Structures of fungal and plant acetohydroxyacid synthases.

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, is a flavin adenine dinucleotide-, thiamine diphosphate- and magnesium-dependent enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids1. It is the target for more than 50 commercial herbicides2. AHAS requires both catalytic and regulatory subunits for maximal activity and functionality. Here we describe structures of the hexadecameric AHAS complexes of Saccharomyces cerevisiae and dodecameric AHAS complexes of Arabidopsis thaliana. We found that the regulatory subunits of these AHAS complexes form a core to which the catalytic subunit dimers are attached, adopting the shape of a Maltese cross. The structures show how the catalytic and regulatory subunits communicate with each other to provide a pathway for activation and for feedback inhibition by branched-chain amino acids. We also show that the AHAS complex of Mycobacterium tuberculosis adopts a similar structure, thus demonstrating that the overall AHAS architecture is conserved across kingdoms.

Inhibitors for metallo-ß-lactamases from the B1 and B3 subgroups provide an avenue to combat a major mechanism of antibiotic resistance.

Metallo-ß-lactamases (MBLs) are a group of Zn(II)-dependent enzymes that pose a major threat to global health. They are linked to an increasing number of multi-drug resistant bacterial pathogens, but no clinically useful inhibitor is yet available. Since ß-lactam antibiotics, which are inactivated by MBLs, constitute ∼65% of all antibiotics used to treat infections, the search for clinically relevant MBL inhibitors is urgent. Here, derivatives of a 2-amino-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile (1a) were synthesised and their inhibitory effects assessed against prominent representatives of the MBL family. Several compounds are potent inhibitors of each MBL tested, making them promising candidates for the development of broad-spectrum drug leads. In particular, compound 5f is highly potent across the MBL family, with Ki values in the low µM range. Furthermore, this compound also appears to display synergy in combination with antibiotics such as penicillin G, cefuroxime or meropenem. This molecule thus represents a promising starting point to develop new drugs to inhibit a major mechanism of antibiotic resistance.

Discovery of a Pyrimidinedione Derivative with Potent Inhibitory Activity against Mycobacterium tuberculosis Ketol-Acid Reductoisomerase.

New drugs aimed at novel targets are urgently needed to combat the increasing rate of drug-resistant tuberculosis (TB). Herein, the National Cancer Institute Developmental Therapeutic Program (NCI-DTP) chemical library was screened against a promising new target, ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway. From this library, 6-hydroxy-2-methylthiazolo[4,5-d]pyrimidine-5,7(4H,6H)-dione (NSC116565) was identified as a potent time-dependent inhibitor of Mycobacterium tuberculosis (Mt) KARI with a Ki of 95.4 nm. Isothermal titration calorimetry studies showed that this inhibitor bound to MtKARI in the presence and absence of the cofactor, nicotinamide adenine dinucleotide phosphate (NADPH), which was confirmed by crystal structures of the compound in complex with closely related Staphylococcus aureus KARI. It is also shown that NSC116565 inhibits the growth of H37Ra and H37Rv strains of Mt with MIC50 values of 2.93 and 6.06 µm, respectively. These results further validate KARI as a TB drug target and show that NSC116565 is a promising lead for anti-TB drug development.

Discovery, Synthesis and Evaluation of a Ketol-Acid Reductoisomerase Inhibitor.

Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway, is a potential drug target for bacterial infections including Mycobacterium tuberculosis. Here, we have screened the Medicines for Malaria Venture Pathogen Box against purified M. tuberculosis (Mt) KARI and identified two compounds that have Ki values below 200 nm. In Mt cell susceptibility assays one of these compounds exhibited an IC50 value of 0.8 µm. Co-crystallization of this compound, 3-((methylsulfonyl)methyl)-2H-benzo[b][1,4]oxazin-2-one (MMV553002), in complex with Staphylococcus aureus KARI, which has 56 % identity with Mt KARI, NADPH and Mg2+ yielded a structure to 1.72 Šresolution. However, only a hydrolyzed product of the inhibitor (i.e. 3-(methylsulfonyl)-2-oxopropanic acid, missing the 2-aminophenol attachment) is observed in the active site. Surprisingly, Mt cell susceptibility assays showed that the 2-aminophenol product is largely responsible for the anti-TB activity of the parent compound. Thus, 3-(methylsulfonyl)-2-oxopropanic acid was identified as a potent KARI inhibitor that could be further explored as a potential biocidal agent and we have shown 2-aminophenol, as an anti-TB drug lead, especially given it has low toxicity against human cells. The study highlights that careful analysis of broad screening assays is required to correctly interpret cell-based activity data.

