Aminopeptidase N (APN/CD13) as a target for anti-cancer agent design

APN is an important zinc dependent metallo-exopeptidase; it has been considered as a suitable target for anti-cancer drug design. In this review we focus on the most effective and the most promising inhibitors of aminopeptidase N. Their binding modes to the enzyme, the attempt to explain the origin of the inhibitory activity, as well as the structure-activity relationship for some of these compounds are discussed. Besides, the structural and electronic requirements of the enzyme active site and the binding pockets, together with the specificity towards the ligands are presented.     

Paracetamol (acetaminophen)-induced toxicity: molecular and biochemical mechanisms, analogues and protective approaches

An overview is presented on the molecular aspects of toxicity due to paracetamol (acetaminophen) and structural analogues. The emphasis is on four main topics, that is, bioactivation, detoxication, chemoprevention, and chemoprotection. In addition, some pharmacological and clinical aspects are discussed briefly. A general introduction is presented on the biokinetics, biotransformation, and structural modification of paracetamol. Phase II biotransformation in relation to marked species differences and interorgan transport of metabolites are described in detail, as are bioactivation by cytochrome P450 and peroxidases, two important phase I enzyme families. Hepatotoxicity is described in depth, as it is the most frequent clinical observation after paracetamol-intoxication. In this context, covalent protein binding and oxidative stress are two important initial (Stage I) events highlighted. In addition, the more recently reported nuclear effects are discussed as well as secondary events (Stage II) that spread over the whole liver and may be relevant targets for clinical treatment. The second most frequent clinical observation, renal toxicity, is described with respect to the involvement of prostaglandin synthase, N-deacetylase, cytochrome P450 and glutathione S-transferase. Lastly, mechanism-based developments of chemoprotective agents and progress in the development of structural analogues with an improved therapeutic index are outlined.     

Inhibition of angiotensin-converting enzyme by angiotensin I analogue peptide inhibitors. A kinetic study

Angiotensin I analogues with a phosphonic acid group replacing the C-terminal carboxyl group were shown to be competitive inhibitors of angiotensin-converting enzyme. This new class of inhibitors was used to study the binding requirements of the angiotensin I-like ligands to the enzyme's active site. These studies indicate that angiotensin-converting enzyme recognizes at least five amino acid residues at the C-terminus of the peptide. The effect of pH on the binding of the most potent inhibitor peptide was compared to Captopril. The two inhibitors showed similar K_i-pH profiles despite their structural differences. Chloride enhanced the binding of the peptide inhibitor at both pH 9.0 and pH 6.5. At pH 9.0 the inhibitor peptide and the anion bind randomly to the enzyme, while at pH 6.5 the mechanism is ordered. In the latter case, the anion binds first to the enzyme.     

Structural requirements of alloxan and ninhydrin for glucokinase inhibition and of glucose for protection against inhibition.

1. In order to elucidate the mechanism underlying the interactions between glucose and alloxan when competing for the sugar binding site of glucokinase from pancreatic B-cells or liver, the structural requirements of the enzyme for inhibition by alloxan and for protection by glucose were determined. 2. With a half-maximal inhibitory concentration of 5 microM, alloxan was the most potent pyrimidine derivative inhibitor of glucokinase. Uramil was a less potent enzyme inhibitor. A variety of other pyrimidine derivatives and related substances were ineffective. 3. Ninhydrin also inhibited glucokinase with a half-maximal inhibitory concentration of 5 microM. Isatin was a slightly less potent enzyme inhibitor. Several other indoline derivatives were ineffective. 4. Only glucose derivatives with a sufficiently bulky substituent in position C-2, such as the glucokinase substrates glucose and mannose and the inhibitors mannoheptulose, glucosamine, and N-acetylglucosamine, protected glucokinase against inhibition by alloxan by binding to the active site of the enzyme. Glucose epimers which differed in other positions did not protect the enzyme against alloxan inhibition. 5. DTT (dithiothreitol) protected glucokinase against inhibition by alloxan and reversed the inhibition of the enzyme induced by alloxan. Thus the mechanism of glucokinase inhibition by alloxan and other inhibitors, such as uramil and ninhydrin, is an oxidation of functionally essential SH groups of the enzyme, where the most reactive keto group of the inhibitor acts as the hydrogen acceptor. The protective action of glucose and several C-2 epimers demonstrates that these functionally essential SH groups are situated in the sugar binding site of the glucokinase. 6. The present results support our contention, that the pancreatic B-cell glucokinase is the major target mediating the inhibition of insulin secretion by alloxan.     

