Ketone-substrate analogues of Clostridium histolyticum collagenases: tight-binding transition-state analogue inhibitors

A series of ketone-substrate analogues has been synthesized for the two classes of collagenases from Clostridium histolyticum and shown to be competitive inhibitors. These compounds have sequences that match those of specific peptide substrates for these enzymes. The best inhibitor is the ketone analogue of cinnamoyl-Leu-Gly-Pro-Pro, which has a KI value of 18 nM for epsilon-collagenase, a class II enzyme. This is the tightest binding inhibitor reported for any collagenase to date. Plots of log KI for the inhibitors vs log KM/kcat for the matched substrates for both collagenases are linear with slopes near unity, indicating that the ketones are transition-state analogues. This strongly implies that the ketone carbon atoms of these inhibitors are tetrahedral when bound to the enzymes.     

Rat liver iodothyronine monodeiodinase. Evaluation of the iodothyronine ligand-binding site

Ligand binding characteristics of rat liver microsomal type I iodothyronine deiodinase were evaluated by measuring dose-response inhibition and apparent Michaelis-Menten or inhibitor constants of iodothyronine analogues to compete as substrates or inhibitors for the natural substrate L-thyroxine. These data show strong correlations with the binding requirements of hormone analogues to serum thyroxine-binding prealbumin since iodothyronine analogues with a negatively charged side chain, a negative charge or hydrogen bonding function in the 4'-position, tetraiodo ring substitution, and a skewed hormone conformation are structural features shared in common which markedly affect enzyme activity and protein binding affinity. 3,3',5'-Triiodo-L-thyronine is the most potent natural substrate (IC50 = 0.3 microM) and tetraiodothyroacetic acid is the most potent inhibitor (IC50 = 0.2 microM). Both thyroxine (T4)-5'- and T4-5-deiodination pathways are inhibited by these potent analogues, providing further evidence for a single enzyme catalyzing the rat liver microsomal deiodination reactions. These data also show that L-hormone analogues are preferentially deiodinated via the T4-5'-deiodination pathway, whereas D-analogues produce products via the T4-5-deiodination pathway. The thyroxine-binding prealbumin complex was used to model the interaction of thyroid hormones with the deiodinase active site. Computer graphic modeling of the prealbumin complex showed that only those analogues which are potent deiodinase inhibitors or substrates can be accommodated in the hormone binding site. This model suggests the design of functionally specific ligands which can modulate peripheral thyroid hormone metabolism and act as antithyroidal drugs.     

Some 6-Aza-5-substituted-2′-deoxyuridines Show Potent and Selective Inhibition of Herpes Simplex Virus Type 1 Thymidine Kinase

Abstract

The synthesis and X-ray crystal structures of a series of 5-substituted-6-aza-2′-deoxyuridines is reported. These nucleoside analogues inhibit the phosphorylation of thymidine by HSV-1 TK but have no effect on the corresponding human enzyme. Detailed examination of one analogue proves it to be a competitive inhibitor of thymidine with a Ki of 0.34 μM and is a very poor substrate. The analogues are not substrates for the enzyme and also do not inhibit the degradation of thymidine by thymidine phosphorylase. Molecular modelling showed that the inhibitors fit well in the active site of HSV-1 TK, provided the conformation of the sugar moiety is the same for thymidine in the complex.     

Cycloeucalenol-obtusifoliol isomerase. Structural requirements for transformation or binding of substrates and inhibitors

The molecular features of 19 synthetic substrates and ground-state analogues of cycloeucalenol, the natural substrate of cycloeucalenol - obtusifoliol isomerase, a membrane-bound enzyme specific to higher plants, and of 9 synthetic carbocationic analogues of the high-energy intermediate occurring during the reaction catalyzed by the isomerase, were related to their ability to be transformed by this enzyme (catalytical competence) and their potency as inhibitors of this enzyme. With substrates and ground-state analogues it has been possible to determine at least two critical domains: significant binding requires the presence of the 3 beta-hydroxyl group on the ring A with the correct stereochemistry together with absence of a 4 beta-methyl group. Moreover initial enzyme-substrate interaction appears to be dependent upon the accessibility of the 3 beta-oxygen. Substitutions on the ring B do not preclude binding whereas they are of great influence on substrate transformation. Modifications of the ring A and other modifications suggest that ground-state and high-energy intermediate analogues bind two different conformations of the isomerase active site.     

