Lead Identification and development against histone deacetylase, carbonic anhydrase, cyclooxygenase-2 and thromboxane prostanoid receptor

Drug discovery is a highly time consuming and costly process. Lead identification and development are key steps in the drug discovery programme. Studies have suggested that a large number of commercially available drugs exhibit deep structural similarity to the lead compounds from which they were developed. Quality lead identification in terms of compounds with high potency and selectivity, favourable physicochemical parameters and invitro Absorption Distribution Metabolism and Excretion (ADME) parameters is the foremost requirement for the success of drug discovery process. In the present work we focus on lead identification for specific pharmaceutically important targets. In our current investigations we exploited the principle of bioisosterism for lead identification across various targets. In particular, we have developed sulfonylurea derivatives as lead compounds for Histone Deacetylase (HDAC) activator and Carbonic Anhydrase (CA) inhibitor. Starting with HDAC, systematic Structure Activity Relationship (SAR) studies were performed using known HDAC inhibitors. SAR studies suggest that a modular architect of zinc binding group (ZBG), linker and cap require for effective HDAC inhibition. A set of a novel compounds with sulfonylurea as zinc binding group (ZBG), alkyl chain as linker and phenyl group as cap were designed. Length of linker chain was optimized using docking studies. All the designed compounds were synthesized by sodium cyanate mediated synthesis of sulfonylurea method. Synthesized compound were screened for its HDAC inhibitory activity using HDAC1 isoform. Interestingly, some of these compounds show HDAC1 activation rather than inhibition. Compound 1 shows approximately 2 fold activation at 100 μM concentration. Numerous controls were performed in order to verify the results and to finally conclude that the designed compounds show HDAC activation rather than inhibition. Molecular docking studies of compound 1 revealed that sulfonylurea group of compound 1 binds with zinc metal present in the active site of HDAC2 (HDAC2 isoform were used because of unavailability of HDAC1 isoform crystal structure). Similarly a new set of compounds were designed with tosylureido group for another pharmaceutically important zinc metalloenzyme, namely carbonic anhydrase. All designed compounds were synthesized by using the same sodium cyanate mediated synthesis of sulfonylurea method. Screening of these compounds for their carbonic anhydrase inhibitory properties using human carbonic anhydrase II (hCAII) led to identification of compounds 12 and 13 with IC50 of approximately 600 nM. A FRET based assay implicates the lead compounds i.e. compound 12 and 13 as binding in proximity to the active site region of the enzyme. Molecular docking studies reveal that compound 12 and 13 bind to residues at the entrance of the active site thereby blocking access and resulting in enzyme inhibition. Based on our results, analogues of the lead compounds were designed and synthesized for Structure Activity Relationship (SAR) and isoform-selectivity studies. Preliminary studies of newly synthesized compound against hCAII inhibitory activity show promising results and its detailed investigation for its isoform selectivity is under way in collaborator’s laboratory. In developing sulfonylurea-derivatives as zinc metalloenzyme modulators, we have expanded the scope of a hitherto unexplored method of synthesis of sulfonylureas from sulfonyl chlorides and amines using sodium cyanate. In our lead identification programme for HDAC, we were able to access aliphatic sulfonylurea using sodium cyanate mediated synthesis of sulfonylurea method. In case of aliphatic sulfonylurea for HDAC i.e. compound 1-5, the scope of reaction was explored using various sulfonylchlorides. Further scope of reaction in term of amine was examined by using various sulfonylchlorides and employing aniline as amine. Reaction of propylamine with ethanesulfonyl chloride yields novel sulfonylureas with aliphatic side chains on both sides. Similarly scope of reaction in term of aromatic sulfonylchloride and amine were studied in our programme for lead identification for CA using the compounds 11-23. Results clearly indicate that nature of both the substrates influence the reaction. In case of electron donating groups on both the substrates, the reaction works well but in case of electron withdrawing group on them, the reaction does not yield desired product. In case of p-chlorophenyl sulphonyl chloride, the reaction shows interesting by-product profile. Isolation and characterization of by-product with the help of 1HNMR, 13CNMR and LCMS led to discovery of novel