Inhibition of monometalated methionine aminopeptidase: inhibitor discovery and crystallographic analysis

Two divalent metal ions are commonly seen in the active-site cavity of methionine aminopeptidase, and at least one of the metal ions is directly involved in catalysis. Although ample structural and functional information is available for dimetalated enzyme, methionine aminopeptidase likely functions as a monometalated enzyme under physiological conditions. Information on structure, as well as catalysis and inhibition, of the monometalated enzyme is lacking. By improving conditions of high-throughput screening, we identified a unique inhibitor with specificity toward the monometalated enzyme. Kinetic characterization indicates a mutual exclusivity in binding between the inhibitor and the second metal ion at the active site. This is confirmed by X-ray structure, and this inhibitor coordinates with the first metal ion and occupies the space normally occupied by the second metal ion. Kinetic and structural analyses of the inhibition by this and other inhibitors provide insight in designing effective inhibitors of methionine aminopeptidase.     

Detection of novel coupled mutations in the katG gene (His276Met, Gln295His and Ser315Thr) in a multidrug-resistant Mycobacterium tuberculosis strain from Chennai, India, and insight into the molecular mechanism of isoniazid resistance using structural bioinformatics approaches

This study reports on the structural basis of drug resistance targeting the katG gene in a multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strain with two novel mutations (His276Met and Gln295His) in addition to the most commonly reported mutation (Ser315Thr). A structural bioinformatics approach was used to predict the structure of the mutant KatG enzyme (MT). Subsequent molecular dynamics and docking studies were performed to explain the mechanism of isoniazid (INH) resistance. The results show significant conformational changes in the structure of MT leading to a change in INH binding residues at the active site, with a significant increase in the inhibition constant (Ki) of 5.67 μm in the mutant KatG-isoniazid complex (MT-INH) compared with the wild-type KatG-isoniazid complex (WT-INH). In the case of molecular dynamics studies, root mean square deviation (RMSD) analysis of the protein backbone in simulated biological conditions revealed an unstable trajectory with higher deviations in MT throughout the simulation process (1 ns). Moreover, root mean square fluctuation (RMSF) analysis revealed an overall increase in residual fluctuations in MT compared with the wild-type KatG enzyme (WT), whilst the INH binding residues of MT showed a decreased fluctuation that can be observed as peak deviations. Hence, the present study suggests that His276Met, Gln295His and Ser315Thr mutations targeting the katG gene result in decreased stability and flexibility of the protein at INH binding residues leading to impaired enzyme function.     

Analogues of ornithine as inhibitors of ornithine decarboxylase. New deductions concerning the topography of the enzyme's active site

Fourteen structural analogues of ornithine were synthesized and evaluated as inhibitors of preparations of the enzyme L-ornithine carboxylase (ODC) (E.C. 4.1.1.17) obtained from rat liver, rat hepatoma cells in culture, or bull prostate. The synthesis of these compounds was achieved either via a Bucherer type reaction or via alkylation of carbanions derived from ethyl acetamidocyanoacetate, methyl isocyanoacetate, benzyl alpha-isocyanopropionate, methylbenzaldimine alanate, and the azlactone derivative of ornithuric acid. (+)-alpha-Methylornithine, which was assigned the L configuration on the basis of rotational criteria, was found to be the most potent reversible inhibitor of ODC among the synthesized compounds. From the degree of inhibition of ODC activity in the presence of the various ornithine analogues, it has been possible to delineate some of the structural features of the substrate L-ornithine which are required for binding to the mammalian ODC active site.     

