Hexose-6-phosphate dehydrogenase blockade reverses prostate cancer drug resistance in xenograft models by glucocorticoid inactivation.

Prostate cancer resistance to next-generation hormonal treatment with enzalutamide is a major problem and eventuates into disease lethality. Biologically active glucocorticoids that stimulate glucocorticoid receptor (GR) have an 11ß-OH moiety, and resistant tumors exhibit loss of 11ß-HSD2, the oxidative (11ß-OH → 11-keto) enzyme that normally inactivates glucocorticoids, allowing elevated tumor glucocorticoids to drive resistance by stimulating GR. Here, we show that up-regulation of hexose-6-phosphate dehydrogenase (H6PD) protein occurs in prostate cancer tissues of men treated with enzalutamide, human-derived cell lines, and patient-derived prostate tissues treated ex vivo with enzalutamide. Genetically silencing H6PD blocks NADPH generation, which inhibits the usual reductive directionality of 11ß-HSD1, to effectively replace 11ß-HSD2 function in human-derived cell line models, suppress the concentration of biologically active glucocorticoids in prostate cancer, and reverse enzalutamide resistance in mouse xenograft models. Similarly, pharmacologic blockade of H6PD with rucaparib normalizes tumor glucocorticoid metabolism in human cell lines and reinstates responsiveness to enzalutamide in mouse xenograft models. Our data show that blockade of H6PD, which is essential for glucocorticoid synthesis in humans, normalizes glucocorticoid metabolism and reverses enzalutamide resistance in mouse xenograft models. We credential H6PD as a pharmacologic vulnerability for treatment of next-generation androgen receptor antagonist-resistant prostate cancer by depleting tumor glucocorticoids.

2-Aryl-3-(6-trifluoromethoxy)benzo[d]thiazole-based thiazolidinone hybrids as potential anti-infective agents: Synthesis, biological evaluation and molecular docking studies.

The search for new antimicrobial agents is greater than ever due to the perpetual threat of multidrug resistance in known pathogens and the relentless emergence of new infections. In this manuscript, ten thiazole-based thiazolidinone hybrids bearing a 6-trifluoromethoxy substituent on the benzothiazole core were synthesized and evaluated against a panel of four bacterial strains Salmonella typhimurium, Staphylococcus aureus, Escherichia coli and Listeria monocytogenes and three resistant strains Pseudomonas aeruginosa, E. coli and MRSA. The evaluation of minimum bactericidal and minimum inhibitory concentrations was accomplished by microdilution assay. As reference compounds ampicillin and streptomycin were employed. All compounds displayed antibacterial efficiencies with MBCs/MICs at 0.25-1 mg/mL and 0.12-1 mg/mL respectively while ampicillin displayed MBCs/MICs at 0.15-0.3 mg/mL and at 0.1-0.2 mg/mL respectively. MICs/MBC of streptomycin varied from 0.05 to 0.15 mg/mL and from 0.1 to 0.3 mg/mL respectively. The best overall effect was observed for compound h4, while compound h1 exhibited the highest effective action against E. coli (MIC/MBC 0.12/0.25 mg/ml) among all tested compounds.

Inhibition of the GDP-d-Mannose Dehydrogenase from Pseudomonas aeruginosa Using Targeted Sugar Nucleotide Probes.

Sufferers of cystic fibrosis are at extremely high risk for contracting chronic lung infections. Over their lifetime, one bacterial strain in particular, Pseudomonas aeruginosa, becomes the dominant pathogen. Bacterial strains incur loss-of-function mutations in the mucA gene that lead to a mucoid conversion, resulting in copious secretion of the exopolysaccharide alginate. Strategies that stop the production of alginate in mucoid Pseudomonas aeruginosa infections are therefore of paramount importance. To aid in this, a series of sugar nucleotide tools to probe an enzyme critical to alginate biosynthesis, guanosine diphosphate mannose dehydrogenase (GMD), have been developed. GMD catalyzes the irreversible formation of the alginate building block, guanosine diphosphate mannuronic acid. Using a chemoenzymatic strategy, we accessed a series of modified sugar nucleotides, identifying a C6-amide derivative of guanosine diphosphate mannose as a micromolar inhibitor of GMD. This discovery provides a framework for wider inhibition strategies against GMD to be developed.

