New Sight: Enzymes as Targets for Drug Development.

In the dynamic realm of medical research, a resounding chord is struck by recent studies that have propelled drug discovery to new horizons across a spectrum of disciplines [...].

Enzymes as Targets for Drug Development II.

Enzymes are viewed as the most desirable targets for drug development by the pharmaceutical community [...].

Development of Hydroxamic Acid Compounds for Inhibition of Metallo-ß-Lactamase from Bacillus anthracis.

The emergence of resistant bacteria takes place, endangering the effectiveness of antibiotics. A reason for antibiotic resistance is the presence of lactamases that catalyze the hydrolysis of ß-lactam antibiotics. An inhibitor of serine-ß-lactamases such as clavulanic acid binds to the active site of the enzymes, thus solving the resistance problem. A pressing issue, however, is that the reaction mechanism of metallo-ß-lactamases (MBLs) hydrolyzing ß-lactam antibiotics differs from that of serine-ß-lactamases due to the existence of zinc ions in the active site of MBLs. Thus, the development of potential inhibitors for MBLs remains urgent. Here, the ability to inhibit MBL from Bacillus anthracis (Bla2) was investigated in silico and in vitro using compounds possessing two hydroxamate functional groups such as 3-chloro-N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)benzamide (Compound 4) and N-hydroxy-4-(7-(hydroxyamino)-7-oxoheptyl)-3-methoxybenzamide (Compound 6). In silico docking and molecular dynamics simulations revealed that both Compounds 4 and 6 were coordinated with zinc ions in the active site, suggesting that the hydroxamate group attached to the aromatic ring of the compound plays a crucial role in the coordination to the zinc ions. In vitro kinetic analysis demonstrated that the mode of inhibitions for Compounds 4 and 6 were a competitive inhibition with Ki values of 6.4 ± 1.7 and 4.7 ± 1.4 kcal/mol, respectively. The agreement between in silico and in vitro investigations indicates that compounds containing dihyroxamate moieties may offer a new avenue to overcome antibiotic resistance to bacteria.

Novel Peptide-Based Inhibitors for Microtubule Polymerization in Phytophthora capsici.

The plant disease Phytophthora blight, caused by the oomycete pathogen Phytophthora capsici, is responsible for major economic losses in pepper production. Microtubules have been an attractive target for many antifungal agents as they are involved in key cellular events such as cell proliferation, signaling, and migration in eukaryotic cells. In order to design a novel biocompatible inhibitor, we screened and identified inhibitory peptides against alpha- and beta-tubulin of P. capsici using a phage display method. The identified peptides displayed a higher binding affinity (nanomolar range) and improved specificity toward P. capsici alpha- and beta-tubulin in comparison to Homo sapiens tubulin as evaluated by fluorometric analysis. One peptide demonstrated the high inhibitory effect on microtubule formation with a nanomolar range of IC50 values, which were much lower than a well-known chemical inhibitor-benomyl (IC50 = 500 µM). Based on these results, this peptide can be employed to further develop promising candidates for novel antifungal agents against Phytophthora blight.

Inhibition of anthrax lethal factor by ssDNA aptamers.

Anthrax is caused by Bacillus anthracis, a bacterium that is able to secrete the toxins protective antigen, edema factor and lethal factor. Due to the high level of secretion from the bacteria and its severe virulence, lethal factor (LF) has been sought as a biomarker for detecting bacterial infection and as an effective target to neutralize toxicity. In this study, we found three aptamers, and binding affinity was determined by fluorescently labeled aptamers. One of the aptamers exhibited high affinity, with a Kd value of 11.0 ±â€¯2.7 nM, along with low cross reactivity relative to bovine serum albumin and protective antigen. The therapeutic functionality of the aptamer was examined by assessing the inhibition of LF protease activity against a mitogen-activated protein kinase kinase. The aptamer appears to be an effective inhibitor of LF with an IC50 value of 15 ±â€¯1.5 µM and approximately 85% cell viability, suggesting that this aptamer provides a potential clue for not only development of a sensitive diagnostic device of B. anthracis infection but also the design of novel inhibitors of LF.