Synthesis and evaluation of a new series of tri-, di-, and mono-N-alkylcarbamylphloroglucinols as bulky inhibitors of acetylcholinesterase

1,3,5-Tri-N-alkylcarbamylphloroglucinols (1-4) are synthesized as a new series of bulky inhibitors of acetylcholinesterase that may block the catalytic triad, the anionic substrate binding site, and the entrance of the enzyme simultaneously. Among three series of phloroglucinol-derived carbamates, tridentate inhibitors 1,3,5-tri-N-alkylcarbamylphloroglucinols (1-4), bidentate inhibitors 3,5-di-N-n-alkylcarbamyloxyphenols (5-8), and monodentate inhibitors 5-N-n-alkylcarbamyloxyresorcinols (9-12), tridentate inhibitors 1-4 are the most potent inhibitors of mouse acetylcholinesterase. When different n-alkylcarbamyl substituents in tridentate inhibitors 1-4 are compared, n-octylcarbamate 1 is the most potent inhibitor of the enzyme. All inhibitors 1-12 are characterized as the pseudo substrate inhibitors of acetylcholinesterase. Thus, tridentate inhibitors 1-4 are supposed to be hydrolyzed to bidentate inhibitors 5-8 after the enzyme catalysis. Subsequently, bidentate inhibitors 5-8 and monodentate inhibitors 9-12 are supposed to yield monodentate inhibitors 9-12 and phloroglucinol, respectively, after the enzyme catalysis. This means that tridentate inhibitors 1-4 may act as long period inhibitors of the enzyme. Therefore, inhibitors 1-4 may be considered as a new methodology to develop the long-acting drug for Alzheimer's disease. Automated dockings of inhibitor 1 into the X-ray crystal structure of acetylcholinesterase suggest that the most suitable configuration of inhibitor 1 to the enzyme binding is the (1,3,5)- (cis,trans,trans)-tricarbamate rotamer. The cis-carbamyl moiety of this rotamer does not bind into the acetyl group binding site of the enzyme but stretches out itself to the entrance. The other two trans-carbmayl moieties of this rotamer bulkily block the tryptophan 86 residue of the enzyme.     

Molecular modelling approaches to the design of acetylcholinesterase inhibitors: new challenges for the treatment of Alzheimer's disease

The interest for acetylcholinesterase as a target for the palliative treatment of Alzheimer's disease has been renewed in the last years owing to the evidences that support the role of this enzyme in accelerating the aggregation and deposition of the beta-amyloid peptide. A large amount of structural information on the acetylcholinesterase enzyme and of its complexes with inhibitors acting at the catalytic site, the peripheral binding site, or both is now available. Based on that, molecular modelling studies can be intensively used to decipher the molecular determinants that mediate the relationship between chemical structure and inhibitory potency. In turn, this knowledge can be exploited to design new compounds leading to more effective cholinergic strategies. At this point, inhibitors able to interact at the peripheral binding site are of particular relevance, as they might disrupt the interactions between the enzyme acetylcholinesterase and the beta-amyloid peptide. Therefore, these compounds might not only ameliorate the cholinergic deficit, but also be capable of slowing down the progression of the disease.     

Probing the subpockets of factor Xa reveals two binding modes for inhibitors based on a 2-carboxyindole scaffold: a study combining structure-activity relationship and X-ray crystallography

Structure-activity relationships within a series of highly potent 2-carboxyindole-based factor Xa inhibitors incorporating a neutral P1 ligand are described with particular emphasis on the structural requirements for addressing subpockets of the factor Xa enzyme. Interactions with the subpockets were probed by systematic substitution of the 2-carboxyindole scaffold, in combination with privileged P1 and P4 substituents. Combining the most favorable substituents at the indole nucleus led to the discovery of a remarkably potent factor Xa inhibitor displaying a K(i) value of 0.07 nM. X-ray crystallography of inhibitors bound to factor Xa revealed substituent-dependent switching of the inhibitor binding mode and provided a rationale for the SAR obtained. These results underscore the key role played by the P1 ligand not only in determining the binding affinity of the inhibitor by direct interaction but also in modifying the binding mode of the whole scaffold, resulting in a nonlinear SAR.     