Novel bis(pyrazole-benzofuran) hybrids possessing piperazine linker: Synthesis of potent bacterial biofilm and MurB inhibitors.

Novel 1,4-bis[(2-(3-(dimethylamino)-1-oxoprop-2-en-1-yl)benzofuran-5-yl)methyl]piperazine was prepared and used as a key synthon for the this study. Therefore, 1,3-dipolar cycloaddition of this synthon with the appropriate hydrazonyl chlorides afforded a new series of bis(1,3,4-trisubstituted pyrazoles), linked via piperazine moiety. Furthermore, it reacted with hydrazine hydrate and phenyl hydrazine individually to afford the corresponding 1,4-bis[(2-(1H-pyrazolyl)benzofuran-5-yl)methyl]piperazines. Different bacterial strains and cell lines were selected to study the in-vitro antibacterial and cytotoxic activities for the new derivatives. 1,4-Bis[((2-(3-acetyl-1-(4-nitrophenyl)-1H-pyrazole-4-yl)carbonyl)benzofuran-5-yl)methyl]piperazine 5e showed the best antibacterial efficacies with MIC/MBC values of 1.2/1.2, 1.2/2.4 and 1.2/2.4 µM against each of E. coli, S. aureus and S. mutans strains, respectively. In addition, the inhibitory activity of some new bis(pyrazoles) as MRSA and VRE inhibitors were studied. Compound 5e gave the best inhibitory activity with MIC/MBC values of 18.1/36.2, 9.0/18.1 and 18.1/18.1 µM, respectively, against MRSA (ATCC:33591 and ATCC:43300) and VRE (ATCC:51575) bacterial strains, respectively. Compound 5e showed more effective biofilm inhibition activities than the reference Ciprofloxacin. It showed IC50 values of 3.0 ± 0.05, 3.2 ± 0.08 and 3.3 ± 0.07 µM against S. aureus, S. mutans and E. coli strains, respectively. Furthermore, experimental study showed excellent inhibitory activities of 1,4-bis[((2-(3-substituted-1-aryl-1H-pyrazole-4-yl)carbonyl)benzofuran-5-yl)methyl]piperazine derivatives, attached to p-NO2 or p-Cl groups, against MurB enzyme. Compound 5e gave the best MurB inhibitory activity with IC50 value of 3.1 µM. The in-silico study was performed to predict the capability of new derivatives as potential inhibitors of MurB enzyme.

Synthesis of novel 1,2-diarylpyrazolidin-3-one-based compounds and their evaluation as broad spectrum antibacterial agents.

There is a continuous need to develop new antibacterial agents with non-traditional mechanisms to combat the nonstop emerging resistance to most of the antibiotics used in clinical settings. We identified novel pyrazolidinone derivatives as antibacterial hits in an in-house library screening and synthesized several derivatives in order to improve the potency and increase the polarity of the discovered hit compounds. The oxime derivative 24 exhibited promising antibacterial activity against E. coli TolC, B. subtilis and S. aureus with MIC values of 4, 10 and 20 µg/mL, respectively. The new lead compound 24 was found to exhibit a weak dual inhibitory activity against both the E. coli MurA and MurB enzymes with IC50 values of 88.1 and 79.5 µM, respectively, which could partially explain its antibacterial effect. A comparison with the previously reported, structurally related pyrazolidinediones suggested that the oxime functionality at position 4 enhanced the activity against MurA and recovered the activity against the MurB enzyme. Compound 24 can serve as a lead for further development of novel and safe antibiotics with potential broad spectrum activity.

