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Prostate cancer starts as a treatable hormone-dependent disease, but often ends in a drug-resistant form called castration-resistant prostate cancer (CRPC). Despite the development of the antiandrogens enzalutamide and abiraterone for CRPC, which target the androgen receptor (AR), drug resistance usually develops within 6 months and metastatic CRPC (mCRPC) leads to lethality. EZH2, found with SUZ12, EED, and RbAP48 in Polycomb repressive complex 2 (PRC2), has emerged as an alternative target for the treatment of deadly mCRPC. Unfortunately, drugs targeting EZH2 have shown limited efficacy in mCRPC. To address these failures, we have developed novel, dual-acting peptide inhibitors of PRC2 that uniquely target the SUZ12 protein component, resulting in the inhibition of both PRC2 canonical and noncanonical functions in prostate cancer. These peptides were found to inhibit not only the EZH2 methylation activity, but also block its positive effect on AR gene expression in prostate cancer cells. Since the peptide effect on AR levels is transcriptional, the inhibitory peptides can block the expression of both full-length AR and its splicing variants including AR-V7, which plays a significant role in the development of drug resistance. This dual-mode action provides the peptides with the capability to kill enzalutamide-resistant CRPC cells. These peptides are also more cytotoxic to prostate cancer cells than the combination of enzalutamide and an EZH2 inhibitory drug, which was recently suggested to be an effective treatment of mCRPC disease. Our data show that such a dual-acting therapeutic approach can be more effective than the existing front-line drug therapies for treating deadly mCRPC. [ABSTRACT FROM AUTHOR]

The use of cell growth‐based assays to identify inhibitory compounds is straightforward and inexpensive, but is also inherently insensitive and somewhat nonspecific. To overcome these limitations and develop a sensitive, specific cell‐based assay, two different approaches were combined. To address the sensitivity limitation, different fluorescent proteins have been introduced into a bacterial expression system to serve as growth reporters. To overcome the lack of specificity, these protein reporters have been incorporated into a plasmid in which they are paired with different orthologs of an essential target enzyme, in this case l‐methionine S‐adenosyltransferase (MAT, AdoMet synthetase). Screening compounds that serve as specific inhibitors will reduce the growth of only a subset of strains, because these strains are identical, except for which target ortholog they carry. Screening several such strains in parallel not only reveals potential inhibitors but the strains also serve as specificity controls for one another. The present study makes use of an existing Escherichia coli strain that carries a deletion of metK, the gene for MAT. Transformation with these plasmids leads to a complemented strain that no longer requires externally supplied S‐adenosylmethionine for growth, but its growth is now dependent on the activity of the introduced MAT ortholog. The resulting fluorescent strains provide a platform to screen chemical compound libraries and identify species‐selective inhibitors of AdoMet synthetases. A pilot study of several chemical libraries using this platform identified new lead compounds that are ortholog‐selective inhibitors of this enzyme family, some of which target the protozoal human pathogen Cryptosporidium parvum. [ABSTRACT FROM AUTHOR]

Prostate cancer starts as a treatable hormone-dependent disease, but often ends in a drug-resistant form called castration-resistant prostate cancer (CRPC). Despite the development of the antiandrogens enzalutamide and abiraterone for CRPC, which target the androgen receptor (AR), drug resistance usually develops within 6 months and metastatic CRPC (mCRPC) leads to lethality. EZH2, found with SUZ12, EED, and RbAP48 in Polycomb repressive complex 2 (PRC2), has emerged as an alternative target for the treatment of deadly mCRPC. Unfortunately, drugs targeting EZH2 have shown limited efficacy in mCRPC. To address these failures, we have developed novel, dual-acting peptide inhibitors of PRC2 that uniquely target the SUZ12 protein component, resulting in the inhibition of both PRC2 canonical and noncanonical functions in prostate cancer. These peptides were found to inhibit not only the EZH2 methylation activity, but also block its positive effect on AR gene expression in prostate cancer cells. Since the peptide effect on AR levels is transcriptional, the inhibitory peptides can block the expression of both full-length AR and its splicing variants including AR-V7, which plays a significant role in the development of drug resistance. This dual-mode action provides the peptides with the capability to kill enzalutamide-resistant CRPC cells. These peptides are also more cytotoxic to prostate cancer cells than the combination of enzalutamide and an EZH2 inhibitory drug, which was recently suggested to be an effective treatment of mCRPC disease. Our data show that such a dual-acting therapeutic approach can be more effective than the existing front-line drug therapies for treating deadly mCRPC. [ABSTRACT FROM AUTHOR]