The inhibition of 5-enolpyruvylshikimate-3-phosphate synthase as a model for development of novel antimicrobials

EPSP synthase (EPSPS) is an essential enzyme in the shikimate pathway, transferring the enolpyruvyl group of phosphoenolpyruvate to shikimate-3-phosphate to form 5-enolpyruvyl-3-shikimate phosphate and inorganic phosphate. This enzyme is composed of two domains, which are formed by three copies of betaalphabetaalphabetabeta-folding units; in between there are two crossover chain segments hinging the nearly topologically symmetrical domains together and allowing conformational changes necessary for substrate conversion. The reaction is ordered with shikimate-3-phosphate binding first, followed by phosphoenolpyruvate, and then by the subsequent release of phosphate and EPSP. N-[phosphomethyl]glycine (glyphosate) is the commercial inhibitor of this enzyme. Apparently, the binding of shikimate-3-phosphate is necessary for glyphosate binding, since it induces the closure of the two domains to form the active site in the interdomain cleft. However, it is somehow controversial whether binding of shikimate-3-phosphate alone is enough to induce the complete conversion to the closed state. The phosphoenolpyruvate binding site seems to be located mainly on the C-terminal domain, while the binding site of shikimate-3-phosphate is located primarily in the N-terminal domain residues. However, recent results demonstrate that the active site of the enzyme undergoes structural changes upon inhibitor binding on a scale that cannot be predicted by conventional computational methods. Studies of molecular docking based on the interaction of known EPSPS structures with (R)- phosphonate TI analogue reveal that more experimental data on the structure and dynamics of various EPSPS-ligand complexes are needed to more effectively apply structure-based drug design of this enzyme in the future.     

Enzyme fragment complementation binding assay for p38alpha mitogen-activated protein kinase to study the binding kinetics of enzyme inhibitors

The majority of protein kinase assays used in drug discovery research are enzyme activity assays. These assays are based on the measurement of phosphorylated protein or peptide substrate, which is the end product of the enzyme reaction. Since most kinase inhibitors are ATP competitive, prediction of the activity of compounds in cellular systems based on potency values in enzyme activity assays is complex, as this should take into account the affinity of the enzyme for ATP and the cellular ATP concentration. The fact that some of the most successful kinase inhibitors, such as STI 571 (imatinib mesylate, Gleevec, Novartis Pharmaceuticals, East Hanover, NJ), act through binding to the inactive isoform of the kinase provides another limitation of enzyme activity assays. Binding assays allow separate measurement of compound affinity to active and inactive kinase and do not require ATP or substrate in the reaction. Recently, a non-radioactive kinase binding assay for p38 mitogen-activated protein kinase has become available from DiscoveRx (Fremont, CA). The assay method, called HitHunter, utilizes enzyme fragment complementation of Escherichia coli beta-galactosidase to generate an assay signal by chemiluminescence. We have reconfigured the commercial assay kit to study the binding kinetics of two known reference inhibitors of the alpha-isoform of p38, the pyridinyl imidazole SB 203580 and the diaryl urea BIRB 796. Our data confirm the slow association kinetics of BIRB 796 as compared to SB 203580, which corresponded with the requirement of a relatively long preincubation time to obtain maximal effect in a cellular assay. Although neither of the two compounds showed preference for either active or inactive p38alpha, our data demonstrate that the HitHunter kinase binding assay can be used to select compounds that specifically target inactive kinase.     