Unraveling the mechanism of l-gulonate-3-dehydrogenase inhibition by ascorbic acid: Insights from molecular modeling.

l-Gulonate dehydrogenase (GuDH) is a crucial enzyme in the non-phosphorylated sugar metabolism or glucuronate-xylulose (GX) pathway. Some naturally occurring compounds inhibit GuDH. Ascorbic acid is one of such inhibitors for GuDH. However, the exact mechanism by which ascorbic acid inhibits GuDH is still unknown. In this study, we try to investigate GuDH inhibition using computational approaches by generating a model for buffalo GuDH. We used this model to perform blind dockings of ascorbic acid to GuDH. Some docked conformations of ascorbic acid bind near Asp39 and have steric clashes with crystal structure conformation of NADH. To assess the dynamic stability of the GuDH-ascorbic acid complex, we performed six molecular dynamics simulations for GuDH, three each in its free form and in complex with ascorbic acid for 50 ns, to obtain 300 ns of trajectories in total. During the simulations, ascorbic acid interacted with several residues nearby Asp39. As Asp39 is an important residue for NADH binding and specificity, the interaction of ascorbic acid near Asp39 hinders further NADH binding and ultimately affects the enzymatic functioning of GuDH. In this study, we analyze these interactions between ascorbic acid and GuDH. Our analysis reveals novel details on the mechanism of GuDH inhibition by ascorbic acid.

In silico identification, synthesis and biological evaluation of novel tetrazole inhibitors of MurB.

In the context of antibacterial drug discovery resurgence, novel therapeutic targets and new compounds with alternative mechanisms of action are of paramount importance. We focused on UDP-N-acetylenolpyruvylglucosamine reductase (i.e. MurB), an underexploited target enzyme that is involved in early steps of bacterial peptidoglycan biosynthesis. On the basis of the recently reported crystal structure of MurB in complex with NADP+ , a pharmacophore model was generated and used in a virtual screening campaign with combined structure-based and ligand-based approaches. To explore chemical space around hit compounds, further similarity search and organic synthesis were employed to obtain several compounds with micromolar IC50 values on MurB. The best inhibitors in the reported series of 5-substituted tetrazol-2-yl acetamides were compounds 13, 26 and 30 with IC50 values of 34, 28 and 25 µm, respectively. None of the reported compounds possessed in vitro antimicrobial activity against Staphylococcus aureus and Escherichia coli.

Genistein inhibits glucocorticoid amplification in adipose tissue by suppression of 11ß-hydroxysteroid dehydrogenase type 1.

Excess glucocorticoids promote visceral obesity, hyperlipidemia, and insulin resistance. The main regulator of intracellular glucocorticoid levels is 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which converts inactive glucocorticoids into bioactive forms such as cortisol in humans and corticosterone in rodents. Hexose-6-phosphate dehydrogenase (H6PD), which is colocalized with 11ß-HSD1 in the intralumen of the endoplasmic reticulum, supplies a crucial coenzyme, NADPH, for full reductase activity of 11ß-HSD1. Therefore, it is possible that inhibition of 11ß-HSD1 will become a considerable medical treatment for metabolic diseases including obesity and diabetes. Genistein, a soy isoflavone, has received attention for its therapeutic potential for obesity, diabetes, and cardiovascular disease, and has been proposed as a promising compound for the treatment of metabolic disorders. However, the mechanisms underlying the pleiotropic anti-obesity effects of genistein have not been fully clarified. Here, we demonstrate that genistein was able to inhibit 11ß-HSD1 and H6PD activities within 10 or 20min, in dose- and time-dependent manners. Inhibition of 11ß-HSD2 activity was not observed in rat kidney microsomes. The inhibition was not reversed by two estrogen receptor antagonists, tamoxifen and ICI182,780. A kinetic study revealed that genistein acted as a non-competitive inhibitor of 11ß-HSD1, and its apparent Km value for 11-dehydrocorticosterone was 0.5µM. Genistein also acted as a non-competitive inhibitor of H6PD, and its apparent Km values for G6P and NADP were 0.9 and 3.3µM, respectively. These results suggest that genistein may exert its inhibitory effect by interacting with these enzymes.