sulonyltriuret compounds. Initial attempts to decrease formation of sulfonyltriuret by varying various reaction parameters such as equivalents of sodium cyanate, time of addition of amine, solvent and temperature were in vain. The formation of sulfonyltriuret in each case is predictable. We thus investigated sodium cyanate mediated method for synthesis of sulfonylurea, as a method for synthesis of sulfonyltriuret. We have successfully isolated and characterized the corresponding sulfonyltriuret in some of the above reaction and the scope of the single-step reaction has also thus been investigated as a method for preparation of sulfonyltriurets. The unique structural features of the sulfonyltriurets, namely the presence of pseudo-peptide bonds and resemblance to triurets indicate potential applications. In the last part of our work we used a Design Multi-target Ligand (DML) approach for drug design. Inspired by bioisosterism of pharmacophores and by combining essential pharmacophores for their cognate targets, we attempted to identify a lead for dual inhibition of cyclooxygenase-2 (COX-2) and Thromboxane Prostanoid (TP) receptor. Such dual inhibitors are still in a nascent stage of development and have been suggested as the next generation of anti-inflammatory and pain-relieving medicines. We combined the essential pharmacophores from an established COX-2 inhibitor (Celecoxib) and a TP receptor antagonist (BM-573) into designing one class of molecules. Designed compounds were retrosynthesized and the synthesis of designed compound using the scheme intermediate C2 was achieved. Reaction of intermediate C2 (possessing primary amine group) with sodium cyanate and tosylchloride (sodium cyanate mediated synthesis of sulfonylurea) towards corresponding desired sulfonylurea derivatives was unsuccessful mainly because of presence of nitro group on one of the phenyl ring in intermediate C2. Since nitro is not an essential pharmacophore for both targets i.e. COX-2 and TP, proposed dual inhibitor was modified without nitro group. Based on the synthetic scheme of intermediate C2, intermediate C15 (without nitro group) were synthesized. Reaction of intermediate C15 (have primary amine group) with sodium cyanate and tosylchloride yield the desired corresponding sulfonylurea derivative. However in the current context we fail to purify desired sulfonylurea from its sulfonyltriuret derivative. In order to provide proof of our hypothesis and to avoid synthetic challenge, we have repurposed desired dual inhibitor by using Terutroban as a known TP receptor antagonist instead of BM-573. Terutroban is a nonprostanoid TP receptor antagonist and sulfonamide is essential pharmacophore. Thus in repurposed dual inhibitor, sulfonylurea group was replaced by sulfonamide and synthesized by treatment of intermediate C15 (possessing primary amine group) with tosylchloride. All synthesized compounds were screened for their COX-2 inhibitory activity. However, compounds bearing this phenomenological design fail to display any activity against the COX-2 target. Since all of these compounds fail to show promising results against the principle target COX-2 we did not screen them for their TP receptor activity. We thereafter explored an entirely bottom-up design strategy using various techniques of computational structure based drug design (SBDD). Starting from the basic scaffold of flavonoid, activity of Design 4 was optimized for its COX-2 activity using docking studies. In the absence of structural information for TP receptor, model for TP receptor was generated based on fold recognition method. Loops were refined using prime and the quality of model was judged by using Ramachandran plot. MMGBSA based binding free energy of a known TP receptor antagonist was predicted and its correlation with experimental activity of the compound was calculated. Enrichment studies were performed to predict ability of docking protocol to distinguish active molecules from inactive ones using a set of 133 decoy compounds. The goodness of hit (GH) score was used to confirm that the docking protocol was capable of distinguishing active molecules from inactive ones. Docking and repeated iterations of structural modification finally led to design 6 which shows good docking score and binding pose in TP receptor. Docking of design 6 in the active site of COX-2 results into better stabilization due to flipping of B ring from hydrophilic to hydrophobic region. MD studies of Design 6 show that the compound is stable in TP receptor over entire simulation of 50 ns and does not diffuse away. The compound designed using the above methodology could be a potential lead as a dual COX-2 inhibitor and TP receptor antagonist and may overcome the side effects associated with COXIBs.by Hadianawala Murtuza ShabbiraliPh.D

