Schistosomiasis: Setting Routes for Drug Discovery

Schistosomiasis is the second most prevalent parasitic disease in the world. Currently, the treatment of this disease relies on a single drug, praziquantel, and due to the identification of resistant parasites, the development of new drugs is urged. The demand for the development of robust high‐throughput parasite screening techniques is increasing as drug discovery research in schistosomiasis gains significance. Here, we review the most common methods used for compound screening in the parasites life stages and also summarize some of the methods that have been recently developed. In addition, we reviewed the methods most commonly implemented to search for promising targets and how they have been used to validate new targets against the parasite Schistosoma mansoni. We also review some promising targets in this parasite and show the main approaches and the major advances that have been achieved by those studies. Moreover, we share our experiences in schistosomiasis drug discovery attained with our S. mansoni drug screening platform establishment

Identification of 6-(piperazin-1-yl)-1,3,5-triazine as a chemical scaffold with broad anti-schistosomal activities

Background: Schistosomiasis, caused by infection with blood fluke schistosomes, is a neglected tropical disease of considerable importance in resource-poor communities throughout the developing world. In the absence of an immunoprophylactic vaccine and due to over-reliance on a single chemotherapy (praziquantel), schistosomiasis control is at risk should drug insensitive schistosomes develop. In this context, application of in silico virtual screening on validated schistosome targets has proven successful in the identification of novel small molecules with anti-schistosomal activity. Methods: Focusing on the Schistosoma mansoni histone methylation machinery, we herein have used RNA interference (RNAi), ELISA-mediated detection of H3K4 methylation, homology modelling and in silico virtual screening to identify a small collection of small molecules for anti-schistosomal testing. A combination of low to high-throughput whole organism assays were subsequently used to assess these compounds’ activities on miracidia to sporocyst transformation, schistosomula phenotype/motility metrics and adult worm motility/oviposition readouts. Results: RNAi-mediated knockdown of smp_138030/smmll-1 (encoding a histone methyltransferase, HMT) in adult worms (~60%) reduced parasite motility and egg production. Moreover, in silico docking of compounds into Smp_138030/SmMLL-1’s homology model highlighted competitive substrate pocket inhibitors, some of which demonstrated significant activity on miracidia, schistosomula and adult worm lifecycle stages together with variable effects on HepG2 cells. Particularly, the effect of compounds containing a 6-(piperazin-1-yl)-1,3,5-triazine core on adult schistosomes recapitulated the results of the smp_138030/smmll-1 RNAi screens. Conclusions: The biological data and the structure-activity relationship presented in this study define the 6-(piperazin-1-yl)-1,3,5-triazine core as a promising starting point in ongoing efforts to develop new urgently needed schistosomicides

A computational framework for structure-based drug discovery with GPU acceleration.

