Kuldeep K. Roy

Novel Carbamates as Orally Active Acetylcholinesterase Inhibitors Found to Improve Scopolamine-Induced Cognition Impairment: Pharmacophore-Based Virtual Screening, Synthesis, and Pharmacology†

A systematic virtual screening (VS) experiment, consisting of the development of 3D-pharmacophore, screening of virtual library, synthesis, and pharmacology, is reported. The predictive pharmacophore model (correlation = 0.955) with one H-bond donor and three hydrophobic features was developed using HypoGen on a training set of 24 carbamates as AChE inhibitors. The model was validated on a test set of 40 carbamates (correlation = 0.844). The pharmacophore-based VS of virtual library led to the identification of novel carbamates as potent AChE inhibitors. The synthesis and pharmacological evaluation of nine carbamates against three diverse assay systems, namely (i) in vitro Ellman method, (ii) in vivo passive avoidance test, and (iii) aldicarb-sensitivity assay, led to the discovery of orally active novel AChE inhibitors which improved scopolamine-induce cognition impairment in Swiss male mice. Finally, two novel lead compounds 85 and 86 are selected as candidate molecules for further optimization.

An investigation of structurally diverse carbamates for acetylcholinesterase (AChE) inhibition using 3D-QSAR analysis.

In order to identify the essential structural features and physicochemical properties for acetylcholinesterase (AChE) inhibitory activity in some carbamate derivatives, the systematic QSAR (Quantitative Structure Activity Relationship) studies (CoMFA, advance CoMFA and CoMSIA) have been carried out on a series of (total 78 molecules) taking 52 and 26 molecules in training and test set, respectively. Statistically significant 3D-QSAR (three-dimensional Quantitative Structure Activity Relationship) models were developed on training set molecules using CoMFA and CoMSIA and validated against test set compounds. The highly predictive models (CoMFA q(2)=0.733, r(2)=0.967, predictive r(2)=0.732, CoMSIA q(2)=0.641, r(2)=0.936, predictive r(2)=0.812) well explained the variance in binding affinities both for the training and the test set compounds. The generated models suggest that steric, electrostatic and hydrophobic interactions play an important role in describing the variation in binding affinity. In particular the carbamoyl nitrogen should be more electropositive; substitutions on this nitrogen should have high steric bulk and hydrophobicity while the amino nitrogen should be electronegative in order to have better activity. These studies may provide important insights into structural variations leading to the development of novel AChE inhibitors which may be useful in the development of novel molecules for the treatment of Alzheimers disease.

Consensus superiority of the pharmacophore-based alignment, over maximum common substructure (MCS): 3D-QSAR studies on carbamates as acetylcholinesterase inhibitors.

In view of the nonavailability of complete X-ray structure of carbamates cocrystallized with AChE enzyme, the 3D-QSAR model development based on cocrystallized conformer (CCBA) as well as docked conformer-based alignment (DCBA) is not feasible. Therefore, the only two alternatives viz. pharmacophore and maximum common substructure-based alignments are left for the 3D-QSAR comparative molecular field analyses (CoMFA) and comparative molecular similarity indices analyses (CoMSIA) model development. So, in the present study, the 3D-QSAR models have been developed using both alignment methods, where CoMFA and CoMSIA models based on pharmacophore-based alignment were in good agreement with each other and demonstrated significant superiority over MCS-based alignment in terms of leave-one-out (LOO) cross-validated q(2) values of 0.573 and 0.723 and the r(2) values of 0.972 and 0.950, respectively. The validation of the best CoMFA and CoMSIA models based on pharmacophore (Hip-Hop)-based alignment on a test set of 17 compounds provided significant predictive r(2) [r(2)(pred(test))] of 0.614 and 0.788, respectively. The contour map analyses revealed the relative importance of steric, electrostatic, and hydrophobicity for AChE inhibition activity. However, hydrophobic factor plays a major contribution to the AChE inhibitory activity modulation which is in strong agreement with the fact that the AChE is having a wide active site gorge (approximately 20 A) occupied by a large number of hydrophobic amino acid residues.

Synthesis and biological evaluation of substituted 4-arylthiazol-2-amino derivatives as potent growth inhibitors of replicating Mycobacterium tuberculosis H37RV

In search of potential therapeutics for tuberculosis, we describe herein synthesis and biological evaluation of some substituted 4-arylthiazol-2-amino derivatives as modified analogues of the antiprotozoal drug Nitazoxanide (NTZ), which has recently been reported as potent inhibitor of Mtb H(37)Rv (Mtb MIC=52.12 μM) with an excellent ability to evade resistance. Among the synthesized derivatives, the two compounds 7a (MIC=15.28 μM) and 7c (MIC=17.03 μM) have exhibited about three times better Mtb growth inhibitory activity over NTZ and are free from any cytotoxicity (Vero CC(50) of 244 and 300 μM respectively). These two compounds represent promising leads for further optimization.

