggested that electrostatic interactions between Arg(643) and the succinyl group competed with the productive binding mechanism. Unlike most enzyme reactions, catalysis by the wild-type enzyme exhibited positive activation entropy. In contrast, the activation entropy for the Y473F variant was negative, suggesting that the tyrosine OH group is involved in stabilizing both the transition state and the water shell at the active site. Importantly, Tyr(473) is also implicated in the formation of the enzyme-substrate complex. The nonlinear Arrhenius plot suggested a greater significance of the oxyanion binding site at physiological temperature. The results indicated that Tyr(473) was more needed at high pH, at high temperature, and with charged substrates exhibiting "internally competitive inhibition.

Prolyl oligopeptidase, a serine peptidase unrelated to trypsin and subtilisin, is implicated in memory disorders and is an important target of drug design. The catalytic competence of the Asp(641) residue of the catalytic triad (Ser(554), Asp(641), His(680)) was studied using the D641N and D641A variants of the enzyme. Both variants displayed 3 orders of magnitude reduction in k(cat)/K(m) for benzyloxycarbonyl-Gly-Pro-2-naphthylamide. Using an octapeptide substrate, the decrease was 6 orders of magnitude, whereas with Z-Gly-Pro-4-nitrophenyl ester there was virtually no change in k(cat)/K(m). This indicates that the contribution of Asp(641) is very much dependent on the substrate-leaving group, which was not the case for the classic serine peptidase, trypsin. The rate constant for benzyloxycarbonyl-Gly-Pro-thiobenzylester conformed to this series as demonstrated by a method designed for monitoring the hydrolysis of thiolesters in the presence of thiol groups. Alkylation of His(680) with Z-Gly-Pro-CH(2)Cl was concluded with similar rate constants for wild-type and D641A variant. However, kinetic measurements with Z-Gly-Pro-OH, a product-like inhibitor, indicated that the His(680) is not accessible in the enzyme variants. Crystal structure determination of these mutants revealed subtle perturbations related to the catalytic activity. Many of these observations show differences in the catalysis between trypsin and prolyl oligopeptidase.

Structure determination of the inactive S554A variant of prolyl oligopeptidase complexed with an octapeptide has shown that substrate binding is restricted to the P4-P2' region. In addition, it has revealed a hydrogen bond network of potential catalytic importance not detected in other serine peptidases. This involves a unique intramolecular hydrogen bond between the P1' amide and P2 carbonyl groups and another between the P2' amide and Nepsilon2 of the catalytic histidine 680 residue. It is argued that both hydrogen bonds promote proton transfer from the imidazolium ion to the leaving group. Another complex formed with the product-like inhibitor benzyloxycarbonyl-glycyl-proline, indicating that the carboxyl group of the inhibitor forms a hydrogen bond with the Nepsilon2 of His(680). Because a protonated histidine makes a stronger interaction with the carboxyl group, it offers a possibility of the determination of the real pK(a) of the catalytic histidine residue. This was found to be 6.25, lower than that of the well studied serine proteases. The new titration method gave a single pK(a) for prolyl oligopeptidase, whose reaction exhibited a complex pH dependence for k(cat)/K(m), and indicated that the observed pK(a) values are apparent. The procedure presented may be applicable for other serine peptidases.

Proteases have a variety of strategies for selecting substrates in order to prevent uncontrolled protein degradation. A recent crystal structure determination of prolyl oligopeptidase has suggested a way for substrate selection involving an unclosed seven-bladed beta-propeller domain. We have engineered a disulfide bond between the first and seventh blades of the propeller, which resulted in the loss of enzymatic activity. These results provided direct evidence for a novel strategy of regulation in which oscillating propeller blades act as a gating filter during catalysis, letting small peptide substrates into the active site while excluding large proteins to prevent accidental proteolysis.

Prolyl oligopeptidase is a large cytosolic enzyme that belongs to a new class of serine peptidases. The enzyme is involved in the maturation and degradation of peptide hormones and neuropeptides, which relate to the induction of amnesia. The 1.4 A resolution crystal structure is presented here. The enzyme contains a peptidase domain with an alpha/beta hydrolase fold, and its catalytic triad (Ser554, His680, Asp641) is covered by the central tunnel of an unusual beta propeller. This domain makes prolyl oligopeptidase an oligopeptidase by excluding large structured peptides from the active site. In this way, the propeller protects larger peptides and proteins from proteolysis in the cytosol. The structure is also obtained with a transition state inhibitor, which may facilitate drug design to treat memory disorders.

Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80,751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. [3H]Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.

The kinetics of inactivation of prolyl endopeptidase by acetyl-Ala-Ala-Pro-CH2Cl were studied by progress-curve methods in the presence of substrate. The kinetic mechanism was found to involve the formation of an initial complex between the enzyme and the chloromethane followed by an inactivation step. The substrate was shown to compete for the formation of the initial complex, indicating that binding at the active site was a prerequisite for inactivation. After reaction of the enzyme with [3H]acetyl-Ala-Ala-Pro-CH2Cl, it was possible to isolate five labelled peptides. Four of these peptides contained a cysteine residue as the site of modification, whereas the fifth peptide contained no cysteine and a histidine residue was identified as the site of modification. This residue (His-680) probably represents the active-site histidine of prolyl endopeptidase.