Falgun Shah

Identification of Novel Malarial Cysteine Protease Inhibitors Using Structure-Based Virtual Screening of a Focused Cysteine Protease Inhibitor Library

Malaria, in particular that caused by Plasmodium falciparum , is prevalent across the tropics, and its medicinal control is limited by widespread drug resistance. Cysteine proteases of P. falciparum , falcipain-2 (FP-2) and falcipain-3 (FP-3), are major hemoglobinases, validated as potential antimalarial drug targets. Structure-based virtual screening of a focused cysteine protease inhibitor library built with soft rather than hard electrophiles was performed against an X-ray crystal structure of FP-2 using the Glide docking program. An enrichment study was performed to select a suitable scoring function and to retrieve potential candidates against FP-2 from a large chemical database. Biological evaluation of 50 selected compounds identified 21 diverse nonpeptidic inhibitors of FP-2 with a hit rate of 42%. Atomic Fukui indices were used to predict the most electrophilic center and its electrophilicity in the identified hits. Comparison of predicted electrophilicity of electrophiles in identified hits with those in known irreversible inhibitors suggested the soft-nature of electrophiles in the selected target compounds. The present study highlights the importance of focused libraries and enrichment studies in structure-based virtual screening. In addition, few compounds were screened against homologous human cysteine proteases for selectivity analysis. Further evaluation of structure-activity relationships around these nonpeptidic scaffolds could help in the development of selective leads for antimalarial chemotherapy.

Structural insights into the Plasmodium falciparum histone deacetylase 1 (PfHDAC-1): A novel target for the development of antimalarial therapy

The histone deacetylase (HDAC) enzyme from Plasmodium falciparum has been identified as a novel target for the development of antimalarial therapy. A ligand-refined homology model of PfHDAC-1 was generated from the crystal structures of human HDAC8 and HDLP using a restraint guided optimization procedure involving the OPLS/GBSA potential setup. The model was extensively validated using protein structure checking tools. A predictive docking study was carried out using a set of known human HDAC inhibitors, which were shown to have in vitro antimalarial activity against the chloroquine sensitive D6 and resistant W2 strains of P. falciparum. Pose validation and score-based active/inactive separation studies provided independent validation of the geometric accuracy and the predictive ability of the generated model. Comparative analysis was carried out with the human HDACs to identify differences in the binding site topology and interacting residues, which might be utilized to develop selective PfHDAC-1 inhibitors.

Expression in Yeast Links Field Polymorphisms in PfATP6 to in Vitro Artemisinin Resistance and Identifies New Inhibitor Classes

BACKGROUND The mechanism of action of artemisinins against malaria is unclear, despite their widespread use in combination therapies and the emergence of resistance. RESULTS Here, we report expression of PfATP6 (a SERCA pump) in yeast and demonstrate its inhibition by artemisinins. Mutations in PfATP6 identified in field isolates (such as S769N) and in laboratory clones (such as L263E) decrease susceptibility to artemisinins, whereas they increase susceptibility to unrelated inhibitors such as cyclopiazonic acid. As predicted from the yeast model, Plasmodium falciparum with the L263E mutation is also more susceptible to cyclopiazonic acid. An inability to knockout parasite SERCA pumps provides genetic evidence that they are essential in asexual stages of development. Thaperoxides are a new class of potent antimalarial designed to act by inhibiting PfATP6. Results in yeast confirm this inhibition. CONCLUSIONS The identification of inhibitors effective against mutated PfATP6 suggests ways in which artemisinin resistance may be overcome.

Computer-aided drug design of falcipain inhibitors: virtual screening, structure-activity relationships, hydration site thermodynamics, and reactivity analysis.

Falcipains (FPs) are hemoglobinases of Plasmodium falciparum that are validated targets for the development of antimalarial chemotherapy. A combined ligand- and structure-based virtual screening of commercial databases was performed to identify structural analogs of virtual screening hits previously discovered in our laboratory. A total of 28 low micromolar inhibitors of FP-2 and FP-3 were identified and the structure-activity relationship (SAR) in each series was elaborated. The SAR of the compounds was unusually steep in some cases and could not be explained by a traditional analysis of the ligand-protein interactions (van der Waals, electrostatics, and hydrogen bonds). To gain further insights, a statistical thermodynamic analysis of explicit solvent in the ligand binding domains of FP-2 and FP-3 was carried out to understand the roles played by water molecules in binding of these inhibitors. Indeed, the energetics associated with the displacement of water molecules upon ligand binding explained some of the complex trends in the SAR. Furthermore, low potency of a subset of FP-2 inhibitors that could not be understood by the water energetics was explained in the context of poor chemical reactivity of the reactive centers of these compounds. The present study highlights the importance of considering energetic contributors to binding beyond traditional ligand-protein interactions.

Design, Synthesis, and Development of Novel Guaianolide-Endoperoxides as Potential Antimalarial Agents

Design and synthesis of a guaianolide-endoperoxide (thaperoxide) 3 was pursued as a new antimalarial lead which was found to be noncytotoxic as compared to the natural product lead thapsigargin 2. Several analogues of 3 were successfully synthesized and found to be comparable to derivatives of artemisinin 1 in in vitro antimalarial assay. Among the synthesized compounds, 22 showed excellent in vitro potency against the cultured parasites (W2 IC(50) = 13 nM) without apparent cytotoxicity. Furthermore, SAR trends in thaperoxide analogues are presented and explained with the help of docking studies in the homology model of PfSERCA(PfATP6).