Inhibition studies of ketol-acid reductoisomerases from pathogenic microorganisms.

Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid (BCAA) biosynthesis pathway, is an emerging target for the discovery of biocides. Here, we demonstrate that cyclopropane-1,1-dicarboxylate (CPD) inhibits KARIs from the pathogens Mycobacterium tuberculosis (Mt) and Campylobacter jejuni (Cj) reversibly with Ki values of 3.03 µM and 0.59 µM, respectively. Another reversible inhibitor of both KARIs, Hoe 704, is more potent than CPD with Ki values of 300 nM and 110 nM for MtKARI and CjKARI, respectively. The most potent inhibitor tested here is N-hydroxy-N-isopropyloxamate (IpOHA). It has a Ki of ~26 nM for MtKARI, but binds rather slowly (kon ~900 M-1s-1). In contrast, IpOHA binds more rapidly (kon ~7000 M-1s-1) to CjKARI and irreversibly.

Rapid discovery of a selective butyrylcholinesterase inhibitor using structure-based virtual screening.

Acetylcholinesterase inhibitors are the mainstay of Alzheimer's disease treatments, despite having only short-term symptomatic benefits and severe side effects. Selective butyrylcholinesterase inhibitors (BuChEIs) may be more effective treatments in late-stage Alzheimer's disease with fewer side effects. Virtual screening is a powerful tool for identifying potential inhibitors in large digital compound databases. This study used structure-based virtual screening combined with physicochemical filtering to screen the InterBioScreen and Maybridge databases for novel selective BuChEIs. The workflow rapidly identified 22 potential hits in silico, resulting in the discovery of a human BuChEI with low-micromolar potency in vitro (IC50 2.4 µM) and high selectivity for butyrylcholinesterase over acetylcholinesterase. The compound was a rapidly reversible BuChEI with mixed-model in vitro inhibition kinetics. The binding interactions were investigated using in silico molecular dynamics and by developing structure-activity relationships using nine analogues. The compound also displayed high permeability in an in vitro model of the blood-brain barrier.

Design, synthesis, biological evaluation and in silico studies of certain aryl sulfonyl hydrazones conjugated with 1,3-diaryl pyrazoles as potent metallo-ß-lactamase inhibitors.

Based on a structure-guided approach, aryl sulfonyl hydrazones conjugated with 1,3-diaryl pyrazoles were designed to target metallo-ß-lactamases (MBLs), using Klebsiella pneumoniaeNDM-1 as a model. The in vitro MBLs inhibition showed remarkable inhibition constant for most of the designed compounds at a low micromolar range (1.5-16.4 µM) against NDM-1, IMP-1 and AIM-1 MBLs. Furthermore, all compounds showed promising antibacterial activity against (K+, K1-K9) resistant clinical isolates of K. pneumoniae and were able to re-sensitize resistant K. pneumoniae (K5) strain towards meropenem and cefalexin. Besides, in vivo toxicity testing exhibited that the most active compound was non-toxic and well tolerated by the experimental animals orally up to 350 mg/kg and up to 125 mg/kg parenterally. The docking experiments on NDM-1 and IMP-1 rationalized the observed in vitro MBLs inhibition activity. Generally, this work presents a fruitful matrix to extend the chemical space for MBLs inhibition. This aids in tackling drug-resistance issues in antibacterial treatment.

The Binding Mode of an ADP Analogue to a Metallohydrolase Mimics the Likely Transition State.

Purple acid phosphatases (PAPs) are members of the large family of metallohydrolases, a group of enzymes that perform a wide range of biological functions, while employing a highly conserved catalytic mechanism. PAPs are found in plants, animals and fungi; in humans they play an important role in bone turnover and are thus of interest for developing treatments for osteoporosis. The majority of metallohydrolases use a metal-bound hydroxide to initiate catalysis, which leads to the formation of a proposed five-coordinate oxyphosphorane species in the transition state. In this work, we crystallized PAP from red kidney beans (rkbPAP) in the presence of both adenosine and vanadate. The in crystallo-formed vanadate analogue of ADP provides detailed insight into the binding mode of a PAP substrate, captured in a structure that mimics the putative fivecoordinate transition state. Our observations not only provide unprecedented insight into the mechanism of metallohydrolases, but might also guide the structure-based design of inhibitors for application in the treatment of several human illnesses.