Biosynthesis of the immunosuppressant immunomycin: the enzymology of pipecolate incorporation

Immunomycin, an immunosuppressant closely related to FK 506, contains a pipecolate residue in amide linkage with an acyl group in its polyketide backbone. An enzyme activating L-pipecolic acid has been isolated from Streptomyces hygroscopicus var. ascomyceticus, which produces immunomycin. Purification results in a monomer of 170 kDa exhibiting N-terminal heterogeneity, apparently arising from proteolysis of a single species. It is a dimer under native conditions. The reaction appears to use an aminoacyl adenylate as an intermediate in the activating reaction, as do most activating enzymes involved in nonribosomal peptide synthesis. A range of pipecolate and proline analogues act as substrates in the pyrophosphate-ATP exchange resulting from the adenylation reaction. Several analogues are inhibitors of the subsequent thioesterification of the enzyme. Antibody raised to the purified enzyme was used to follow antigen during the course of fermentation. Maximal levels of antigen are found when synthesis of immunomycin is maximal. Ten of twelve immunomycin nonproducing mutants lack detectable pipecolate-activating enzyme in Western blots. From the enzymatic characteristics, substrate specificity, and immunological properties, we propose that we have isolated the enzyme responsible for activating pipecolic acid for immunomycin biosynthesis.     

Interaction of rat glutathione S-transferases 7-7 and 8-8 with gamma-glutamyl- or glycyl-modified glutathione analogues.

Analogues of GSH in which either the gamma-glutamyl or the glycyl moiety is modified were synthesized and tested as both substrates for and inhibitors of glutathione S-transferases (GSTs) 7-7 and 8-8. Acceptor substrates for GST 7-7 were 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) and for GST 8-8 CDNB, ETA and 4-hydroxynon-trans-2-enal (HNE). The relative ability of each combination of enzyme and GSH analogue to catalyse the conjugation of all acceptor substrates was similar with the exception of the combination of GST 7-7 and gamma-L-Glu-L-Cys-L-Asp, which used CDNB but not ETA as acceptor substrate. In general, GST 7-7 was better than GST 8-8 in utilizing these analogues as substrates, and glycyl analogues were better than gamma-glutamyl analogues as both substrates and inhibitors. These results are compared with those obtained earlier with GSH analogues and GST isoenzymes 1-1, 2-2, 3-3 and 4-4 [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] and the implications with respect to the nature of their active sites are discussed.     

Adenosine deaminase prefers a distinct sugar ring conformation for binding and catalysis: kinetic and structural studies

Several recent X-ray crystal structures of adenosine deaminase (ADA) in complex with various adenosine surrogates have illustrated the preferred mode of substrate binding for this enzyme. To define more specific structural details of substrate preferences for binding and catalysis, we have studied the ADA binding efficiencies and deamination kinetics of several synthetic adenosine analogues in which the furanosyl ring is biased toward a particular conformation. NMR solution studies and pseudorotational analyses were used to ascertain the preferred furanose ring puckers (P, nu(MAX)) and rotamer distributions (chi and gamma) of the nucleoside analogues. It was shown that derivatives which are biased toward a "Northern" (3'-endo, N) sugar ring pucker were deaminated up to 65-fold faster and bound more tightly to the enzyme than those that preferred a "Southern" (2'-endo, S) conformation. This behavior, however, could be modulated by other structural factors. Similarly, purine riboside inhibitors of ADA that prefer the N hemisphere were more potent inhibitors than S analogues. These binding propensities were corroborated by detailed molecular modeling studies. Docking of both N- and S-type analogues into the ADA crystal structure coordinates showed that N-type substrates formed a stable complex with ADA, whereas for S-type substrates, it was necessary for the sugar pucker to adjust to a 3'-endo (N-type) conformation to remain in the ADA substrate binding site. These data outline the intricate structural details for optimum binding in the catalytic cleft of ADA.     

Cytoplasmic steps of peptidoglycan biosynthesis

The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid and (4) D-glutamic acid and dipeptide D-alanyl-D-alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.     