Mechanism of repression of 11ß-hydroxysteroid dehydrogenase type 1 by growth hormone in 3T3-L1 adipocytes.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is an NADPH-dependent reductase that converts cortisone to cortisol in adipose tissue. We previously reported that GH and IGF-I decrease 11ß-HSD1 activity and mRNA levels in adipocytes. Hexose-6-phosphate dehydrogenase (H6PDH) is involved in the production of NADPH, which is a coenzyme for 11ß-HSD1. The aim of the present study was to clarify further the mechanism of repression of 11ß-HSD1 activity by GH using linsitinib, an IGF-I receptor inhibitor. The suppression of 11ß-HSD1 mRNA by IGF-I was attenuated in the presence of 1 µM linsitinib (17.2% vs. 53.3% of basal level, P<0.05). 11ß-HSD1 mRNA levels in cells treated with GH in the presence of 1 µM linsitinib were not different from those in absence of linsitinib (35.9% vs. 33.9%). The increase in IGF-I mRNA levels with GH and 1 µM linsitinib was not different from that in the absence of linsitinib (359% vs. 347%). H6PDH mRNA levels were significantly decreased in cells treated with IGF-I for 8 and 24 h (55.6% and 33.7%, P<0.05). In the presence of 1 µM linsitinib, there was no repression of H6PDH mRNA (111.4%). H6PDH mRNA levels were significantly decreased in cells treated with GH in the absence of linsitinib for 24 h (55.9%, P<0.05), but not for 8 h (89.5%). The presence of 1 µM linsitinib also prevented repression of H6PDH mRNA by GH over 24 h (107.8%). These results suggest that GH directly represses 11ß-HSD1 mRNA rather than acting via the IGF-I receptor, and that GH represses H6PDH through locally produced IGF-I.

Biological pretreatment with a cellobiose dehydrogenase-deficient strain of Trametes versicolor enhances the biofuel potential of canola straw.

The use of Trametes versicolor as a biological pretreatment for canola straw was explored in the context of biofuel production. Specifically, the effects on the straw of a wild-type strain (52J) and a cellobiose dehydrogenase (CDH)-deficient strain (m4D) were investigated. The xylose and glucose contents of the straw treated with 52J were significantly reduced, while only the xylose content was reduced with m4D treatment. Lignin extractability was greatly improved with fungal treatments compared to untreated straw. Saccharification of the residue of the m4D-treated straw led to a significant increase in proportional glucose yield, which was partially attributed to the lack of cellulose catabolism by m4D. Overall, the results of this study indicate that CDH facilitates cellulose access by T. versicolor. Furthermore, treatment of lignocellulosic material with m4D offers improvements in lignin extractability and saccharification efficacy compared to untreated biomass without loss of substrate due to fungal catabolism.

Identification of hotspot regions of MurB oxidoreductase enzyme using homology modeling, molecular dynamics and molecular docking techniques.

Despite the availability of effective chemotherapy and a moderately protective vaccine, new anti-tuberculosis agents are urgently needed to decrease the global incidence of tuberculosis (TB) disease. The MurB gene belongs to the bacterial cell wall biosynthesis pathway and is an essential drug target in Mycobacterium tuberculosis (Mtb) that has no mammalian counterparts. Here, we present an integrated approach involving homology modeling, molecular dynamics and molecular docking studies on Mtb-MurB oxidoreductase enzyme. A homology model of Mtb-MurB enzyme was built for the first time in order to carry out structure-based inhibitor design. The accuracy of the model was validated using different techniques. The molecular docking study on this enzyme was undertaken using different classes of well known MurB inhibitors. Estimation of binding free energy by docking analysis indicated the importance of Tyr155, Arg156, Ser237, Asn241 and His304 residues within the Mtb-MurB binding pocket. Our computational analysis is in good agreement with experimental results of site-directed mutagenesis. The present study should therefore play a guiding role in the experimental design of Mtb-MurB inhibitors for in vitro/in vivo analysis.

Electrochemical evidence of self-substrate inhibition as functions regulation for cellobiose dehydrogenase from Phanerochaete chrysosporium.

