Identification of Novel Non-secosteroidal Vitamin D Receptor Agonists with Potent Cardioprotective Effects and devoid of Hypercalcemia.

Vitamin D regulates many biological processes, but its clinical utility is limited by its hypercalcemic effect. Using a virtual screening platform to search novel chemical probes that activate the vitamin D signaling, we report discovery of novel non-steroidal small-molecule compounds that activate the vitamin D receptor (VDR), but are devoid of hypercalcemia. A lead compound (known as VDR 4-1) demonstrated potent transcriptional activities in a VDR reporter gene assay, and significantly ameliorated cardiac hypertrophy in cell culture studies and in animal models. VDR 4-1 also effectively suppressed secondary hyperparathyroidism in 1α-hydroxylase knockout mice. In contrast to 1α,25-dihydroxyvitamin D3 (1,25-D3 or calcitriol), a naturally occurring VDR agonist, VDR 4-1 therapy even at high doses did not induce hypercalcemia. These findings were accompanied by a lack of upregulation of calcium transport genes in kidney and in the gut providing a mechanism for the lack of hypercalcemia. Furthermore, VDR 4-1 therapy significantly suppressed cardiac hypertrophy and progression to heart failure in both vitamin D deficient and normal mice without inducing significant hypercalcemia. In conclusion, we have identified a unique VDR agonist compound with beneficial effects in mouse models of hyperparathyroidism and heart failure without inducing significant hypercalcemia.

Inhibition of serum and glucocorticoid regulated kinase-1 as novel therapy for cardiac arrhythmia disorders.

Alterations in sodium flux (INa) play an important role in the pathogenesis of cardiac arrhythmias and may also contribute to the development of cardiomyopathies. We have recently demonstrated a critical role for the regulation of the voltage-gated sodium channel NaV1.5 in the heart by the serum and glucocorticoid regulated kinase-1 (SGK1). Activation of SGK1 in the heart causes a marked increase in both the peak and late sodium currents leading to prolongation of the action potential duration and an increased propensity to arrhythmia. Here we show that SGK1 directly regulates NaV1.5 channel function, and genetic inhibition of SGK1 in a zebrafish model of inherited long QT syndrome rescues the long QT phenotype. Using computer-aided drug discovery coupled with in vitro kinase assays, we identified a novel class of SGK1 inhibitors. Our lead SGK1 inhibitor (5377051) selectively inhibits SGK1 in cultured cardiomyocytes, and inhibits phosphorylation of an SGK1-specific target as well as proliferation in the prostate cancer cell line, LNCaP. Finally, 5377051 can reverse SGK1's effects on NaV1.5 and shorten the action potential duration in induced pluripotent stem cell (iPSC)-derived cardiomyocytes from a patient with a gain-of-function mutation in Nav 1.5 (Long QT3 syndrome). Our data suggests that SGK1 inhibitors warrant further investigation in the treatment of cardiac arrhythmias.

Discovery of small molecule inhibitors to Krüppel-like factor 10 (KLF10): implications for modulation of T regulatory cell differentiation.

The Krüppel-like family of transcription factors (KLFs) constitute a subfamily of C2H2-type zinc finger proteins with distinct cell-type expression patterns and regulate functional aspects of cell growth and differentiation, activation, or development. KLF10 has been previously shown to critically regulate the acquisition of CD4+CD25+ T regulatory cell differentiation and function, an effect important to the maintenance of self-tolerance, immune suppression, and tumor immunosurveillance. To date, there are no selective pharmacological inhibitors to KLF10. Herein, we report on the discovery of first-in-class small molecule compounds that inhibit the KLF10-DNA interaction interface using computer-aided drug design (CADD) screens of chemical libraries. Interrogation of a "druggable" pocket in the second zinc finger of KLF10 revealed three small molecules, #48, #48-15, and #15-09, with similar scaffolds and binding patterns. Each of these small molecules inhibited KLF10-DNA binding and transcriptional activity, conversion of CD4+CD25- T cells to CD4+CD25+ T regulatory cells, and KLF10 target gene expression. Taken together, these findings support the feasibility of using CADD with functional assays to identify small molecules that target members of the KLF subfamily of transcription factors to regulate biological functions in health and disease. We hope these novel compounds will serve as useful mechanistic probes for KLF10-mediated effects and T regulatory cell biology.