Coupling of isoprenoid triflates with organoboron nucleophiles: synthesis and biological evaluation of geranylgeranyl diphosphate analogues

The Suzuki coupling reaction has been used to introduce a methyl group derived from commercially available methylboronic acid into a vinyl triflate. This has led to a concise synthesis of all-trans-geranylgeraniol, with the key step being the palladium-catalyzed, silver-mediated methylation of triflate to give ethyl geranylgeranoate. This coupling protocol has also been used to produce the novel geranylgeranyl diphosphate (GGPP) analogue 3-phenyl-3-desmethylgeranylgeranyl diphosphate (3-PhGGPP, ). Our previously developed organocuprate coupling protocol has been used to introduce the cyclopropyl and tert-butyl moieties into the 3-position of vinyl triflate. The four GGPP analogues 3-vinyl-3-desmethylgeranylgeranyl diphosphate (3-vGGPP, ), 3-cyclopropyl-3-desmethylgeranylgeranyl diphosphate (3-cpGGPP, ), 3-tert-butyl-3-desmethyl-geranylgeranyl diphosphate (3-tbGGPP, ), and were then evaluated as potential inhibitors of recombinant yeast protein-geranylgeranyl transferase I (PGGTase I). The potential mechanism-based inhibitors 3-vGGPP and 3-cpGGPP did not exhibit time-dependent inactivation of PGGTase I. Instead, both analogues were alternative substrates, in accord with the interaction of the corresponding farnesyl analogues 3-vFPP and 3-cpFPP with PFTase. The tert-butyl and phenyl analogues were not substrates, but were instead competitive inhibitors of PGGTase I. Note that all four of the GGPP analogues were bound less tightly by the enzyme than the natural substrate, in contrast to the behavior of the 3-substituted FPP analogues.     

Discovering potent and selective reversible inhibitors of enzymes in complex proteomes

To realize the promise of genomics-based therapeutics, new methods are needed to accelerate the discovery of small molecules that selectively modulate protein activity. Toward this end, advances in combinatorial synthesis have provided unprecedented access to large compound libraries of considerable structural complexity and diversity, shifting the bottleneck in drug discovery to the development of efficient screens for protein targets. Screening for reversible enzyme inhibitors typically requires extensive target-specific work, including protein expression and purification, as well as the development of specific substrate assays. Here we report a proteomic method for the discovery of reversible enzyme inhibitors that avoids these steps. We show that competitive profiling of a library of candidate serine hydrolase inhibitors in complex proteomes with activity-based chemical probes identifies nanomolar reversible inhibitors of several enzymes simultaneously, including the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH), triacylglycerol hydrolase (TGH) and an uncharacterized membrane-associated hydrolase that lacks known substrates. The strategy tests inhibitors against numerous enzymes in parallel, assigning both potency and selectivity factors to each agent. In this way, promiscuous inhibitors were readily rejected in favor of equally potent compounds with 500-fold or greater selectivity for their targets.     

Polymerase-dependent DNA synthesis from phosphoramidate-activated nucleotides

Nucleoside triphosphate mimetics, which are substrates for polymerases, can be used in the enzymatic synthesis of nucleic acids. Alternatively, they might also become reversible or irreversible enzyme inhibitors. In order to analyze the effects of 5'-phosphoramidate modification of deoxynucleotide in DNA synthesis, 3-phosphono-L-Ala-dNMP (N = A, T, or G) were evaluated as substrates of HIV-1 RT, Vent (exo(-)), and Therminator polymerase, respectively. The DNA-dependent DNA polymerase activity is significantly higher for Vent exo(-) polymerase than for HIV-1 RT, which is reflected by the capacity of Vent exo(-) polymerase to efficiently synthesize DNA without stalling effects. In addition, Vent (exo(-)) polymerase proved to be more accurate than Therminator polymerase, based on Watson-Crick base-pairing. The optimal yield (88%-97%) of full-length elongation can be obtained in 60 minutes by Vent (exo(-)) polymerase at 0.025 U/μL, with the phosphoramidate analogues as substrates. These data led us to conclude that the optimal pyrophosphate mimetic for the enzyme-catalyzed synthesis of DNA is polymerase dependent.     

Sugar derivatives as new 6-phosphogluconate dehydrogenase inhibitors selective for the parasite Trypanosoma brucei

Sugar derivatives mimicking compounds which take part in the catalysed reaction have been assayed as alternative substrates and/or competitive inhibitors of 6-phosphogluconate dehydrogenase from Trypanosoma brucei and sheep liver. Phosphonate analogues have been synthesised and the new compound 5-deoxy-5-phosphono-D-arabinonate shows good selectivity towards the parasite enzyme. A number of 4-carbon and 5-carbon aldonates are strong inhibitors of the parasite enzyme with K(i) values below the substrate K(m) and some acyl derivatives are also potent inhibitors. At least five of the compounds showing a significant selectivity for the parasite enzyme represent leads for trypanocidal drugs against this recently validated target.     