Visualization of the Reaction Trajectory and Transition State in a Hydrolytic Reaction Catalyzed by a Metalloenzyme.

Metallohydrolases are a vast family of enzymes that play crucial roles in numerous metabolic pathways. Several members have emerged as targets for chemotherapeutics. Knowledge about their reaction mechanisms and associated transition states greatly aids the design of potent and highly specific drug leads. By using a high-resolution crystal structure, we have probed the trajectory of the reaction catalyzed by purple acid phosphatase, an enzyme essential for the integrity of bone structure. In particular, the transition state is visualized, thus providing detailed structural information that may be exploited in the design of specific inhibitors for the development of new anti-osteoporotic chemotherapeutics.

Crystal Structures of Staphylococcus aureus Ketol-Acid Reductoisomerase in Complex with Two Transition State Analogues that Have Biocidal Activity.

Ketol-acid reductoisomerase (KARI) is an NAD(P)H and Mg2+ -dependent enzyme of the branched-chain amino acid (BCAA) biosynthesis pathway. Here, the first crystal structures of Staphylococcus aureus (Sa) KARI in complex with two transition state analogues, cyclopropane-1,1-dicarboxylate (CPD) and N-isopropyloxalyl hydroxamate (IpOHA) are reported. These compounds bind competitively and in multi-dentate manner to KARI with Ki values of 2.73 µm and 7.9 nm, respectively; however, IpOHA binds slowly to the enzyme. Interestingly, intact IpOHA is present in only ≈25 % of binding sites, whereas its deoxygenated form is present in the remaining sites. This deoxy form of IpOHA binds rapidly to Sa KARI, but with much weaker affinity (Ki =21 µm). Thus, our data pinpoint the origin of the slow binding mechanism of IpOHA. Furthermore, we propose that CPD mimics the early stage of the catalytic reaction (preceding the reduction step), whereas IpOHA mimics the late stage (after the reduction took place). These structural insights will guide strategies to design potent and rapidly binding derivatives of these compounds for the development of novel biocides.

Claisen rearrangements of benzyl vinyl ethers: theoretical investigation of mechanism, substituent effects, and regioselectivity.

Recently we reported the aromatic Claisen rearrangements of benzyl ketene acetals, which form one of the few examples of aromatic Claisen rearrangements involving benzyl vinyl ethers (as opposed to allyl aryl ethers, which are the usual substrates for aromatic Claisen rearrangements). Theoretical calculations predict that these rearrangements proceed via a concerted [3,3]-sigmatropic transition state, which is similar in geometry to the TS for the Claisen rearrangement of an allyl aryl ether but has a 4 kcal mol-1 higher barrier. The effects of donor (OMe) and acceptor (CN) substituents on the kinetics of the [3,3]-rearrangement mirror those reported for allyl vinyl ethers: the largest substituent effects are seen for 1-OMe, 2-OMe, 2-CN, and 4-CN substituents, which lower the barrier by 5-9 kcal mol-1. Substituents on the aromatic ring have smaller effects on the barrier (≤2 kcal mol-1). The regioselectivities of Claisen rearrangements of meta-substituted benzyl ketene acetals favour 1,2,3-trisubstituted products in preference to the less sterically congested 1,2,4-isomers due to favourable orbital interactions in the 1,2,3 transition state.

Diethylalkylsulfonamido(4-methoxyphenyl)methyl)phosphonate/phosphonic acid derivatives act as acid phosphatase inhibitors: synthesis accompanied by experimental and molecular modeling assessments.

Purple acid phosphatases (PAPs) are binuclear metallo-hydrolases that have been isolated from various mammals, plants, fungi and bacteria. In mammals, PAP activity is associated with bone resorption and can lead to bone metabolic disorders such as osteoporosis; thus human PAP is an attractive target to develop anti-osteoporotic drugs. The aim of the present study was to investigate inhibitory effect of synthesized diethylalkylsulfonamido(4-methoxyphenyl)methyl)phosphonate/phosphonic acid derivatives as potential red kidney bean PAP (rkbPAP) inhibitors accompanied by experimental and molecular modeling assessments. Enzyme kinetic data showed that they are good rkbPAP inhibitors whose potencies improve with increasing alkyl chain length. Hexadecyl derivatives, as most potent compounds (Ki = 1.1 µM), inhibit rkbPAP in the mixed manner, while dodecyl derivatives act as efficient noncompetitive inhibitor. Also, analysis by molecular modeling of the structure of the rkbPAP-inhibitor complexes reveals factors, which may be important for the determination of inhibition specificity.