Withanolide A and asiatic acid modulate multiple targets associated with amyloid-beta precursor protein processing and amyloid-beta protein clearance.

Alzheimer's disease (AD) is a progressive, neurodegenerative disease histochemically characterized by extracellular deposits of amyloid beta (Abeta) protein and intracellular neurofibrillary tangles of hyperphosphorylated tau protein. AD is considered to be a complex, multifactorial syndrome, with numerous causal factors contributing to its pathogenesis. Thus, for any novel therapeutic molecule to have a "disease-modifying" effect on AD, it must be able to modulate multiple, synergistic targets simultaneously. In this context, we have studied two compounds of plant origin [withanolide A (1) and asiatic acid (2)] for their potential activities against multiple targets associated with Abeta pathways (BACE1, ADAM10, IDE, and NEP). BACE1 is a rate-limiting enzyme in the production of Abeta from amyloid-beta precursor protein (AbetaPP), while ADAM10 is involved in non-amyloidogenic processing of AbetaPP. IDE and NEP are two of the prominent enzymes involved in effectively degrading Abeta. It was found that both 1 and 2 significantly down-regulated BACE1 and also up-regulated ADAM10 in primary rat cortical neurons. In addition, 1 significantly up-regulated IDE levels, which may help in degrading excess Abeta from the AD brain. On the basis of the data obtained, the two multifunctional compounds may prove valuable in developing novel, effective therapeutics for the prevention and treatment of AD-associated amyloid pathology.

Successful applications of computer aided drug discovery: moving drugs from concept to the clinic.

Drug discovery and development is an interdisciplinary, expensive and time-consuming process. Scientific advancements during the past two decades have changed the way pharmaceutical research generate novel bioactive molecules. Advances in computational techniques and in parallel hardware support have enabled in silico methods, and in particular structure-based drug design method, to speed up new target selection through the identification of hits to the optimization of lead compounds in the drug discovery process. This review is focused on the clinical status of experimental drugs that were discovered and/or optimized using computer-aided drug design. We have provided a historical account detailing the development of 12 small molecules (Captopril, Dorzolamide, Saquinavir, Zanamivir, Oseltamivir, Aliskiren, Boceprevir, Nolatrexed, TMI-005, LY-517717, Rupintrivir and NVP-AUY922) that are in clinical trial or have become approved for therapeutic use.

Binding of lipoic acid induces conformational change and appearance of a new binding site in methylglyoxal modified serum albumin.

The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.

A comprehensive analysis of the thermodynamic events involved in ligand-receptor binding using CoRIA and its variants.

Quantitative Structure-Activity Relationships (QSAR) are being used since decades for prediction of biological activity, lead optimization, classification, identification and explanation of the mechanisms of drug action, and prediction of novel structural leads in drug discovery. Though the technique has lived up to its expectations in many aspects, much work still needs to be done in relation to problems related to the rational design of peptides. Peptides are the drugs of choice in many situations, however, designing them rationally is a complicated task and the complexity increases with the length of their sequence. In order to deal with the problem of peptide optimization, one of our recently developed QSAR formalisms CoRIA (Comparative Residue Interaction Analysis) is being expanded and modified as: reverse-CoRIA (rCoRIA) and mixed-CoRIA (mCoRIA) approaches. In these methodologies, the peptide is fragmented into individual units and the interaction energies (van der Waals, Coulombic and hydrophobic) of each amino acid in the peptide with the receptor as a whole (rCoRIA) and with individual active site residues in the receptor (mCoRIA) are calculated, which along with other thermodynamic descriptors, are used as independent variables that are correlated to the biological activity by chemometric methods. As a test case, the three CoRIA methodologies have been validated on a dataset of diverse nonamer peptides that bind to the Class I major histocompatibility complex molecule HLA-A*0201, and for which some structure activity relationships have already been reported. The different models developed, and validated both internally as well as externally, were found to be robust with statistically significant values of r(2) (correlation coefficient) and r(2)(pred) (predictive r(2)). These models were able to identify all the structure activity relationships known for this class of peptides, as well uncover some new relationships. This means that these methodologies will perform well for other peptide datasets too. The major advantage of these approaches is that they explicitly utilize the 3D structures of small molecules or peptides as well as their macromolecular targets, to extract position-specific information about important interactions between the ligand and receptor, which can assist the medicinal and computational chemists in designing new molecules, and biologists in studying the influence of mutations in the target receptor on ligand binding.