Inhibition of Bacillus anthracis metallo-ß-lactamase by compounds with hydroxamic acid functionality.

Metallo-ß-lactamases (MBLs) that catalyze hydrolysis of ß-lactam antibiotics are an emerging threat due to their rapid spread. A strain of the bacterium Bacillus anthracis has its ability to produce and secrete a MBL, referred to Bla2. To address this challenge, novel hydroxamic acid-containing compounds such as 3-(heptyloxy)-N-hydroxybenzamide (compound 4) and N-hydroxy-3-((6-(hydroxyamino)-6-oxohexyl)oxy)benzamide (compound 7) were synthesized. Kinetic analysis of microbial inhibition indicated that the both sides of hydroxamic acids containing compound 7 revealed a reversible, competitive inhibition with a Ki value of 0.18 ± 0.06 µM. The result has reflected that the both sides of dihydroxamic acids in a molecule play a crucial role in the binding affinity rather than monohydroxamic containing compound 4 which was unable to inhibit Bla2. In addition, in silico analysis suggested that compound 7 was coordinated with a zinc ion in the active site of enzyme. These observations suggest that the dihydroxamic acid-containing compound may be a promising drug candidate, and a further implication for designing new inhibitors of Bla2.

Inhibitory Effect of Bridged Nucleosides on Thermus aquaticus DNA Polymerase and Insight into the Binding Interactions.

Modified nucleosides have the potential to inhibit DNA polymerases for the treatment of viral infections and cancer. With the hope of developing potent drug candidates by the modification of the 2',4'-position of the ribose with the inclusion of a bridge, efforts were focused on the inhibition of Taq DNA polymerase using quantitative real time PCR, and the results revealed the significant inhibitory effects of 2',4'-bridged thymidine nucleoside on the polymerase. Study on the mode of inhibition revealed the competitive mechanism with which the 2',4'-bridged thymidine operates. With a Ki value of 9.7 ± 1.1 µM, the 2',4'-bridged thymidine proved to be a very promising inhibitor. Additionally, docking analysis showed that all the nucleosides including 2',4'-bridged thymidine were able to dock in the active site, indicating that the substrate analogs reflect a structural complementarity to the enzyme active site. The analysis also provided evidence that Asp610 was a key binding site for 2',4'-bridged thymidine. Molecular dynamics (MD) simulations were performed to further understand the conformational variations of the binding. The root-mean-square deviation (RMSD) values for the peptide backbone of the enzyme and the nitrogenous base of the inhibitor stabilized within 0.8 and 0.2 ns, respectively. Furthermore, the MD analysis indicates substantial conformational change in the ligand (inhibitor) as the nitrogenous base rotated anticlockwise with respect to the sugar moiety, complemented by the formation of several new hydrogen bonds where Arg587 served as a pivot axis for binding formation. In conclusion, the active site inhibition of Taq DNA polymerase by 2',4'-bridged thymidine suggests the potential of bridged nucleosides as drug candidates.

Arabidopsis thaliana Nfu2 accommodates [2Fe-2S] or [4Fe-4S] clusters and is competent for in vitro maturation of chloroplast [2Fe-2S] and [4Fe-4S] cluster-containing proteins.