Design and development of sulfonylurea derivatives as zinc metalloenzyme modulators

Sulfonamide derivatives are an important class of zinc metalloenzyme inhibitors. While the sulfonylurea group is a bioisoster of sulfonamide, their effect on the modulation of metalloenzymes has never been studied. In the present work we synthesize and screen new sulfonylurea derivatives towards the zinc metalloenzymes, human Carbonic Anhydrase II (hCA II) and histone deacetylase 1 (HDAC 1). Specific sulfonylurea derivatives are found to inhibit hCA II with IC50 in the nano molar to micro molar range. Docking studies indicate the binding of the inhibitors to the mouth of the active site cavity thereby blocking access to the enzyme. Surprisingly sulfonylurea derivatives exhibit activation of HDAC 1 rather than inhibition. The activation of HDAC 1 is not uniform across the derivatives tested suggesting a specific mode that depends on structural features of the compounds. Extensive research has been performed on HDAC inhibitors due to their potential as anti-cancer agents, however relatively little has been reported in terms of HDAC activation. In this work, we present the distinctly divergent behavior of the same class of molecules namely sulfonylurea derivatives with respect to hCA II and HDAC 1 and attempt to provide an understanding of the same.by Murtuza Hadianawala and Bhaskar Datt

Implication of sulfonylurea derivatives as prospective inhibitors of human carbonic anhydrase II

Selective carbonic anhydrase (CA) inhibitors have gained a lot of importance owing to the implication of specific isoforms of CA in certain diseases like glaucoma, leukemia, cystic fibrosis, and epilepsy. A novel class of sulfonylurea derivatives was synthesized from corresponding sulfonyl chlorides and amines. Compounds with different pendant moieties in the sulfonylurea derivatives show significant interactions with human carbonic anhydrase II (CAII). In vitro evaluation of the sulfonylurea derivatives revealed that three compounds possess admirable inhibitory activity against CAII. Compounds containing methyl (G2), isopropyl (G4) and o-tosyl (G5) groups displayed IC50 (109-137 μm) for CAII. Fluorescence binding and cytotoxicity studies revealed that these compounds are showing good binding affinity (18-34 μM) to CAII and non- toxic to human cells. Further, molecular docking studies of G2, G4 and G5 with CAII showed that these compounds fit nicely in the active site of CAII. Molecular dynamics simulation studies of these compounds complexed with CAII showed that essential interactions were maintained up to 50 ns of simulation. These results indicate the promising nature of the sulfonylurea scaffold towards CAII inhibition and opens scope of hit to-lead optimization for discovery of effective drugs against CAII-associated disorders.by Danish Idrees, Murtuza Hadianawala, Amarjyoti Das Mahapatra, Bhaskar Datta, Sonam Roy, Shahzaib Ahamad, Parvez Khan and Md.Imtiyaz Hassa

Molecular docking, molecular modeling, and molecular dynamics studies of azaisoflavone as dual COX-2 inhibitors and TP receptor antagonists

Designed multi-target ligand (DML) is an emerging strategy for the development of new drugs and involves the engagement of multiple targets with the same moiety. In the context of NSAIDs it has been suggested that targeting the thromboxane prostanoid (TP) receptor along with cyclooxygenase-2 (COX-2) may help to overcome cardiovascular (CVS) complications associated with COXIBs. In the present work, azaisoflavones were studied for their COX-2 and TP receptor binding activities using structure based drug design (SBDD) techniques. Flavonoids were selected as a starting point based on their known COX-2 inhibitory and TP receptor antagonist activity. Iterative design and docking studies resulted in the evolution of a new class scaffold replacing the benzopyran-4-one ring of flavonoids with quinolin-4-one. The docking and binding parameters of these new compounds are found to be promising in comparison to those of selective COX-2 inhibitors, such as SC-558 and celecoxib. Owing to the lack of structural information, a model for the TP receptor was generated using a threading base alignment method with loop optimization performed using an ab initio method. The model generated was validated against known antagonists for TP receptor using docking/MMGBSA. Finally, the molecules that were designed for selective COX-2 inhibition were docked into the active site of the TP receptor. Iterative structural modifications and docking on these molecules generated a series which displays optimum docking scores and binding interaction for both targets. Molecular dynamics studies on a known TP receptor antagonist and a designed molecule show that both molecules remain in contact with protein throughout the simulation and interact in similar binding modes.by Murtuza Hadianawala, Amarjyoti Das, Mahapatra Jitender, K. Yadav and Bhaskar Datt