Design of inhibitors of the MurF enzyme of Streptococcus pneumoniae using docking, 3D-QSAR, and de Novo design.

The biosynthetic pathway for formation of the bacterial cell wall (peptidoglycan) presents an attractive target for intervention. This is exploited by many of the clinically useful antibiotics, which inhibit enzymes involved in the later stages of peptidoglycan synthesis. MurF is one of the four amide bond-forming enzymes (d-alanyl-d-alanine ligating enzyme) that catalyzes the ATP-dependent formation of UDP-MurNAc-tripeptide. In the present study, several MurF inhibitors were docked into the active site of MurF to explore their binding modes and also to gain an insight into the crucial ligand-receptor interactions at the molecular level. The final selection of the "bioactive" conformation of every ligand was influenced by consensus scoring in which various independent scoring functions such as GoldScore, ChemScore, HINT score and X-CScore were employed. Subsequently, 3D-QSAR studies using comparative molecular field analysis (CoMFA) and the new approach comparative residue interaction analysis (CoRIA) have been carried out on the enzyme-inhibitor complexes obtained by docking and postscoring analysis. Finally, new inhibitors have been designed using the de novo approach of Ludi, and the activities of the most promising hits have been predicted with the CoMFA and CoRIA models.

CoMFA study of distamycin analogs binding to the minor-groove of DNA: a unified model for broad-spectrum activity.

A 3D-QSAR analysis has been carried out by comparative molecular field analysis (CoMFA) on a series of distamycin analogs that bind to the DNA of drug-resistant bacterial strains MRSA, PRSP and VSEF. The structures of the molecules were derived from the X-ray structure of distamycin bound to DNA and were aligned using the Database alignment method in Sybyl. Statistically significant CoMFA models for each activity were generated. The CoMFA contours throw light on the structure activity relationship (SAR) and help to identify novel features that can be incorporated into the distamycin framework to improve the activity. Common contours have been gleaned from the three models to construct a unified model that explains the steric and electrostatic requirements for antimicrobial activity against the three resistant strains.

Pharmacophore modeling in drug discovery and development: an overview.

Pharmacophore mapping is one of the major elements of drug design in the absence of structural data of the target receptor. The tool initially applied to discovery of lead molecules now extends to lead optimization. Pharmacophores can be used as queries for retrieving potential leads from structural databases (lead discovery), for designing molecules with specific desired attributes (lead optimization), and for assessing similarity and diversity of molecules using pharmacophore fingerprints. It can also be used to align molecules based on the 3D arrangement of chemical features or to develop predictive 3D QSAR models. This review begins with a brief historical overview of the pharmacophore evolution followed by a coverage of the developments in methodologies for pharmacophore identification over the period from inception of the pharmacophore concept to recent developments of the more sophisticated tools such as Catalyst, GASP, and DISCO. In addition, we present some very recent successes of the widely used pharmacophore generation methods in drug discovery.

The B(OH)-NH Analog Is a Surrogate for the Amide Bond (CO-NH) in Peptides: An ab Initio Study.