It is established that Yersinia pestis, the causative agent of bubonic plague, recently evolved from enteropathogenic Yersinia pseudotuberculosis by undergoing chromosomal degeneration while acquiring two unique plasmids that facilitate tissue invasion (pPCP) and dissemination by fleabite (pMT). Thereafter, plague bacilli spread from central Asia to sylvatic foci throughout the world. These epidemic isolates exhibit a broad host range including man as opposed to enzootic (pestoides) variants that remain in ancient reservoirs where infection is limited to muroid rodents. Cells of Y. pseudotuberculosis are known to express glucose-6-phosphate dehydrogenase (Zwf) and aspartase (AspA); these activities are not detectable in epidemic Y. pestis due to missense mutations (substitution of proline for serine at amino position 155 of Zwf and leucine for valine at position 363 of AspA). In this study, functional Zwf was found in pestoides strains E, F and G but not seven other enzootic isolates; enzymic activity was associated with retention of serine at amino acid position 155. Essentially, full AspA activity occurred in pestoides isolates where valine (pestoides A, B, C and D) or serine (pestoides E, F, G and I) occupied position 363. Reduced activity occurred in strains Angola and A16, which contained phenylalanine at this position. The [k.sub.cat] but not [K.sub.m] of purified AspA from strain Angola was significantly reduced. In this context, aspA of the recently described attenuated enzootic microtus biovar encodes active valine at position 363, further indicating that functional AspA is a biomarker for avirulence of Y. pestis in man.

Systemic infections from fungal organisms are becoming increasingly difficult to treat as drug resistance continues to emerge. To substantially expand the antifungal drug landscape new compounds must be identified and developed with novel modes of action against previously untested drug targets. Most drugs block the activity of their targets through reversible, noncovalent interactions. However, a significant number of drugs form irreversible, covalent bonds with their selected targets. While more challenging to develop, these irreversible inactivators offer some significant advantages as novel antifungal agents. Vinyl sulfones contain a potentially reactive functional group that could function as a selective enzyme inactivator, and members of this class of compounds are now being developed as inactivators against an antifungal drug target. The enzyme aspartate semialdehyde dehydrogenase (ASADH) catalyzes a key step in an essential microbial pathway and is essential for the survival of every microorganism examined. A series of vinyl sulfones have been designed, guided by molecular modeling and docking studies to enhance their affinity for fungal ASADHs. These newly synthesized compounds have been examined against this target enzyme from the pathogenic fungal organism Candida albicans. Vinyl sulfones containing complementary structural elements inhibit this enzyme with inhibition constants in the low‐micromolar range. These inhibitors have also led to the rapid and irreversible inactivation of this enzyme, and show some initial selectivity when compared to the inactivation of a bacterial ASADH. The best inactivators will serve as lead compounds for the development of potent and selective antifungal agents. [ABSTRACT FROM AUTHOR]

It is established that cells of Yersinia pestis, the causative agent of bubonic plague, excrete L-aspartic acid at the expense of exogenous L-glutamic acid during expression of the low-calcium response. Results of enzymic analysis provided here suggest that a previously defined deficiency of aspartase (AspA) accounts for this phenomenon rather than an elevated oxaloacetate pool. The only known distinction between most sequenced isolates of aspA from Y. pestis and the active gene in Yersinia pseudotuberculosis (the immediate progenitor of Y. pestis) is a single base transversion (G-C[right arrow]T-A) causing replacement of leucine (encoded by UUG) for valine (encoded by GUG) at amino acid position 363. The gene from Y. pestis KIM possesses a unique second transversion (G-C[right arrow]T-A) at amino acid 146 causing substitution of aspartic acid (encoded by GAU) with tyrosine (encoded by UAU). We show in this study that Y. pestis expresses aspA as cross-reacting immunological material (CRIM). Functional and inactive aspA of Y. pseudotuberculosis PB1 and Y. pestis KIM, respectively, were then cloned and expressed in AspA-deficient Escherichia coil. After purification to near homogeneity, the products were subjected to biochemical analysis and found to exhibit similar secondary, tertiary and quaternary (tetrameric) structures as well as comparable Michaelis constants for L-aspartic acid. However, the [K.sub.cat] of the Y. pestis CRIM of strain KIM is only about 0.1% of that determined for the active AspA of Y. pseudotuberculosis. Return of valine for leucine at position 363 of the Y. pestis enzyme restored normal turnover ([k.sub.cat] 86 [+ or -] 2 [s.sup.-1]) provided that the amino acid substitution at position 146 was also reversed. These observations have important implications for understanding the nature of the stringent low-calcium response of Y. pestis and its role in promoting acute disease.