Li, Hongjian.Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.Includes bibliographical references (p. 132-156).Abstracts in English and Chinese.Abstract --- p.iAbstract in Chinese --- p.iiiAcknowledgement --- p.ivChapter 1 --- Introduction --- p.1Chapter 1.1 --- Motivation --- p.2Chapter 1.2 --- Objective --- p.2Chapter 1.3 --- Method --- p.3Chapter 1.4 --- Outline --- p.4Chapter 2 --- Background --- p.7Chapter 2.1 --- Overview of the Pharmaceutical Industry --- p.7Chapter 2.2 --- The Process of Modern Drug Discovery --- p.10Chapter 2.2.1 --- Development of an Innovative Idea --- p.10Chapter 2.2.2 --- Establishment of a Project Team --- p.11Chapter 2.2.3 --- Target Identification --- p.11Chapter 2.2.4 --- Hit Identification --- p.12Chapter 2.2.5 --- Lead Identification --- p.13Chapter 2.2.6 --- Lead Optimization --- p.14Chapter 2.2.7 --- Clinical Trials --- p.14Chapter 2.3 --- Drug Discovery via Computational Means --- p.15Chapter 2.3.1 --- Structure-Based Virtual Screening --- p.16Chapter 2.3.2 --- Computational Synthesis of Potent Ligands --- p.20Chapter 2.3.3 --- General-Purpose Computing on GPU --- p.23Chapter 3 --- Approximate Matching of DNA Patterns --- p.26Chapter 3.1 --- Problem Definition --- p.27Chapter 3.2 --- Motivation --- p.28Chapter 3.3 --- Background --- p.30Chapter 3.4 --- Method --- p.32Chapter 3.4.1 --- Binary Representation --- p.32Chapter 3.4.2 --- Agrep Algorithm --- p.32Chapter 3.4.3 --- CUDA Implementation --- p.34Chapter 3.5 --- Experiments and Results --- p.39Chapter 3.6 --- Discussion --- p.44Chapter 3.7 --- Availability --- p.45Chapter 3.8 --- Conclusion --- p.47Chapter 4 --- Structure-Based Virtual Screening --- p.50Chapter 4.1 --- Problem Definition --- p.51Chapter 4.2 --- Motivation --- p.52Chapter 4.3 --- Medicinal Background --- p.52Chapter 4.4 --- Computational Background --- p.59Chapter 4.4.1 --- Scoring Function --- p.59Chapter 4.4.2 --- Optimization Algorithm --- p.65Chapter 4.5 --- Method --- p.68Chapter 4.5.1 --- Scoring Function --- p.69Chapter 4.5.2 --- Inactive Torsions --- p.72Chapter 4.5.3 --- Optimization Algorithm --- p.73Chapter 4.5.4 --- C++ Implementation Tricks --- p.74Chapter 4.6 --- Data --- p.75Chapter 4.6.1 --- Proteins --- p.75Chapter 4.6.2 --- Ligands --- p.76Chapter 4.7 --- Experiments and Results --- p.77Chapter 4.7.1 --- Program Validation --- p.77Chapter 4.7.2 --- Virtual Screening --- p.81Chapter 4.8 --- Discussion --- p.89Chapter 4.9 --- Availability --- p.90Chapter 4.10 --- Conclusion --- p.91Chapter 5 --- Computational Synthesis of Ligands --- p.92Chapter 5.1 --- Problem Definition --- p.93Chapter 5.2 --- Motivation --- p.93Chapter 5.3 --- Background --- p.94Chapter 5.4 --- Method --- p.97Chapter 5.4.1 --- Selection --- p.99Chapter 5.4.2 --- Mutation --- p.102Chapter 5.4.3 --- Crossover --- p.102Chapter 5.4.4 --- Split --- p.103Chapter 5.4.5 --- Merging --- p.104Chapter 5.4.6 --- Drug Likeness Testing --- p.104Chapter 5.5 --- Data --- p.105Chapter 5.5.1 --- Proteins --- p.105Chapter 5.5.2 --- Initial Ligands --- p.107Chapter 5.5.3 --- Fragments --- p.107Chapter 5.6 --- Experiments and Results --- p.109Chapter 5.6.1 --- Binding Conformation --- p.112Chapter 5.6.2 --- Free Energy and Molecule Weight --- p.115Chapter 5.6.3 --- Execution Time --- p.116Chapter 5.6.4 --- Support for Phosphorus --- p.116Chapter 5.7 --- Discussion --- p.120Chapter 5.8 --- Availability --- p.123Chapter 5.9 --- Conclusion --- p.123Chapter 5.10 --- Personal Contribution --- p.124Chapter 6 --- Conclusion --- p.125Chapter 6.1 --- Conclusion --- p.125Chapter 6.2 --- Future Work --- p.128Chapter A --- Publications --- p.130Chapter A.1 --- Conference Papers --- p.130Chapter A.2 --- Journal Papers --- p.131Bibliography --- p.13