Captopril analogues as metallo-ß-lactamase inhibitors.

A number of captopril analogues were synthesised and tested as inhibitors of the metallo-ß-lactamase IMP-1. Structure-activity studies showed that the methyl group was unimportant for activity, and that the potencies of these inhibitors could be best improved by shortening the length of the mercaptoalkanoyl side-chain. Replacing the thiol group with a carboxylic acid led to complete loss of activity, and extending the length of the carboxylate group led to decreased potency. Good activity could be maintained by substituting the proline ring with pipecolic acid.

'Click' assembly of glycoclusters and discovery of a trehalose analogue that retards Aß40 aggregation and inhibits Aß40-induced neurotoxicity.

Osmolytes have been proposed as treatments for neurodegenerative proteinopathies including Alzheimer's disease. However, for osmolytes to reach the clinic their efficacy must be improved. In this work, copper(I)-catalyzed azide-alkyne cycloaddition chemistry was used to synthesize glycoclusters bearing six copies of trehalose, lactose, galactose or glucose, with the aim of improving the potency of these osmolytes via multivalency. A trehalose glycocluster was found to be superior to monomeric trehalose in its ability to retard the formation of amyloid-beta peptide 40 (Aß40) fibrils and protect neurons from Aß40-induced cell death.

Binuclear metallohydrolases: complex mechanistic strategies for a simple chemical reaction.

Binuclear metallohydrolases are a large family of enzymes that require two closely spaced transition metal ions to carry out a plethora of hydrolytic reactions. Representatives include purple acid phosphatases (PAPs), enzymes that play a role in bone metabolism and are the only member of this family with a heterovalent binuclear center in the active form (Fe(3+)-M(2+), M = Fe, Zn, Mn). Other members of this family are urease, which contains a di-Ni(2+) center and catalyzes the breakdown of urea, arginase, which contains a di-Mn(2+) center and catalyzes the final step in the urea cycle, and the metallo-ß-lactamases, which contain a di-Zn(2+) center and are virulence factors contributing to the spread of antibiotic-resistant pathogens. Binuclear metallohydrolases catalyze numerous vital reactions and are potential targets of drugs against a wide variety of human disorders including osteoporosis, various cancers, antibiotic resistance, and erectile dysfunctions. These enzymes also tend to catalyze more than one reaction. An example is an organophosphate (OP)-degrading enzyme from Enterobacter aerogenes (GpdQ). Although GpdQ is part of a pathway that is used by bacteria to degrade glycerolphosphoesters, it hydrolyzes a variety of other phosphodiesters and displays low levels of activity against phosphomono- and triesters. Such a promiscuous nature may have assisted the apparent recent evolution of some binuclear metallohydrolases to deal with situations created by human intervention such as OP pesticides in the environment. OP pesticides were first used approximately 70 years ago, and therefore the enzymes that bacteria use to degrade them must have evolved very quickly on the evolutionary time scale. The promiscuous nature of enzymes such as GpdQ makes them ideal candidates for the application of directed evolution to produce new enzymes that can be used in bioremediation and against chemical warfare. In this Account, we review the mechanisms employed by binuclear metallohydrolases and use PAP, the OP-degrading enzyme from Agrobacterium radiobacter (OPDA), and GpdQ as representative systems because they illustrate both the diversity and similarity of the reactions catalyzed by this family of enzymes. The majority of binuclear metallohydrolases utilize metal ion-activated water molecules as nucleophiles to initiate hydrolysis, while some, such as alkaline phosphatase, employ an intrinsic polar amino acid. Here we only focus on catalytic strategies applied by the former group.

Identification and characterization of an unusual metallo-ß-lactamase from Serratia proteamaculans.

Metallo-ß-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing ß-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL. A full length amino acid sequence alignment indicates overall homology to B3-type MBLs, but also suggests considerable variations in the active site, notably among residues that are relevant to metal ion binding. Steady-state kinetic measurements further indicate functional differences and identify two relevant pK a values for catalysis (3.8 for the enzyme-substrate complex and 7.8 for the free enzyme) and a preference for penams with modest reactivity towards some cephalosporins. An analysis of the metal ion content indicates the presence of only one zinc ion per active site in the resting enzyme. In contrast, kinetic data suggest that the enzyme may operate as a binuclear enzyme, and it is thus proposed that a catalytically active di-Zn(2+) center is formed only once the substrate is present.