The reaction mechanism of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium, adsorbed on graphite electrodes, was investigated by following its catalytic reaction with cellobiose registered in both direct and mediated electron transfer modes between the enzyme and the electrode. A wall-jet flow through amperometric cell housing the CDH-modified graphite electrode was connected to a single line flow injection system. In the present study, it is proven that cellobiose, at concentrations higher than 200 microM, competes for the reduced state of the FAD cofactor and it slows down the transfer of electrons to any 2e(-)/H(+) acceptors or further to the heme cofactor, via the internal electron transfer pathway. Based on and proven by electrochemical results, a kinetic model of substrate inhibition is proposed and supported by the agreement between simulation of plots and experimental data. The implications of this kinetic model, called pseudo-ping-pong mechanism, on the possible functions CDH are also discussed. The enzyme exhibits catalytic activity also for lactose, but in contrast to cellobiose, this sugar does not inhibit the enzyme. This suggests that even if some other substrates are coincidentally oxidized by CDH, however, they do not trigger all the possible natural functions of the enzyme. In this respect, cellobiose is regarded as the natural substrate of CDH.

Evaluation of the metS and murB loci for antibiotic discovery using targeted antisense RNA expression analysis in Bacillus anthracis.

The biowarfare-relevant bacterial pathogen Bacillus anthracis contains two paralogs each of the metS and murB genes, which encode the important antibiotic target functions methionyl-tRNA synthetase and UDP-N-acetylenolpyruvoylglucosamine reductase, respectively. Empirical screens were conducted to detect and characterize gene fragments of each of these four genes that could cause growth reduction of B. anthracis when inducibly expressed from a plasmid-borne promoter. Numerous such gene fragments that were overwhelmingly in the antisense orientation were identified for the metS1 and murB2 alleles, while no such orientation bias was seen for the metS2 and murB1 alleles. Gene replacement mutagenesis was used to confirm the essentiality of the metS1 and murB2 alleles, and the nonessentiality of the metS2 and murB1 alleles, for vegetative growth. Induced transcription of RNA from metS1 and murB2 antisense-oriented gene fragments resulted in specific reduction of mRNA of their cognate genes. Attenuation of MetS1 enzyme expression hypersensitized B. anthracis cells to a MetS-specific antimicrobial compound but not to other antibiotics that affect cell wall assembly, fatty acid biosynthesis, protein translation, or DNA replication. Antisense-dependent reduction of MurB2 enzyme expression caused hypersensitivity to beta-lactam antibiotics, a synergistic response that has also been noted for the MurA-specific antibiotic fosfomycin. These experiments form the basis of mode-of-action detection assays that can be used in the discovery of novel MetS- or MurB-specific antibiotic drugs that are effective against B. anthracis or other gram-positive bacterial pathogens.

Pyrazolidine-3,5-diones and 5-hydroxy-1H-pyrazol-3(2H)-ones, inhibitors of UDP-N-acetylenolpyruvyl glucosamine reductase.

A series of pyrazolidine-3,5-dione and 5-hydroxy-1H-pyrazol-3(2H)-one inhibitors of Escherichia coli UDP-N-acetylenolpyruvyl glucosamine reductase (MurB) has been prepared. The 5-hydroxy-1H-pyrazol-3(2H)-ones show low micromolar IC(50) values versus E. coli MurB and submicromolar minimal inhibitory concentrations (MIC) against Staphylococcus aureus GC 1131, Enterococcus faecalis GC 2242, Streptococcus pneumoniae GC 1894, and E. coli GC 4560 imp, a strain with increased outer membrane permeability. None of these compounds show antimicrobial activity against Candida albicans, a marker of eukaryotic toxicity. Moreover, these compounds inhibit peptidoglycan biosynthesis, as assessed by measuring the amount of soluble peptidoglycan produced by Streptococcus epidermidis upon incubation with compounds. A partial least squares projection to latent structures analysis shows that improving MurB potency and MIC values correlate with increasing lipophilicity of the C-4 substituent of the 5-hydroxy-1H-pyrazol-3(2H)-one core. Docking studies using FLO and PharmDock produced several binding orientations for these molecules in the MurB active site.