Human aspartoacylase is cloned and expressed in Pichia pastoris and the properties of this highly purified and fully active enzyme are examined. The chelation studies to remove the zinc have resulted in a reversible loss of catalytic activity, thereby establishing aspartoacylase as a zinc metalloenzyme.

The structural analysis of an enzymatic reaction intermediate affords a unique opportunity to study a catalytic mechanism in extraordinary detail. Here we present the structure of a tetrahedral intermediate in the catalytic cycle of aspartate-[beta]-semialdehyde dehydrogenase (ASADH) from Haemophilus influenzae at 2.0-[Angstrom] resolution. ASADH is not found in humans, yet its catalytic activity is required for the biosynthesis of essential amino acids in plants and microorganisms. Diaminopimelic acid, also formed by this enzymatic pathway, is an integral component of bacterial cell walls, thus making ASADH an attractive target for the development of new antibiotics. This enzyme is able to capture the substrates aspartate-[beta]-semialdehyde and phosphate as an active complex that does not complete the catalytic cycle in the absence of NADP. A distinctive binding pocket in which the hemithioacetal oxygen of the bound substrate is stabilized by interaction with a backbone amide group dictates the R stereochemistry of the tetrahedral intermediate. This pocket, reminiscent of the oxyanion hole found in serine proteases, is completed through hydrogen bonding to the bound phosphate substrate.

The channeling of the substrate beta-aspartyl phosphate between aspartokinase and aspartate semialdehyde dehydrogenase in Escherichia coli is descrbed. Data suggest that aspartate semialdehyde dehydrogenase may facilitate the channeling of the intermediates between the nonconsecutive reactions of the enzyme.

Research examines the nonconsecutive reaction catalysis of the bifunctional aspartokinase-homoserine dehydrogenase isoenzymes from Escherichia coli as well as synthesized hybrid enzymes. Results show that in the hybrids, the kinase activity is sensitive to lysine and dehydrogenase is regulated by both threonine and lysine. The kinase domain is stabler than dehydrogenase domain.

Research has been conducted on the F65A/Y131C murine alpha-carbonic anhydrase V. The crystal structure of this anhydrase has been investigated in examining the catalytic proton shuttle features and the results are reported.

Research has been conducted on the aspartate-beta-semialdehyde dehydrogenase. The structure of this dehydrogenase from Escherichia coli has been investigated via crystallography in the presence of its coenzyme and substrate analogue and the results are reported.

Researchers have investigated potential active site residues for L-aspartate ammonia-lyase from Escherichia coli on the basis of their proximity to a cleft which contains the functionally significant lysine 327. It was possible to use site-directed mutagenesis to test the five most likely residues in the assumed active site cleft. Arg 29 was assigned a substrate binding role, and it was concluded that the beta-carboxyl group of the substrate was the most likely site of interaction.

Researchers have succeeded in identifying the structure of aspartase from Escherichia coli to 2.8 angstrom. Sequence alignments of the fumarase superfamily and mutagenesis studies have made it possible to locate the likely active site of aspartase in a region which contains side chains from three different subunits. The high substrate specificity of aspartase indicates that the enzyme must bind both the alpha- and beta-carboxylate groups.

The substrate specificity of homoserine kinase was analyzed by utilizing a series of structural analogs of L-homoserine exhibiting site-directed mutagenesis in specific functional groups. Sequence homology studies of the structural analogs of L-homoserine during homoserine kinase-catalyzed reactions indicated the role of arginine-234 in the binding of the carboxyl group. Furthermore, the beta- and gamma-carbons of the hydroxyl group were phosphorylated by homoserine kinase which can also accommodate large hydrophobic groups at the carboxyl end of the substrate.