The conformational preferences of N-methyl-methylboronamide (NMB), a B(OH)-NH analog of the amide CO-NH in natural peptides, have been investigated at the Hartree-Fock; Becke's three-parameter exchange functional and the gradient-corrected functional of Lee, Yang, and Parr; and second-order Møller-Plesset levels of theory with the 6-31+G* basis set. The minima, saddle points, and rotation barriers on the potential energy surface of NMB have been located and the energy barriers estimated. Besides the global minimum, there are three local minima within 2.0 kcal mol(-)(1) of the global minimum characterized by specific ω and τ torsion values. The energy barriers for rotation about the "ω angle" are 16.4-18.8 kcal mol(-)(1) and are a consequence of the double-bond character of the B-N bond as revealed by natural bond orbitals calculations. The "ω angle" and the ω rotation barrier are nearly the same as those seen in natural peptides. The τ rotation barriers (B-O bond) are relatively low because of the single-bond character of the B-O bond. Ala-BON, the Ala-dipeptide derived from NMB, has been constructed as a model peptide to study the conformational preferences about the φ and ψ torsion angles. The study reveals a strong preference for α-helix, type-II ß-turn, 2.27 ribbon, and antiparallel ß-sheet conformations, and mirror images of both type-II ß-turn and 2.27 ribbon motifs whose φ and ψ values fall in the "disfavored regions" of the Ramachandran map. Thus, the replacement of the carbonyl group by B-OH retains the geometry and barrier around the "ω angle" and induces a strong preference for regular secondary structure motifs and also structures with positive φ values. This makes the B(OH)-NH analog an important surrogate for the peptide bond, with the additional advantage of stability to proteolytic enzymes.

Comparative residue interaction analysis (CoRIA): a 3D-QSAR approach to explore the binding contributions of active site residues with ligands.

A novel approach termed comparative residue-interaction analysis (CoRIA), emphasizing the trends and principles of QSAR in a ligand-receptor environment has been developed to analyze and predict the binding affinity of enzyme inhibitors. To test this new approach, a training set of 36 COX-2 inhibitors belonging to nine families was selected. The putative binding (bioactive) conformations of inhibitors in the COX-2 active site were searched using the program DOCK. The docked configurations were further refined by a combination of Monte Carlo and simulated annealing methods with the Affinity program. The non-bonded interaction energies of the inhibitors with the individual amino acid residues in the active site were then computed. These interaction energies, plus specific terms describing the thermodynamics of ligand-enzyme binding, were correlated to the biological activity with G/PLS. The various QSAR models obtained were validated internally by cross validation and boot strapping, and externally using a test set of 13 molecules. The QSAR models developed on the CoRIA formalism were robust with good r (2), q (2) and r (pred) (2) values. The major highlights of the method are: adaptation of the QSAR formalism in a receptor setting to answer both the type (qualitative) and the extent (quantitative) of ligand-receptor binding, and use of descriptors that account for the complete thermodynamics of the ligand-receptor binding. The CoRIA approach can be used to identify crucial interactions of inhibitors with the enzyme at the residue level, which can be gainfully exploited in optimizing the inhibitory activity of ligands. Furthermore, it can be used with advantage to guide point mutation studies. As regards the COX-2 dataset, the CoRIA approach shows that improving Coulombic interaction with Pro528 and reducing van der Waals interaction with Tyr385 will improve the binding affinity of inhibitors.

The ω, φ, and ψ Space of N-Hydroxy-N-methylacetamide and N-Acetyl-N '-hydroxy-N '-methylamide of Alanine and Their Boron Isosteres.