Profiling the structural determinants for the selectivity of representative factor-Xa and thrombin inhibitors using combined ligand-based and structure-based approaches.

The current study deciphers the combined ligand- and structure-based computational insights to profile structural determinants for the selectivity of representative diverse classes of FXa-selective and thrombin-selective as well as dual FXa-thrombin high affinity inhibitors. The thrombin-exclusive insertion 60-loop (D-pocket) was observed to be one of the most notable recognition sites for the known thrombin-selective inhibitors. Based on the topological comparison of four common active-site pockets (S1-S4) of FXa and thrombin, the greater structural disparity was observed in the S4-pocket, which was more symmetrical (U-shaped) in FXa as compared to thrombin mainly due to the presence of L99 and I174 residues in latter in place of Y99 and F174 respectively in former protease. The S2 pocket forming partial roof at the entry of 12 Å deep S1-pocket, with two extended β-sheets running antiparallel to each other by undergoing U-turn (∼180̊), has two conserved glycine residues forming H-bonds with the bound ligand for governing ligand binding affinity. The docking, scoring, and binding pose comparison of the representative high-affinity and selective inhibitors into the active sites of FXa and thrombin revealed critical residues (S214, Y99, W60D) mediating selectivity through direct- and long-range electrostatic interactions. Interestingly, most of the thrombin-selective inhibitors attained S-shaped conformation in thrombin, while FXa-selective inhibitors attained L-shaped conformations in FXa. The role of residue at 99th position of FXa and thrombin toward governing protease selectivity was further substantiated using molecular dynamics simulations on the wild-type and mutated Y99L FXa bound to thrombin-selective inhibitor 2. Furthermore, predictive CoMFA (FXa q² = 0.814; thrombin q² = 0.667) and CoMSIA (FXa q² = 0.807; thrombin q² = 0.624) models were developed and validated (FXa r²(test) = 0.823; thrombin r(2)(test) = 0.816) to feature molecular determinants of ligand binding affinity using the docking-based conformational alignments (DBCA) of 141 (88(train)+53(test)) and 39 (27(train)+11(test)) nonamidine class of potent FXa (0.004 ≤ K(i) (nM) ≤ 4700) and thrombin (0.001 ≤ K(i) (nM) ≤ 940) inhibitors, respectively. Interestingly, the ligand-based insights well corroborated with the structure-based insights in terms of the role of steric, electrostatic, and hydrophobic parameters for governing the selectivity for the two proteases. The new computational insights presented in this study are expected to be valuable for understanding and designing potent and selective antithrombotic agents.

Structural basis for the β-adrenergic receptor subtype selectivity of the representative agonists and antagonists.

The β(3)-adrenegic receptor (β(3)-AR) selectivity over β(1)- and β(2)-ARs has been the most important aspect for successful therapeutic agents for obesity and type-II diabetes, as the concomitant activation of β(1)- and β(2)-ARs would lead to undesirable side effects, such as increased heart rate. In order to explore the structural basis for the β-AR subtype selectivity of agonists and anatagonists, a three-dimensional structure of until date unresolved β(3)-AR has been modeled, compared with the resolved X-ray structures of β(1)- and β(2)-ARs, and used to study its stereoselective binding with until-date known diverse classes of representative agonists and antagonist. The obtained binding structures and calculated prime molecular mechanics-generalized Born surface area (MM-GBSA) binding free energies consistently reveal that while the subtype selectivity is strongly governed by the residues present in the extracellular ends of TM3, TM5, TM6, TM7 helices and of the ECL2 domain, the binding affinity is governed by the conserved residues present in the deep pocket limiting the degree of conformational and rotational freedoms to the bound ligand. The study demonstrates that the key structural requirements for the β(3)-selectivity are: (i) a negatively ionizable group (NIG) for direct interaction with β(3)-specific residue R315(6.58), (ii) a linker (9-10 Å length) between the protonated amine and NIG, and (iii) a substituted aryl ring directly attached to the β-hydroxyl carbon. The new computational insights acquired in this study are expected to be valuable in structure-based rational design of high-affinity agonists and antagonists with pronounced β(3)-selectivity for successful therapeutic agents for type-II diabetes and obesity.