Rationalization of physicochemical characters and docking of 3-alkoxy-5-phenoxy-N-thiazolyl benzamide analogs toward glucokinase activator activity

Diabetes mellitus is a chronic metabolic disorder involving the dysregulation of glucose metabolism, β-cell dysfunction, and impaired insulin sensitivity. Glucokinase (GK) promotes glycogen synthesis, while it enhances insulin secretion from pancreatic β-cells. In this study, we focused on molecular modeling study of 3-alkoxy-5-phenoxy-N-thiazolyl benzamide analogs with reference to structural requirements. The amalgamated best fit consensus scoring function showed coefficient of determination (0.927), leave-one-out cross-validated squared correlation coefficient (0.865), and external predictivity value (0.763). The binding of 3-alkoxy-5-phenoxy-N-thiazolyl benzamide analogs to glucokinase enzyme was explored with the help of docking. The most stable ligand–enzyme complex of compound TR-2 showed that the NH of the benzamide make key hydrogen bonds with the backbone C=O of Arg63. The phenoxy moiety on the 5th position of benzene ring occupies the hydrophobic space on the allosteric binding site constituted from Met210, Met235, Cys220, and Tyr214. One of the oxygen of methylsulfonyl group forms hydrogen bond with NE2 of Gln98 and phenyl ring and the aromatic ring of Tyr215 are perpendicular to each other, which probably increase potency due to van der Waals interactions.The structural insights gleaned from the study could be usefully employed to design activators with a much more enhanced potency.     

CONFORMATIONAL ENERGY CALCULATIONS OF ENZYME-SUBSTRATE INTERACTIONS. II. Computation of the Binding Energy for Substrates in the Active Site of α-Chymotrypsin

Empirical energy calculations are carried out to study the binding of the substrates N-acetyl-L-phenylalanine amide, N-acetyl-L-tyrosine amide, N-formyl-L-tyrosine α-mide, and N-acetyl-L-tryptophan amide to the enzyme molecule α-chymotrypsin. As an initial probe of the active-site surface, the low-energy conformations of the phenylalanine substrate (see Part I) are allowed to approach the enzyme by overall rotational and translational motion until a minimum is achieved in the enzyme-substrate interaction energy. Four regions of good binding are found near the active site, the one of lowest energy being that in which the ring is oriented in the cleft of the enzyme. This cleft binding site is the same as the binding position found by X-ray diffraction for the virtual substrate N-formyl-L-tryptophan; the other three favorable binding regions might not be detected by X-ray analysis, possibly because they are blocked in the dimer structure of the crystalline enzyme. Further investigation of the most favorable (cleft) binding site indicates that left-handed α-helical backbone conformations of the substrate do not form stable enzyme-substrate complexes, while the equatorial seven-membered ring, as well as the right-handed α-helical and β and γ backbone conformations (the latter three of which commonly occur in proteins) bind well. The calculated binding energies of all four substrates correlate with experimental binding constants.     

NMR screening applied to the fragment-based generation of inhibitors of creatine kinase exploiting a new interaction proximate to the ATP binding site

Using an in-house fragment NMR library, we identified a set of ligands that bind rabbit muscular creatine kinase, an enzyme involved in key ATP-dependent processes. The ligands docked to the crystal structures of creatine kinase indicated that a phenylfuroic acid could enter into a pocket adjacent to the nucleotide binding site. This fragment served as an anchor to develop in silico a series of potential inhibitors which could partly access the nucleotide binding site. The short synthesis of only four derivatives provided entirely novel hit compounds that reversibly inhibit creatine kinase at micromolar concentrations with a mixed ATP-competitive/noncompetitive mechanism in agreement with the structural model of the inhibited enzyme. These initial biologically active compounds are novel and modular and exploit a new interaction proximate to the ATP binding site.     

Phorbol esters and neurotransmitter release: more than just protein kinase C?