3,5-dioxopyrazolidines, novel inhibitors of UDP-N- acetylenolpyruvylglucosamine reductase (MurB) with activity against gram-positive bacteria.

A series of 3,5-dioxopyrazolidines was identified as novel inhibitors of UDP-N-acetylenolpyruvylglucosamine reductase (MurB). Compounds 1 to 3, which are 1,2-bis(4-chlorophenyl)-3,5-dioxopyrazolidine-4-carboxamides, inhibited Escherichia coli MurB, Staphyloccocus aureus MurB, and E. coli MurA with 50% inhibitory concentrations (IC50s) in the range of 4.1 to 6.8 microM, 4.3 to 10.3 microM, and 6.8 to 29.4 microM, respectively. Compound 4, a C-4-unsubstituted 1,2-bis(3,4-dichlorophenyl)-3,5-dioxopyrazolidine, showed moderate inhibitory activity against E. coli MurB, S. aureus MurB, and E. coli MurC (IC50s, 24.5 to 35 microM). A fluorescence-binding assay indicated tight binding of compound 3 with E. coli MurB, giving a dissociation constant of 260 nM. Structural characterization of E. coli MurB was undertaken, and the crystal structure of a complex with compound 4 was obtained at 2.4 A resolution. The crystal structure indicated the binding of a compound at the active site of MurB and specific interactions with active-site residues and the bound flavin adenine dinucleotide cofactor. Peptidoglycan biosynthesis studies using a strain of Staphylococcus epidermidis revealed reduced peptidoglycan biosynthesis upon incubation with 3,5-dioxopyrazolidines, with IC50s of 0.39 to 11.1 microM. Antibacterial activity was observed for compounds 1 to 3 (MICs, 0.25 to 16 microg/ml) and 4 (MICs, 4 to 8 microg/ml) against gram-positive bacteria including methicillin-resistant S. aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.

Inactivation of GDP-mannose dehydrogenase from Pseudomonas aeruginosa by penicillic acid identifies a critical active site loop.

The pathogenic bacterium Pseudomonas aeruginosa synthesizes alginate as one of a group of virulence factors that are produced during infections. The enzyme GDP-mannose dehydrogenase catalyzes the committed step in alginate biosynthesis. We show here that penicillic acid is an irreversible inactivator of GDP-mannose dehydrogenase. Inactivation occurs with a rate constant of 0.39+/-0.01 mM(-1) min(-1) at pH 8.0, and does not exhibit saturation behavior. Partial protection from inactivation is afforded by GDP-mannose, but not by the other substrate, NAD+. GMP and NAD+ together provide complete protection against inactivation. Analysis by mass spectrometry confirmed that the enzyme is alkylated at multiple cysteine residues by penicillic acid, including Cys 213, Cys 246, and the active site cysteine, Cys 268. However, the pH dependence of the inactivation rate suggested that alkylation of a single cysteine residue is sufficient to inactivate the enzyme. The C268A mutant protein was also susceptible to inactivation by penicillic acid. The presence of NAD+ and GMP provided partial protection of Cys 246 and Cys 268, and almost complete protection of Cys 213. Cys 213 is located on a helix that forms part of the binding pocket for GDP-mannose, and forms a hydrogen bond with Asn 252. Asn 252 is located on a loop that surrounds GDP-mannose. The C213A mutant enzyme exhibits a Vmax that is 1.8-fold greater than the wild-type enzyme, suggesting that the interaction between Cys 213 and Asn 252 helps to hold the loop in place during catalysis, and that opening the loop to release product is partially rate-limiting. Cys 246 is adjacent to the GDP-mannose binding loop, and its alkylation may also interfere with loop movement.

Structural and functional characterization of rabbit and human L-gulonate 3-dehydrogenase.