The developmental patterns of lysosomal enzyme activities during Ca++-induced sporangium formation in Achlya bisexualis

The present paper describes intracellular changes in α-mannosidase specific activity during Ca⁺⁺-induced sporangium formation in the water mold Achlya bisexualis. The enzyme, which is concentrated in a cellular fraction with lysosome-like characteristics, undergoes a four-fold increase during sporangium differentiation. Addition of cycloheximide (100 μg/ml) or actinomycin D (10 μg/ml) at any time during the developmental sequence prevents further increase in the enzyme activity. These data suggest that coincident RNA and protein synthesis are essential for the accumulation of enzyme activity. Mixing of cell extracts from different developmental stages provides evidence that activators or inhibitors of the enzyme activity are not responsible for the enzyme activity evident at the different stages.     

Prohormone-substrate peptide sequence recognition by peptidylglycine α-amidating monooxygenase and its reflection in increased glycolate inhibitor potency

The interactions of nineteen peptide substrates and fifteen analogous peptidomimetic glycolate inhibitors with human peptidylglycine α-amidating monooxygenase (PAM) have been investigated. The substrates and inhibitors are the prohormones of calcitonin and oxytocin and their analogues. PAM both secreted into the medium by and extracted from DMS53 small lung carcinoma cells has been studied. The results show that recognition of the prooxytocin and procalcitonin peptide sequences by the enzyme extends more than four and five amino acid residues, respectively, from their C-termini. This substrate sequence recognition is mirrored by increased inhibitor potency with increased peptide length in the glycolate peptidomimetics. Substitution of the C-terminal penultimate glycine and proline residues of prooxytocin and procalcitonin and their analogues with phenylalanine increases the enzyme binding affinity. However, this changes the binding mode from one that depends on peptide sequence recognition to another primarily determined by the phenylalanine moiety, for both the substrates and analogous glycolate inhibitors.     

Recent progresses on synthesized LuxS inhibitors: A mini-review

Design and synthesis of LuxS enzyme inhibitors otherwise known as S-ribosylhomocysteine analogues, to target quorum sensing in bacteria, has been considerably developed within the last decade. This review presents which molecules have been synthesized to target LuxS enzyme in other words inhibitors of S-ribosylhomocysteinase. It reports their tested biological activity as LuxS inhibitors when available. A systematic overview has been conducted by searching PubMed, Medline, and The Cochrane Library and data extraction of all synthesized S-ribosylhomocysteine analogues has been collected. This mini-review shows limited data to date on this area and should continue to be studied.     

Synthesis and evaluation of N8-acetylspermidine analogues as inhibitors of bacterial acetylpolyamine amidohydrolase

Polyamines are small essential polycations involved in many biological processes. Enzymes of polyamine metabolism have been extensively studied and are attractive drug targets. Nevertheless, the reversible acetylation of polyamines remains poorly understood. Although eukaryotic N⁸-acetylspermidine deacetylase activity has already been detected and studied, the specific enzyme responsible for this activity has not yet been identified. However, a zinc deacetylase from Mycoplana ramosa, acetylpolyamine amidohydrolase (APAH), has been reported to use various acetylpolyamines as substrates. The recently solved crystal structure of this polyamine deacetylase revealed the formation of an ‘L’-shaped active site tunnel at the dimer interface, with ideal dimensions and electrostatic properties for accommodating narrow, flexible, cationic polyamine substrates. Here, we report the design, synthesis, and evaluation of N⁸-acetylspermidine analogues bearing different zinc binding groups as potential inhibitors of APAH. Most of the synthesized compounds exhibit modest potency, with IC₅₀ values in the mid-micromolar range, but compounds bearing hydroxamate or trifluoromethylketone zinc binding groups exhibit enhanced inhibitory potency in the mid-nanomolar range. These inhibitors will enable future explorations of acetylpolyamine function in both prokaryotes and eukaryotes.     

Chemical synthesis of S-ribosyl-L-homocysteine and activity assay as a LuxS substrate

Bacterial quorum sensing is mediated by autoinducers, small signaling molecules generated by bacteria. It has been proposed that the LuxS enzyme converts S-ribosyl-L-homocysteine to 4,5-dihydroxy-2,3-pentanedione, the precursor of autoinducer 2 (AI-2). We report here a chemical synthesis of S-ribosyl-L-homocysteine and its analogue using Mitsunobu coupling. Chemically synthesized ribosylhomocysteine has been confirmed as a substrate for LuxS in both an enzyme assay and a whole cell quorum sensing assay. The chemical entities of products from the LuxS reaction were also established. Several ribosylhomocysteine analogues have been tested as LuxS inhibitors.     