Synthesis of a series of carbon-14 labeled tetrahydropyrido[4,3-d]pyrimidin-4(3H)-ones.

A series of tetrahydropyrido[4,3-d]pyrimidin-4(3H)-ones labeled with carbon-14 in the 2-position of pyrimidinone moiety were prepared as part of a 3-step sequence from benz[amidino-(14) C]amidine hydrochloride as a key synthetic intermediate.

Cyclodextrin-crosslinked poly(acrylic acid): adhesion and controlled release of diflunisal and fluconazole from solid dosage forms.

The controlled release of diflunisal and fluconazole from tablets made of novel polymers, poly(acrylic acid) (PAA) crosslinked with either ß-cyclodextrin (ßCD) or hydroxypropyl-ßCD (HPßCD), was investigated and Carbopol 934P (Carbopol) was used as a highly crosslinked PAA for comparison. Diflunisal strongly associates with ßCD-PAA and HPßCD-PAA polymers (Ka of 486 and 6,055 M(-1) respectively); thus, it was physically mixed into the conjugates and also precomplexed to identify whether decomplexation has any influence on release kinetics. Fluconazole has poor complexing ability (Ka of 34 M(-1) with HPßCD-PAA); thus, it was only tested as a physical mixture. Swelling and adhesion studies were conducted on all tablet combinations and adhesivity of the CD-PAA polymer tablets was maintained. Diflunisal release was much slower from HPßCD-PAA tablets than from ßCD-PAA, suggesting that a higher degree of complexation retards release. The precomplexed diflunisal release was also slower than the physically mixed diflunisal of the corresponding conjugate. The release closely followed zero-order kinetics for HPßCD-PAA, but was more sigmoidal for ßCD-PAA and especially Carbopol. Conversely, poorly associating fluconazole released in almost exactly the same way across both polymers and Carbopol, indicating that the release kinetics of poorly associating drugs are not influenced by the presence of cyclodextrins. In view of the varying profiles and release rates shown with diflunisal for the different polymers, the fluconazole data support the concept that adequate complexation can indeed modulate the release kinetics of drugs.

Optimization of an α-aminonaphthylmethylphosphonic acid inhibitor of purple acid phosphatase using rational structure-based design approaches.

Purple acid phosphatases (PAPs) are ubiquitous binuclear metallohydrolases that have been isolated from various animals, plants and some types of fungi. In humans and mice, elevated PAP activity in osteoclasts is associated with osteoporosis, making human PAP an attractive target for the development of anti-osteoporotic drugs. Based on previous studies focusing on phosphonate scaffolds, as well as a new crystal structure of a PAP in complex with a derivative of a previously synthesized α-aminonaphthylmethylphosphonic acid, phosphonates 24-40 were designed as new PAP inhibitor candidates. Subsequent docking studies predicted that all of these compounds are likely to interact strongly with the active site of human PAP and most are likely to interact strongly with the active site of pig PAP. The seventeen candidates were synthesized with good yields and nine of them (26-28, 30, 33-36 and 38) inhibit in the sub-micromolar to nanomolar range against pig PAP, with 28 and 35 being the most potent mammalian PAP inhibitors reported with Ki values of 168 nM and 186 nM, respectively. This study thus paves the way for the next stage of drug development for phosphonate inhibitors of PAPs as anti-osteoporotic agents.

Penicillin inhibitors of purple acid phosphatase.

Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have a multitude of biological functions and are found in fungi, bacteria, plants and animals. In mammals, PAP activity is linked with bone resorption and over-expression can lead to bone disorders such as osteoporosis. PAP is therefore an attractive target for the development of drugs to treat this disease. A series of penicillin conjugates, in which 6-aminopenicillanic acid was acylated with aromatic acid chlorides, has been prepared and assayed against pig PAP. The binding mode of most of these conjugates is purely competitive, and some members of this class have potencies comparable to the best PAP inhibitors yet reported. The structurally related penicillin G was shown to be neither an inhibitor nor a substrate for pig PAP. Molecular modelling has been used to examine the binding modes of these compounds in the active site of the enzyme and to rationalise their activities.