L-Gulonate 3-dehydrogenase (GDH) catalyzes the NAD(+)-linked dehydrogenation of L-gulonate into dehydro-L-gulonate in the uronate cycle. In this study, we isolated the enzyme and its cDNA from rabbit liver, and found that the cDNA is identical to that for rabbit lens lambda-crystallin except for lacking a codon for Glu(309). The same cDNA species, but not the lambda-crystallin cDNA with the codon for Glu(309), was detected in the lens, which showed the highest GDH activity among rabbit tissues. In addition, recombinant human lambda-crystallin that lacks Glu(309) displays enzymatic properties similar to rabbit GDH. These data indicate that GDH is recruited as lambda-crystallin without gene duplication. An outstanding feature of GDH is modulation of its activity by low concentrations of P(i), which decreases the catalytic efficiency in a dose dependent manner. P(i) also protects the enzyme against both thermal and urea denaturation. Kinetic analysis suggests that P(i) binds to both the free enzyme and its NAD(H)-complex in the sequential ordered mechanism. Furthermore, we examined the roles of Asp(36), Ser(124), His(145), Glu(157 )and Asn(196) in the catalytic function of rabbit GDH by site-directed mutagenesis. The D36R mutation leads to a switch in favor of NADP(H) specificity, suggesting an important role of Asp(36) in the coenzyme specificity. The S124A mutation decreases the catalytic efficiency 500-fold, and the H145Q, N196Q and N195D mutations result in inactive enzyme forms, although the E157Q mutation produces no large kinetic alteration. Thus, Ser(124), His(145) and Asn(196) may be critical for the catalytic function of GDH.

Multiconformational states in phosphoglycerate dehydrogenase.

Phosphoglycerate dehydrogenase (PGDH) catalyzes the first step in the serine biosynthetic pathway. In lower plants and bacteria, the PGDH reaction is regulated by the end-product of the pathway, serine. The regulation occurs through a V(max) mechanism with serine binding and inhibition occurring in a cooperative manner. The three-dimensional structure of the serine inhibited enzyme, determined by previous work, showed a tetrameric enzyme with 222 symmetry and an unusual overall toroidal appearance. To characterize the allosteric, cooperative effects of serine, we identified W139G PGDH as an enzymatically active mutant responsive to serine but not in a cooperative manner. The position of W139 near a subunit interface and the active site cleft suggested that this residue is a key player in relaying allosteric effects. The 2.09 A crystal structure of W139G-PGDH, determined in the absence of serine, revealed major quaternary and tertiary structural changes. Contrary to the wildtype enzyme where residues encompassing residue 139 formed extensive intersubunit contacts, the corresponding residues in the mutant were conformationally flexible. Within each of the three-domain subunits, one domain has rotated approximately 42 degrees relative to the other two. The resulting quaternary structure is now in a novel conformation creating new subunit-to-subunit contacts and illustrates the unusual flexibility in this V(max) regulated enzyme. Although changes at the regulatory domain interface have implications in other enzymes containing a similar regulatory or ACT domain, the serine binding site in W139G PGDH is essentially unchanged from the wildtype enzyme. The structural and previous biochemical characterization of W139G PGDH suggests that the allosteric regulation of PGDH is mediated not only by changes occurring at the ACT domain interface but also by conformational changes at the interface encompassing residue W139.

Combinatorial enzymatic assay for the screening of a new class of bacterial cell wall inhibitors.

We have developed a screening assay by thin-layer chromatography (TLC) to identify inhibitors for the bacterial essential enzymes MurA, -B, and -C. Libraries of compounds were synthesized using the mix-and-split combinatorial chemistry approach. Screening of the pooled compounds using the developed assay revealed the presence of many pools active in vitro. Pools of interest were tested for antibacterial activity. Individual molecules in the active pools were synthesized and retested with the TLC assay and with bacteria. We focused on the best five compounds for further analysis. They were tested for inhibition on each of the three enzymes separately, and showed no inhibition of MurA or MurB activity but were all inhibitors of MurC enzyme. This approach yielded interesting lead compounds for the development of novel antibacterial agents.