Design, Synthesis, and Docking Studies of Novel Benzimidazoles for the Treatment of Metabolic Syndrome

In addition to lowering blood pressure, telmisartan, an angiotensin (AT(1)) receptor blocker, has recently been shown to exert pleiotropic effects as a partial agonist of nuclear peroxisome proliferator-activated receptor gamma (PPAR gamma). On the basis of these findings and docking pose similarity between telmisartan and rosiglitazone in PPAR gamma active site, two classes of benzimidazole derivatives were designed and synthesized as dual PPAR gamma agonist/angiotensin II antagonists for the possible treatment of metabolic syndrome. Compound 4, a bisbenzimidazole derivative showed the best affinity for the AT(1) receptor with a K(i) = 13.4 nM, but it was devoid of PPAR gamma activity. On the other hand 9, a monobenzimidazole derivative, showed the highest activity in PPAR gamma transactivation assay (69% activation) with no affinity for the AT(1) receptor. Docking studies lead to the designing of a molecule with dual activity, 10, with moderate PPARgamma activity (29%) and affinity for the AT(1) receptor (K(i) = 2.5 microM).

Inhibitors of SARS-3CLpro: Virtual Screening, Biological Evaluation and Molecular Dynamics Simulation Studies

SARS-CoV from the coronaviridae family has been identified as the etiological agent of Severe Acute Respiratory Syndrome (SARS), a highly contagious upper respiratory disease that reached epidemic status in 2002. SARS-3CL(pro), a cysteine protease indispensible to the viral life cycle, has been identified as one of the key therapeutic targets against SARS. A combined ligand and structure-based virtual screening was carried out against the Asinex Platinum collection. Multiple low micromolar inhibitors of the enzyme were identified through this search, one of which also showed activity against SARS-CoV in a whole cell CPE assay. Furthermore, multinanosecond explicit solvent simulations were carried out using the docking poses of the identified hits to study the overall stability of the binding site interactions as well as identify important changes in the interaction profile that were not apparent from the docking study. Cumulative analysis of the evaluated compounds and the simulation studies led to the identification of certain protein-ligand interaction patterns which would be useful in further structure based design efforts.

Computational Approaches for the Discovery of Cysteine Protease Inhibitors Against Malaria and SARS

Cysteine proteases are implicated in a variety of human physiological processes and also form an essential component of the life cycle of a number of pathogenic protozoa and viruses. The present review highlights the drug design approaches utilized to understand the mechanism of inhibition and discovery of inhibitors against protozoal cysteine protease, falcipain (a cysteine protease of P. falciparum which causes malaria), and viral cysteine protease, SARS-CoV M(pro) (a cysteine protease of severe acute respiratory syndrome corona virus). The article describes rational approaches for the design of inhibitors and focuses on a variety of structure as well as ligand-based modeling strategies adopted for the discovery of the inhibitors. Also, the key features of ligand recognition against these targets are accentuated. Although no apparent similarities exist between viral and protozoal cysteine proteases discussed here, the goal is to provide examples of rational drug design approaches adopted to design inhibitors against these proteases. The current review would be of interest to scientists engaged in the development of drug design strategies to target the cysteine proteases present in mammals and other lower order organisms.

Design, synthesis and biological evaluation of novel benzothiazole and triazole analogs as falcipain inhibitors

We describe the design, combinatorial library synthesis and biological evaluation of compounds with benzothiazole and triazole cores as inhibitors of falcipain, cysteine proteases of the malaria parasite Plasmodium falciparum. These classes were originally discovered by structure-based virtual screening of a focused cysteine protease inhibitor library. Fifteen structural analogs of both series showed moderate inhibition of falcipain-2. Two compounds, 41 and 42, were predicted by docking studies to interact with polar residues buried in the S2 pockets of falcipain-2 and -3, and these compounds inhibited both enzymes. Compound 41 also demonstrated activity against chloroquine-resistant cultured P. falciparum parasites at the lower micromolar concentration. Evaluation of 41 and 42 against mammalian cysteine proteases of papain family suggest these polar residues of the S2 pocket may not be important for the design of selective inhibitors against falcipain.

Synthesis, biological evaluation, hydration site thermodynamics, and chemical reactivity analysis of α-keto substituted peptidomimetics for the inhibition of Plasmodium falciparum.

A new series of peptidomimetic pseudo-prolyl-homophenylalanylketones were designed, synthesized and evaluated for inhibition of the Plasmodium falciparum cysteine proteases falcipain-2 (FP-2) and falcipain-3 (FP-3). In addition, the parasite killing activity of these compounds in human blood-cultured P. falciparum was examined. Of twenty-two (22) compounds synthesized, one peptidomimetic comprising a homophenylalanine-based α-hydroxyketone linked Cbz-protected hydroxyproline (39) showed the most potency (IC50 80 nM against FP-2 and 60 nM against FP-3). In silico analysis of these peptidomimetic analogs offered important protein-ligand structural insights including the role, by WaterMap, of water molecules in the active sites of these protease isoforms. The pseudo-dipeptide 39 and related compounds may serve as a promising direction forward in the design of competitive inhibitors of falcipains for the effective treatment of malaria.