PURINE NUCLEOSIDE PHOSPHORYLASES AS BIOCATALYSTS AND PHARMACOLOGICAL TARGETS

A purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) was successfully exploited to catalyze the \u201cone-pot, one-enzyme\u201d regio- and stereoselective transfer of \u3b2-D-ribose from a proper sugar donor (7-methylguanosine iodide) to a library of 6-substituted purine acceptors, resulting in the \u201cin batch\u201d synthesis of 24 ribonucleosides. Transglycosylation conversions confirmed the broad tolerance and the potential of AhPNP as biocatalyst, providing the necessary information to undertake the preparative synthesis of 6-modified purine nucleosides. [1] AhPNP was then immobilized in a stainless steel column resulting in a stable and active bioreactor (AhPNP-IMER, Immobilized Enzyme Reactor) that, upon on-line connection to a semi-preparative HPLC system, was used to run transglycosylations in a flow mode. In such a set-up, biotransformation, on-line monitoring and product purification occurred in a single integrated platform, thus allowing the preparation of five nucleoside analogues at a mg scale (52-89% yield). [2] As a step forward, a \u201cone-pot, two-enzyme\u201d strategy was applied by coupling AhPNP-IMER with an analogous bioreactor based on a uridine phosphorylase from Clostridium perfringens (CpUP), immobilized in a monolith column. The on-line apparatus obtained by connecting CpUP-IMER and AhPNP-IMER in series was tested in the synthesis of adenosine, 2\u2019-deoxyadenosine and arabinosyladenine from uridine, 2\u2019-deoxyuridine and arabinosyluracyl as sugar donors, respectively. The corresponding nucleobases were transformed into the products in 90-95% conversion over 1 h for the ribosyl and 2\u2019-deoxyribosyl derivatives, and 20% conversion after 5 h for arabinosyladenine. [3] Furthermore, a new LC-ESI-MS/MS method was set up to evaluate the inhibition activity of 8-substituted purine ribonucleosides toward the PNP from Mycobacterium tuberculosis (MtPNP), as well as the selectivity against the microbial enzyme with respect to the corresponding human one (HsPNP). The corresponding enzymatic assay, based on the phosphorolysis of inosine, proved to be very convenient in terms of time as well as of target amount. A small library of seven 8-substituted purine ribonucleosides were screened, not exerting any significant effect up to 1 mM, with 8-bromoguanosine and 8-methylaminoguanosine being the only exceptions at 500 mM as weak inhibitors. [4] Finally, the chemical synthesis of a series of 8- and N2-substituted inosinic and guanylic acids as potential ligands of the human GPR17 receptor was carried out, starting from studies aided by molecular modeling on a homology model of the target. The molecules were prepared by 5\u2019-phosphorylation of properly 8- and N2-modified/protected inosine or guanosine. Owing to the scarce nucleophilicity of the exocyclic NH2 group of guanosine, the 2-position of the purine ring was activated as a bromo derivative, whose displacement with the proper amine afforded the desired N2-alkylated products. On the contrary, N2-acylations were carried out through nitrogen functionalization with a proper acyl chloride or anhydride. An additional 2\u2019,3\u2019-O-isopropylidene group was inserted in all the N2-functionalized nucleotides. Binding assays on GPR17 will be carried out. [1] D. Ubiali, C. F. Morelli, M. Rabuffetti, G. Cattaneo, I. Serra, T. Bavaro, A. M. Albertini, G. Speranza Curr. Org. Chem. 2015, 19, 2220-2225; [2] E. Calleri, G. Cattaneo, M. Rabuffetti, I. Serra, T. Bavaro, G. Massolini, G. Speranza, D. Ubiali Adv. Synth. Catal. 2015, 357, 2520-2528; [3] G. Cattaneo, M. Rabuffetti, G. Speranza, T. Kupfer, B. Peters, G. Massolini, D. Ubiali, E. Calleri Submitted 2017; [4] G. Cattaneo, D. Ubiali, E. Calleri, M. Rabuffetti, G. C. Hofner, K. T. Wanner, M. C. De Moraes, L. K. B Martinelli, D. S. Santos, G. Speranza Anal. Chim. Acta 2016, 943, 89-97

Drug Discovery

Natural products are a constant source of potentially active compounds for the treatment of various disorders. The Middle East and tropical regions are believed to have the richest supplies of natural products in the world. Plant derived secondary metabolites have been used by humans to treat acute infections, health disorders and chronic illness for tens of thousands of years. Only during the last 100 years have natural products been largely replaced by synthetic drugs. Estimates of 200 000 natural products in plant species have been revised upward as mass spectrometry techniques have developed. For developing countries the identification and use of endogenous medicinal plants as cures against cancers has become attractive. Books on drug discovery will play vital role in the new era of disease treatment using natural products