Substituted 1,2,3,4-tetrahydroquinolin-6-yloxypropanes as β3-adrenergic receptor agonists: Design, synthesis, biological evaluation and pharmacophore modeling

In search of potent beta(3)-adrenergic receptor agonists, a series of novel substituted 1,2,3,4-tetrahydroquinolin-6-yloxypropanes has been synthesized and evaluated for their beta(3)-adrenergic receptor agonistic activity (ranging from -17.73% to 90.64% inhibition at 10 microM) using well established Human SK-N-MC neuroblastoma cells model. Four molecules viz. 11, 15, 22 and 23 showed beta(3)-AR agonistic IC(50) value of 0.55, 0.59, 1.18 and 1.76 microM, respectively. These four candidates have been identified as possible leads for further development of beta(3)-adrenergic receptor agonists for obesity and Type-II diabetes pharmacotherapy. The free OH and NH functions are found to be essential for beta(3)-adrenergic receptor agonistic activity. Among the synthesized beta(3)-adrenergic receptor agonists having 1,2,3,4-tetrahydroquinoline scaffold, the N-benzyl group is found to be superior over N-arylsulfonyl group. A putative pharmacophore model has been modeled considering the above four active molecules which distinguishes well between the active and inactive molecules.

Activation of the γ-Aminobutyric Acid Type B (GABAB) Receptor by Agonists and Positive Allosteric Modulators

Since the discovery of the GABA(B) agonist and muscle relaxant baclofen, there have been substantial advancements in the development of compounds that activate the GABA(B) receptor as agonists or positive allosteric modulators. For the agonists, most of the existing structure-activity data apply to understanding the role of substituents on the backbone of GABA as well as replacing the carboxylic acid and amine groups. In the cases of the positive allosteric modulators, the allosteric binding site(s) and structure-activity relationships are less well-defined; however, multiple classes of molecules have been discovered. The recent report of the X-ray structure of the GABA(B) receptor with bound agonists and antagonists provides new insights for the development of compounds that bind the orthosteric site of this receptor. From a therapeutic perspective, these data have enabled efforts in drug discovery in areas of addiction-related behavior, the treatment of anxiety, and the control of muscle contractility.

Synthesis and Anti-Renal Fibrosis Activity of Conformationally Locked Truncated 2-Hexynyl-N6-Substituted-(N)-Methanocarba-nucleosides as A3 Adenosine Receptor Antagonists and Partial Agonists

Truncated N6-substituted-(N)-methanocarba-adenosine derivatives with 2-hexynyl substitution were synthesized to examine parallels with corresponding 4′-thioadenosines. Hydrophobic N6 and/or C2 substituents were tolerated in A3AR binding, but only an unsubstituted 6-amino group with a C2-hexynyl group promoted high hA2AAR affinity. A small hydrophobic alkyl (4b and 4c) or N6-cycloalkyl group (4d) showed excellent binding affinity at the hA3AR and was better than an unsubstituted free amino group (4a). A3AR affinities of 3-halobenzylamine derivatives 4f–4i did not differ significantly, with Ki values of 7.8–16.0 nM. N6-Methyl derivative 4b (Ki = 4.9 nM) was a highly selective, low efficacy partial A3AR agonist. All compounds were screened for renoprotective effects in human TGF-β1-stimulated mProx tubular cells, a kidney fibrosis model. Most compounds strongly inhibited TGF-β1-induced collagen I upregulation, and their A3AR binding affinities were proportional to antifibrotic effects; 4b was most potent (IC50 = 0.83 μM), indicating its potential as a good therapeutic candidate for treating renal fibrosis.

Identification and characterisation of small-molecule inhibitors of Rv3097c-encoded lipase (LipY) of Mycobacterium tuberculosis that selectively inhibit growth of bacilli in hypoxia

The mycobacterial Rv3097c-encoded lipase LipY is considered as a true lipase involved in the hydrolysis of triacylglycerol stored in lipid inclusion bodies for the survival of dormant mycobacteria. To date, orlistat is the only known LipY inhibitor. In view of the important emerging role of this enzyme, a search for small-molecule inhibitors of LipY was made, leading to the identification of some new compounds (8a-8d, 8f, 8h and 8i) with potent inhibitory activities against recombinant LipY, with no cytotoxicity [50% inhibitory concentration (CC(50)) ≥ 500 μg/mL]. The compounds 6a, 8c and 8f potently inhibited (>90%) the growth of Mycobacterium tuberculosis H37Rv grown under hypoxia (oxygen-depleted condition) but had no effect on aerobically grown bacilli, suggesting that these new small molecules are highly selective towards the growth inhibition of hypoxic cultures of M. tuberculosis and hence provide new leads for combating latent tuberculosis.