The conformational space of N-hydroxy-N-methylacetamide [CH3-CO-N(OH)CH3, NMAOH] and its boron isostere [CH3-CO-B(OH)CH3, BMAOH] has been studied by quantum chemical methods. The potential energy surface of NMAOH and BMAOH has been built at the HF, B3LYP, and MP2 levels of theory with the 6-31+G* basis set. The minima and transition states for rotations about various torsional angles have been located, and the energy barriers have been estimated. The global minimum energy structure of both peptides exhibits an intramolecular hydrogen bond between the carbonyl oxygen and the hydroxyl group, imparting a conformational rigidity to the peptides. The omega rotation barrier is lower in the boron isostere than in NMAOH. The difference in the rotation barrier has been attributed to second-order orbital interactions, like negative hyperconjugation, as revealed by NBO calculations. In contrast, the rotation barrier around the torsion angle tau (torsion governing rotation about the N-O and B-O bonds) is relatively higher in the boron analogue. This difference is due to the double bond character in the B-O bond as opposed to the N-O bond which has the character of a single bond. As an extension, N-acetyl-N'-hydroxy-N'-methylamide of alanine (Ala-NOH) and its boron isostere (Ala-BOH) have been adopted as model peptides to study the conformational preferences about the φ and ψ torsion angles. The study reveals a strong preference for a Type I beta turn as well as inclinations for a left-handed alpha helix, for positive phi torsions, and for extended psi conformations for Ala-NOH; Ala-BOH, on the other hand, shows a leaning toward positive phi and extended psi, with no preference for any regular secondary structure motifs. The replacement of nitrogen by boron changes the electronic and conformational properties of the peptide, extending greater flexibility around the omega angle, a strong preference for positive phi values, and a shift in the site of nucleophilic attack from the carbonyl group to boron.

Stationary Points on the PES of N-Methoxy Peptides and Their Boron Isosteres: An Ab Initio Study.

The conformational space of N-methoxy-N-methylacetamide [CH3-CO-N(OCH3)CH3, NMA-NOM] and its boron isostere [CH3-CO-B(OCH3)CH3, BMA-BOM] has been studied at the HF, B3LYP, and MP2 levels of theory with the 6-31+G* basis set. The minima, saddle points, and rotation barriers on the PES of these molecules have been located, and the energy barriers estimated. The omega rotation barrier is relatively lower in the boron isostere than in NMA-NOM. The difference in the rotation barrier has been attributed to second-order orbital interactions, like negative hyperconjugation, as revealed by NBO calculations. As an extension, N-acetyl-N'-methoxy-N'-methylamide of alanine (Ala-NOM) and its boron isostere (B-Ala-BOM) have been adopted as model peptides to study the conformational preferences about the φ and ψ torsion angles. The study reveals a strong preference for conformations of type-V beta turn and left-handed α-helix for Ala-NOM. B-Ala-BOM, on the other hand, favors conformations of type-Va beta turn, mirror image of Poly-L-Pro II helix, and structures with positive φ and extended ψ. The replacement of nitrogen by boron changes the electronic and conformational properties of the peptide, extends greater flexibility around the omega angle, induces a strong preference for positive phi values, and shifts the site of nucleophilic attack from the carbonyl group to boron.

Comparative protein modeling of methionine S-adenosyltransferase (MAT) enzyme from Mycobacterium tuberculosis: a potential target for antituberculosis drug discovery.

Mycobacterium tuberculosis (Mtb) is a successful pathogen that overcomes the numerous challenges presented by the immune system of the host. In the last 40 years few anti-TB drugs have been developed, while the drug-resistance problem is increasing; there is thus a pressing need to develop new anti-TB drugs active against both the acute and chronic growth phases of the mycobacterium. Methionine S-adenosyltransferase (MAT) is an enzyme involved in the synthesis of S-adenosylmethionine (SAM), a methyl donor essential for mycolipid biosynthesis. As an anti-TB drug target, Mtb-MAT has been well validated. A homology model of MAT has been constructed using the X-ray structures of E. coli MAT (PDB code: 1MXA) and rat MAT (PDB code: 1QM4) as templates, by comparative protein modeling principles. The resulting model has the correct stereochemistry as gauged from the Ramachandran plot and good three-dimensional (3D) structure compatibility as assessed by the Profiles-3D score. The structurally and functionally important residues (active site) of Mtb-MAT have been identified using the E. coli and rat MAT crystal structures and the reported point mutation data. The homology model conserves the topological and active site features of the MAT family of proteins. The differences in the molecular electrostatic potentials (MEP) of Mtb and human MAT provide evidences that selective and specific Mtb-MAT inhibitors can be designed using the homology model, by the structure-based drug design approaches.