Nfu-type proteins are essential in the biogenesis of iron-sulfur (Fe-S) clusters in numerous organisms. A number of phenotypes including low levels of Fe-S cluster incorporation are associated with the deletion of the gene encoding a chloroplast-specific Nfu-type protein, Nfu2 from Arabidopsis thaliana (AtNfu2). Here, we report that recombinant AtNfu2 is able to assemble both [2Fe-2S] and [4Fe-4S] clusters. Analytical data and gel filtration studies support cluster/protein stoichiometries of one [2Fe-2S] cluster/homotetramer and one [4Fe-4S] cluster/homodimer. The combination of UV-visible absorption and circular dichroism and resonance Raman and Mössbauer spectroscopies has been employed to investigate the nature, properties, and transfer of the clusters assembled on Nfu2. The results are consistent with subunit-bridging [2Fe-2S](2+) and [4Fe-4S](2+) clusters coordinated by the cysteines in the conserved CXXC motif. The results also provided insight into the specificity of Nfu2 for the maturation of chloroplastic Fe-S proteins via intact, rapid, and quantitative cluster transfer. [2Fe-2S] cluster-bound Nfu2 is shown to be an effective [2Fe-2S](2+) cluster donor for glutaredoxin S16 but not glutaredoxin S14. Moreover, [4Fe-4S] cluster-bound Nfu2 is shown to be a very rapid and efficient [4Fe-4S](2+) cluster donor for adenosine 5'-phosphosulfate reductase (APR1), and yeast two-hybrid studies indicate that APR1 forms a complex with Nfu2 but not with Nfu1 and Nfu3, the two other chloroplastic Nfu proteins. This cluster transfer is likely to be physiologically relevant and is particularly significant for plant metabolism as APR1 catalyzes the second step in reductive sulfur assimilation, which ultimately results in the biosynthesis of cysteine, methionine, glutathione, and Fe-S clusters.

Kinetic characterization of a slow-binding inhibitor of Bla2: thiomaltol.

The increasing prevalence of drug resistant bacteria is a pandemic problem. Metallo-ß-lactamases (MBLs) are one of the main causes of drug resistance due to hydrolysis of ß-lactam antibiotics. Thus, the development of effective inhibitors of MBLs remains urgent. The compound thiomaltol was used as a lead compound to investigate its ability to inhibit metallo-ß-lactamase from Bacillus anthracis (Bla2), which causes anthrax. Kinetic evaluation with nitrocefin as a substrate indicates that thiomaltol inhibits Bla2 in a time-dependent manner with an IC(50) value of 290 µM after 20 min preincubation. Progress curve analysis and reversibility tests suggest that thiomaltol is a reversible, slow-binding inhibitor with a K(i) of 85 ± 30 µM. Furthermore, studies on the modality of inhibition and in silico analysis indicate thiomaltol to be a competitive inhibitor. The results demonstrate that thiomaltol is a promising lead compound for slow binding inhibitor design of Bla2.

Homologous Alkalophilic and Acidophilic L-Arabinose isomerases reveal region-specific contributions to the pH dependence of activity and stability.

To study the pH dependence of l-arabinose isomerase (AI) activity and stability, we compared homologous AIs with their chimeras. This study demonstrated that an ionizable amino acid near the catalytic site determines the optimal pH (pH(opt)) for activity, whereas the N-terminal surface R residues play an important role in determining the pH(opt) for stability.

Proteolytic assay-based screening identifies a potent inhibitor of anthrax lethal factor.

Anthrax lethal factor (LF), a Zn(2+)-dependent metalloprotease, is a key virulence component of anthrax toxin. Here, we used proteolytic assay-based screening to identify novel LF inhibitors from a naturally extracted chemical library. The screening identified four compounds that inhibited in vitro proteolytic activity of LF with an IC(50) of low micromolar range (11-20 µM). Three of these compounds were toxic to the mouse macrophage-like cell line, RAW 264.7. Compound 200 was non-toxic, however, and successfully protected Raw 264.7 cells from a lethal toxin challenge with an IC(50) of 39.2 µM. We also identified possible binding modes of compound 200 by molecular docking.

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803.

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E(m)) value of -165mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E(m) value of -220mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.

Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit.

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3µM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1µM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7Å distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.

The oxidation state of active site thiols determines activity of saccharopine dehydrogenase at low pH.