Methylthioadenosine/S-adenosylhomocysteine nucleosidase, a critical enzyme for bacterial metabolism

The importance of methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase in bacteria has started to be appreciated only in the past decade. A comprehensive analysis of its various roles here demonstrates that it is an integral component of the activated methyl cycle, which recycles adenine and methionine through S-adenosylmethionine (SAM)-mediated methylation reactions, and also produces the universal quorum-sensing signal, autoinducer-2 (AI-2). SAM is also essential for synthesis of polyamines, N-acylhomoserine lactone (autoinducer-1), and production of vitamins and other biomolecules formed by SAM radical reactions. MTA, SAH and 5'-deoxyadenosine (5'dADO) are product inhibitors of these reactions, and are substrates of MTA/SAH nucleosidase, underscoring its importance in a wide array of metabolic reactions. Inhibition of this enzyme by certain substrate analogues also limits synthesis of autoinducers and hence causes reduction in biofilm formation and may attenuate virulence. Interestingly, the inhibitors of MTA/SAH nucleosidase are very effective against the Lyme disease causing spirochaete, Borrelia burgdorferi, which uniquely expresses three homologous functional enzymes. These results indicate that inhibition of this enzyme can affect growth of different bacteria by affecting different mechanisms. Therefore, new inhibitors are currently being explored for development of potential novel broad-spectrum antimicrobials.     

Design and synthesis of inhibitors incorporating beta -amino acids of metalloendopeptidase EC 3.4.24.15.

Endopeptidase EC 3.4.24.15 (EP 24.15) is a thermolysin-like metalloendopeptidase which is expressed widely throughout the body, with the highest concentrations in the brain, pituitary and testis. While the precise role of EP 24.15 remains unknown, it is thought to participate in the regulated metabolism of a number of specific neuropeptides. Of the limited number of inhibitors described for EP 24.15, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-amino benzoate (CFP) is the most widely studied. CFP is a potent and specific inhibitor, but is unstable in vivo due to its cleavage between the alanine and tyrosine residues by the enzyme neprilysin (EP 24.11). The cpp-Ala-Ala N-terminal product of this cleavage is a potent inhibitor of angiotensin converting enzyme, which further limits the use of CFP in vivo. To generate specific inhibitors of EP 24.15 that are resistant to in vivo proteolysis by EP 24.11, beta-amino acids have been incorporated into the structure of CFP. We have prepared racemic mixtures of beta-amino acids containing proteogenic side chains, which are 9-fluorenylmethoxycarbonyl (Fmoc)-protected, and several analogues of CFP containing beta-amino acids have been synthesized by solid phase peptide synthesis. The results of stability and inhibitory studies of these new analogues show that the incorporation of beta-amino acids adjacent to the scissile bond can indeed stabilize the peptides against cleavage by EP 24.11 and still inhibit EP 24.15. The results obtained in these studies demonstrate the potential of these amino acids in the synthesis of peptidomimetics and in the design of new stable and specific therapeutics.     

Substrate analogues and divalent cations as inhibitors of glutamate decarboxylase from Escherichia coli

To examine the idea that glutamate decarboxylase from E. coli can be a convenient source for the study of the effects of compounds on GABA synthesis in the nervous system, a series of substrate analogues and divalent cations were tested as potential inhibitors of the bacterial enzyme. Those analogues exhibiting inhibitor activity did so in a competitive manner. The most effective inhibitors were 3-mercaptopropionic acid, 4-bromoisophthalic acid and isophthalic acid which exhibited Ki values of 0.13 mM, 0.22 mM and 0.31 mM, respectively. Eight other analogues produced lesser degrees of inhibition. In addition, seven divalent metal cations were tested as inhibitors of the enzyme. However, only Hg2+, Cd2+, Cu2+ and Zn2+ were effective at a concentration of 0.1mM. When these results were compared to the patterns of inhibition of glutamate decarboxylase from mouse brain, certain differences in the manner in which the enzymes responded to the inhibitors, emerged. Consequently, the bacterial decarboxylase may not be a good model for the study of drug action on brain GABA synthesis.