This review focuses on the effects of phorbol esters and the role of phorbol ester receptors in the secretion of neurotransmitter substances. We begin with a brief background on the historical use of phorbol esters as tools to decipher the role of the enzyme protein kinase C in signal transduction cascades. Next, we illustrate the structural differences between active and inactive phorbol esters and the mechanism by which the binding of phorbol to its recognition sites (C1 domains) on a particular protein acts to translocate that protein to the membrane. We then discuss the evidence that the most important nerve terminal receptor for phorbol esters (and their endogenous counterpart diacylglycerol) is likely to be Munc13. Indeed, Munc13 and its invertebrate homologues are the main players in priming the secretory apparatus for its critical function in the exocytosis process.     

QSAR study of Phenoxypyrimidine Derivatives as Potent Inhibitors of p38 Kinase using different chemometric tools

Selective inhibition of the CSBP/p38 mitogen-activated protein kinase pathway may be an attractive target for the development of therapeutic agents to treat chronic inflammatory disease. A series of Phenoxypyrimidine Derivatives as Potent Inhibitors of p38 Kinase was subjected to quantitative structure-activity relationship (QSAR) analysis to find the structural requirements for ligand binding. A collection of chemometrics methods including multiple linear regression (MLR), factor analysis-based multiple linear regression (FA-MLR), principal component regression and partial least squared combined with genetic algorithm for variable selection (GA-PLS) were employed to make connections between structural parameters and enzyme inhibition. The results revealed the significant roles of steric effect, hydrogen bonding and electronic properties on the p38 inhibitory activity of the studied molecules. The most significant QSAR model, obtained by GA-PLS, could explain and predict 98% and 87% of variances in the pIC(50) data, respectively.     

A contribution to the mechanism of action of SAD-128

SAD-128 was found to be an effective protector of acetylcholinesterase against inhibition by soman, due to its ability to function as a reversible inhibitor and allosteric modifier of the AChE active site. It also attenuated aging of the soman-inhibited enzyme. In order to study the connection between some of these effects of SAD-128 and structural changes in acetylcholinesterase and/or the membrane to which the enzyme is bound, the influences of SAD-128 on the EPR spectra of the spin labelled enzyme and of the membrane were studied under various conditions and the results correlated with the kinetic parameters. SAD-128 increases the fluidity of human erythrocyte membranes but not that of the Torpedo marmorata electric organ. Similarly, the binding properties of membrane acetylcholinesterase for SAD-128, expressed in terms of the Hill coefficient, differ for the two preparations. Some structural changes in the enzyme active site were also observed in the presence of SAD-128. The high protective effect of SAD-128 against AChE inhibition was confirmed by the EPR method regardless of the organophosphorus inhibitor tested. On the other hand, the effect of SAD-128 on the retardation of irreversible inhibition of the enzyme essentially depends on the inhibitor used. From present results it can be concluded that the protective effects of SAD-128 against inhibition of m-AChE are related to the structural changes of the active site and can be additionally moderated by the microviscosity changes of the membrane.     

Inhibitors of protein: geranylgeranyl transferases

The enzyme protein:geranylgeranyl transferase-1 (PGGT-1 or GGTase-I) catalyzes the geranylgeranylation of cysteine residues near the C-termini of a variety of proteins, including most monomeric GTP binding precursor proteins belonging to the Rho, Rac and Rap subfamilies. These proteins are involved in signaling pathways controlling important processes such as cell differentiation and growth. In the framework of the development of therapeutics against disorders associated with aberrant cell proliferation, the interference with these signal transduction cascades has been a major focus of investigation. For instance inhibitors of PGGT-1 have shown promise in the treatment of cancer, smooth muscle hyperplasia as well as parasitic infections, such as malaria. In this review, structural and mechanistic aspects of the protein:geranylgeranyl transferases are discussed as well as their importance with respect to the terpene metabolism. An extensive summary of reported inhibitors of PGGT-1, classified as natural products, peptide substrate (Ca(1)a(2)L box), terpene substrate (geranylgeranyl pyrophosphate) and others, is presented. The few known inhibitors of the other geranylgeranylating enzyme, protein:geranylgeranyl transferase-2 (PGGT-2), are also included.