A study, to identify residues that play an important role in the catalytic activity of aspartate-beta-semialdehyde dehydrogenase (ASA DH), by site -directed mutagenesis, compares ASA DH structure with D-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) to select correct targets for mutagenesis. ASA DH catalyzes the oxidative phosphorylation of an aldehyde substrate and has an essential cysteine residue that is involved in enzymatic reaction.

Site-directed mutagenic study of the L-aspartase enzyme of Escherichia coli suggests that the cysteine-273 residue is modified by the treatment of L-aspartase with L-aspartate-beta-semialdehyde. Thus, cysteine-273 serves as an active-site nucleophile that enables attack of an activated double bond in the enzyme-bound product involved in catalysis in L-aspartase.

Substrate analogue L-aspartate beta-semialdehyde (L-ASA) is a mechanism-based inactivator of L-aspartase from Escherichia coli that catalyzes the deamination of L-ASA to give fumaric acid semialdehyde (FAA) and NH(sub 4)(super +). L-ASA acts as a mechanism-based inactivator by binding at the active site of the enzyme. FAA binds more cohesively to the enzyme-activator-metal ion complex than to the free enzyme form. This confirms the coordination in binding between the activator site and the active site of L-aspartase.

The kinetics of formation and isomerization of phosphato complexes of uridine 5'-di and triphosphates to platinum(II) were reported. The resulting products were characterized with high performance liquid chromatogrphy, circular dichroism and two-dimensional nuclear magnetic resonance spectroscopy. The results show that the isomerization proceeds through a rate-limiting ring-opening. When the chelate ring is opened, deligation and phosphate hydrolysis can also occur, resulting in free UDP, UMP and orthophosphate bound to platinum.

The substrate specificity of aspartokinase I from Escherichia coli was examined by kinetic, isotope labeling and nuclear magnetic resonance studies. It was shown that the alpha-amino group is an essential binding group. In addition, beta-derivatized structural analogues, such as beta-amide, beta-hydroxamate andbeta-esters, were found to be alternative substrates. These beta-derivatized analogues are capable of binding to aspartokinase through a reversal of regiospecificity. The result raises the possibility of using these alternative substrates to synthesize antimetabolites for the enzymes that are downstream inthe aspartate biosynthetic pathway.

The specificity of the anion binding site of aspartate-beta-semialdehyde dehydrogenase was investigated. The objective was to identify alternative substrates that can substitute for phosphate and competitive inhibitors with the ability to stabilize the acyl enzyme intermediate to allow structural characterization. Several oxyanions interact with L-aspartate-beta-semialdehyde dehydrogenase either as an alternative substrate or as a competitive inhibitor.

Potent inhibitors of an essential microbial enzyme have been shown to be effective growth inhibitors of Candida albicans, a pathogenic fungus. C. albicans is the main cause of oropharyngeal candidiasis, and also causes invasive fungal infections, including systemic sepsis, leading to serious complications in immunocompromised patients. As the rates of drug‐resistant fungal infections continue to rise novel antifungal treatments are desperately needed. The enzyme aspartate semialdehyde dehydrogenase (ASADH) is critical for the functioning of the aspartate biosynthetic pathway in microbes and plants. Because the aspartate pathway is absent in humans, ASADH has the potential to be a promising new target for antifungal research. Deleting the asd gene encoding for ASADH significantly decreases the survival of C. albicans, establishing this enzyme as essential for this organism. Previously developed ASADH inhibitors were tested against several strains of C. albicans to measure their possible therapeutic impact. The more potent inhibitors show a good correlation between enzyme inhibitor potency and fungal growth inhibition. Growth curves generated by incubating different C. albicans strains with varying enzyme inhibitor levels show significant slowing of fungal growth by these inhibitors against each of these strains, similar to the effect observed with a clinical antifungal drug. The most effective inhibitors also demonstrated relatively low cytotoxicity against a human epithelial cell line. Taken together, these results establish that the ASADH enzyme is a promising new target for further development as a novel antifungal treatment against C. albicans and related fungal species. [ABSTRACT FROM AUTHOR]