The evolution of antibacterial chemotherapy: targeting RecA to sabotage antibiotic tolerance and resistance mechanisms

Antibiotic resistant bacteria are rendering the current supply of available antibacterial drugs ineffective at an alarming rate and there is a dearth of novel drug targets for the treatment of bacterial infectious diseases. New strategies are required to combat pathogenic bacteria and in this context RecA has emerged as an intriguing candidate for inhibition studies. In the bacterial kingdom, the RecA protein is a highly conserved recombinase enzyme that mediates DNA repair and horizontal gene transfer and across all species it almost uniformly regulates the SOS response to DNA damage. Recent evidence suggests that these RecA-controlled processes are responsible for an increased tolerance to antibiotic chemotherapy and they up-regulate pathways which ultimately lead to full-fledged antibiotic resistance. We propose targeting RecA with small molecules to sabotage the molecular mechanisms which are believed to cause antibiotic chemotherapy to fail. Towards the goal of validating RecA as an important and novel target for the chemotherapeutic treatment of bacterial infectious diseases we have studied the interaction of metal-dithiols, nucleotide analogs and drug-like small molecules with the RecA protein. Upon activation RecA binds ssDNA and performs ATP hydrolysis, therefore we observed either a reduction of RecA-ssDNA binding or ATP hydrolysis in the presence of potential inhibitors using fast and efficient screening assays. As the size and complexity of the compound libraries increased in our studies, the methods we employed to identify inhibitors evolved to meet the demand they imposed. In all, more than 64,000 compounds were assayed against RecA and we identified several lead structures which were active against RecA in Escherichia coli cell cultures. We demonstrate that cell-permeable inhibitors of RecA are capable of abrogating the SOS response and potentiate the toxicity of bactericidal antibiotics, e.g. ciprofloxacin. The results of this study suggests that RecA may serve as a novel antibacterial drug target not belonging to any class of currently prescribed antibiotics, and which has not previously been examined in this regard

Structure-based design of P2X receptor small molecule modulators

P2X4 and P2X7 receptors have gained increasing significance as drug targets for their involvement in neurotransmission, pain, cancer, inflammation and immunity. To date, numerous P2X7 antagonists have been developed, yet relatively few P2X4 antagonists have been reported, and no data is available regarding their binding sites on the receptor. Using in silico techniques, we attempted to identify novel P2X4 and P2X7 modulators, and combined experimental and structural data to explore allosteric pockets with future potential for drug design. Two virtual screenings of commercially available drug-like compounds performed in the human P2X4 homology model led to the selection, purchase and biological evaluation via calcium influx assay of 42 compounds. While no compound with significant antagonist activity at human P2X4 was found, multiple compounds abolished ATP-induced YO-PRO dye uptake at human P2X7, including Compound 25 (IC50 value of 8 μM). Further 27 structural analogues to Compound 25 were purchased and assayed, expanding the number of active antagonists and offering an insight in the structure-activity relationship. In parallel, a mutagenesis study of human and rat P2X4 receptors based on (i) species-specific pharmacology and (ii) the recently published panda P2X7 crystal structures led us to identify the allosteric binding site for the P2X4-selective antagonist BX-430. The pocket was then used to perform a new virtual screen, identifying 20 potential ‘hit’ candidates for future biological evaluation. Finally, we performed docking simulations of the positive P2X4 allosteric modulator ivermectin and the partially selective P2X7 agonist 2’(3’)-4-O-benzoylbenzoyl A TP (BzA TP), with good correlation between binding conformations and previously published pharmacological data. In conclusion, although no novel human P2X4 antagonist has been identified, this work led to (i) the discovery of an allosteric site not previously described in human P2X4, (ii) the identification of a novel P2X7 antagonist with micromolar potency and (iii) and increased understanding of the molecular basis for subtype-specific modulator potency at P2X4 and P2X7 receptors