Saccharopine dehydrogenase catalyzes the NAD-dependent conversion of saccharopine to generate L-lysine and α-ketoglutarate. A disulfide bond between cysteine 205 and cysteine 249, in the vicinity of the dinucleotide-binding site, is observed in structures of the apoenzyme, while a dithiol is observed in a structure with AMP bound, suggesting preferential binding of the dinucleotide to reduced enzyme. Mutation of C205 to S gave increased values of V/E(t) and V/KE(t) at pH 7 compared to wild type. Primary deuterium and solvent deuterium kinetic isotope effects suggest the catalytic pathway, which includes the hydride transfer and hydrolysis steps, contributes more to rate limitation in C205S, but the rates of the two steps relative to one another remain the same. There is a large increase in the rate constants V1/E(t) and V1/K(NAD)Et at pH values below 7 compared to WT. Data indicate the low pH increase in activity results from a decreased sensitivity of the C205S mutant enzyme to the protonation state of an enzyme group with a pK(a) of about 7, likely responsible for a pH-dependent conformational change. Reduction of WT and C205S mutant enzymes with TCEP gives equal activities at pH 6, consistent with the increased activity observed for the C205S mutant enzyme.

Purification development and characterization of the zinc-dependent metallo-ß-lactamase from Bacillus anthracis.

Metallo-ß-lactamase from Bacillus anthracis (Bla2) catalyzes the hydrolysis of ß-lactam antibiotics which are commonly prescribed to combat bacterial infections. Bla2 contributes to the antibiotic resistance of this bacterium. An understanding of it is necessary to design potential inhibitors that can be introduced with current antibiotics for effective eradication of anthrax infections. We have purified Bla2 using Ni(2+)-affinity chromatography with over 140-fold increase in activity with a yield of 3.5%. The final specific activity was 19,000 units/mg. Purified Bla2 displays different K ( m ), V ( max ), and (k ( cat ) /K (M)) with penicillin G and cephalexin as substrates and is also sensitive to pH, with maximum activity between pH 7.0-9.0. The IC(50) (50% inhibition concentration) value of EDTA against Bla2 is 630 nM, which can be understood by observing its three-dimensional interaction with the enzyme.

Screening and characterization of high-affinity ssDNA aptamers against anthrax protective antigen.

The protective antigen (PA) of Bacillus anthracis is a secreted protein that functions as a critical virulence factor. Protective antigen has been selected as a biomarker in detecting bacterial infection. The in vitro selection method, systematic evolution of ligands by exponential enrichment (SELEX), was used to find single-stranded DNAs that were tightly bound to PA. After 8 rounds of the SELEX process with PA, 4 different oligonucleotides (referred to as aptamers) that contain a 30-residue ssDNA sequence were identified. Dissociation constant (K(d)) values with Cy3-attached aptamers were determined via fluorophotometry to be within a nanomolar range. The authors attempted to visualize the detection of PA using an aptamer-based enzyme-linked immunosorbent assay method, which has proven to be successful within a nanomolar K(d) value range. Furthermore, 2 of the 4 aptamers exhibited specificity to PA against bovine serum albumin and bovine serum. The results of this study demonstrate the analytical potential of an oligonucleotide-based biosensor for a wide variety of applications, particularly in diagnosing disease through specific protein biomarkers.

Redox properties of a thioredoxin-like Arabidopsis protein, AtTDX.

AtTDX is an enzyme present in Arabidopsis thaliana which is composed of two domains, a thioredoxin (Trx)-motif containing domain and a tetratricopeptide (TPR)-repeat domain. This enzyme has been shown to function as both a thioredoxin and a chaperone. The midpoint potential (E(m)) of AtTDX was determined by redox titrations using the thiol-specific modifiers, monobromobimane (mBBr) and mal-PEG. A NADPH/Trx reductase (NTR) system was used both to validate these E(m) determination methods and to demonstrate that AtTDX is an electron-accepting substrate for NTR. Titrations of full-length AtTDX revealed the presence of a single two-electron couple with an E(m) value of approximately -260 mV at pH 7.0. The two cysteines present in a typical, conserved Trx active site (WCGPC), which are likely to play a role in the electron transfer processes catalyzed by AtTDX, have been replaced by serines by site-directed mutagenesis. These replacements (i.e., C304S, C307S, and C304S/C307S) resulted in a complete loss of the redox process detected using either the mBBr or mal-PEG method to monitor disulfide/dithiol redox couples. This result supports the conclusion that the couple with an E(m) value of -260 mV is a disulfide/dithiol couple involving Cys304 and Cys307. Redox titrations for the separately-expressed Trx-motif containing C-domain also revealed the presence of a single two-electron couple with an E(m) value of approximately -260 mV at 20°C. The fact that these two E(m) values are identical, provides additional support for assignment of the redox couple to a disulfide/dithiol involving C304 and C307. It was found that, while the disulfide/dithiol redox chemistry of AtTDX was not affected by increasing the temperature to 40°C, no redox transitions were observed at 50°C and higher temperatures. In contrast, Escherichia coli thioredoxin was shown to remain redox-active at temperatures as high as 60°C. The temperature-dependence of the AtTDX redox titration is similar to that observed for the redox activity of the protein in enzymatic assays.

Overexpression of rice isoflavone reductase-like gene (OsIRL) confers tolerance to reactive oxygen species.

Isoflavone reductase is an enzyme involved in isoflavonoid biosynthesis in plants. However, rice isoflavone reductase-like gene (OsIRL, accession no. AY071920) has not been unraveled so far. Here, we have characterized its behavior in response to oxidizing agents. Using Northern and Western blot analyses, the OsIRL gene and protein were shown to be down-regulated in young seedling roots treated with reduced glutathione (GSH) and diphenyleneiodonium (DPI), known quenchers of reactive oxygen species (ROS). The OsIRL transcript level in rice suspension-cultured cells was also found to be induced by oxidants such as hydrogen peroxide (H(2)O(2)), ferric chloride (FeCl(3)), methyl viologen (MV) and glucose/glucose oxidase (G/GO), but down-regulated when co-treated with GSH. Furthermore, to investigate whether overexpression of OsIRL in transgenic rice plants promotes resistance to ROS, we generated transgenic rice lines overexpressing the OsIRL gene under an abscisic acid (ABA) inducible promoter. Results showed that the OsIRL transgenic rice line activated by ABA treatment was tolerant against MV and G/GO-induced stress in rice leave and suspension-cultured cells. Our results strongly suggest the involvement of OsIRL in homeostasis of ROS.

Ternary protein complex of ferredoxin, ferredoxin:thioredoxin reductase, and thioredoxin studied by paramagnetic NMR spectroscopy.

In oxygenic photosynthetic cells, carbon metabolism is regulated by a light-dependent redox signaling pathway through which the light signal is transmitted in the form of electrons via a redox chain comprising ferredoxin (Fd), ferredoxin:thioredoxin reductase (FTR), and thioredoxin (Trx). Trx affects the activity of a variety of enzymes via dithiol oxidation and reduction reactions. FTR reduces an intramolecular disulfide bridge of Trx, and Trx reduction involves a transient cross-link with FTR. NMR spectroscopy was used to investigate the interaction of Fd, FTR, and an m-type Trx. NMR titration experiments indicate that FTR uses distinct sites to bind Fd and Trx simultaneously to form a noncovalent ternary complex. The orientation of Trx-m relative to FTR was determined from the intermolecular paramagnetic broadening caused by the [4Fe-4S] cluster of FTR. Two models of the noncovalent binary complex of FTR/Trx-m based on the paramagnetic distance restraints were obtained. The models suggest that either a modest or major rotational movement of Trx must take place when the noncovalent binary complex proceeds to the covalent complex. This study demonstrates the complementarity of paramagnetic NMR and X-ray diffraction of crystals in the elucidation of dynamics in a transient protein complex.