Metabolomic analyses of the malaria parasite after inhibition of polyamine biosynthesis

Malaria, a disease transmitted by female mosquitoes, has plagued the world for many centuries. The disease is associated with high mortality rates, severe poverty, and economic burden. These are factors which hamper effective eradication of the disease. Drug resistant forms of the parasite have caused increasing concerns and questioned the longevity of current effective antimalarials. Efforts are therefore aimed at the identification and exploitation of essential parasite proteins as potential drug targets. The polyamine pathway of Plasmodium falciparum is an exploitable pathway which contains two distinct, chemically validated drug targets; a bifunctional PfAdoMetDC-ODC protein and PfSpdSyn. These enzymes ensure intricate regulation of polyamine production and the pathway contains various distinctive features which could be selectively targetable from the mammalian counterpart pathways. However, inhibition of polyamine production through the use of specific enzyme inhibitors has revealed various compensatory responses that negate the efficacy of these inhibitors. An account is given of the metabolomic fluctuations in the parasite during inhibition of polyamine biosynthesis. From co-inhibited P. falciparum extracts, it could be demonstrated that the characteristic growth-arrest coincided with the depletion in spermidine, the metabolic product of PfSpdSyn. The co-inhibition strategy therefore emphasised the importance of spermidine biosynthesis by PfSpdSyn. Moreover, adenosyl-related metabolite levels were not disrupted during polyamine depletion, supporting the notion that these metabolites are intricately recycled within the parasites. The identified metabolic compensatory mechanisms have further potential for exploitation, and can strategically be combined with polyamine biosynthesis inhibition to ensure parasitic attenuation. In addition, several novel inhibitors were previously computationally identified, based on a dynamic receptor-based pharmacophore model of PfSpdSyn. The in vitro inhibiting activity of these compounds was determined against PfSpdSyn. Results from the in vitro experiments supported the in silico predictions, and emphasized the supportive role of pharmacophore modelling has for the identification of novel inhibitors. The research contributed in understanding parasitic polyamine metabolite regulation, and will aid in the future optimization of therapeutic strategies, aimed at exploitation of the polyamine pathway as a potential antimalarial drug target. CopyrightDissertation (MSc)--University of Pretoria, 2009.Biochemistryunrestricte

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

Plasmodial enzymes in metabolic pathways as therapeutic targets and contemporary strategies to discover new antimalarial drugs: a review

Malaria continues to pose imminent threat to the world population, as the mortality rate associated with this disease remains high. Current treatment relies on antimalarial drugs such as Artemisinin Combination Therapy (ACT) are still effective throughout the world except in some places, where ACT-resistance has been reported, thus necessitating novel approaches to develop new anti-malarial therapy. In the light of emerging translational research, several plasmodial targets, mostly proteins or enzymes located in the parasite’s unique organelles, have been extensively explored as potential candidates for the development of novel antimalarial drugs. By targeting the metabolic pathways in mitochondrion, apicoplast or cytoplasm of Plasmodium, the possibility to discover new drugs is tremendous, as they have potentials as antimalarial therapeutic targets. This literature review summarizes pertinent information on plasmodial targets, especially enzymes involved in specific metabolic pathways, and the strategies used to discover new antimalarial drugs. © 2019, University of Malaya. All rights reserved

Synthetic approaches to the understanding of DNA nucleobase methylation

DNA methylation is a major source of genetic variation and cancer. Methylation occurs when nucleophilic DNA bases react with methylating agent methyl methanesulfonate (MMS), dimethyl sulfate (DMS), N-methyl-N-nitrosourea (MNU), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), etc. N7-methyl-2\u27-deoxyguanosine (N7-methyl-dG, or 7MedG) adduct is the most abundant DNA methylation products for most methylating agents. DNA polymerase actions on 7MedG are difficult to study due to its instability against ring- opening hydrolysis and deglycosyl ati on. Oligonucleotides containing a single chemical adduct of 7MedG cannot be chemically synthesized. In addition, 7MedG is unstable in vivo due to the presence of DNA repair enzymes. This work explores the possibility of using stable analogues of N7-methyl-dG to study the polymerase bypass. The chemical synthesis of N7-methyl-9-deaza- dG (7Me9CdG) has been developed and the nucleoside has been successfully incorporated into oligonucleotides. Thermal melting studies show that replacement of dG by 7Me9CdG only slightly decreases DNA duplex stability. Replication of the DNA templates containing 7Me9CdG and the related 7- methyl -7-deaza- dG (7Me7CdG) and 7-deaza- dG (7CdG) by Klenow fragment of E. coli DNA polymerase I is examined. The misincorporation frequencies on the 7Me9CdG, 7Me7CdG, and 7CdG templates are comparable to the dG template, although the 7-methyl group slows down the turnover rate of the polymerase when dCTP is incorporated. The stability of 7Me9CdG and 7Me7CdG against the actions of formamidopyrimidine DNA N- glycosylase (Fpg) and human alkyladenine DNA Glycosylase (hAAG) are also studied. 7Me9CdG is stable in the presence of both enzymes. In contrast, 7Me7CdG is cleaved by Fpg, and possibly by hAAG but in an extremely slow rate. This work demonstrates that 7Me9CdG is a better analogue than 7Me7CdG for future cellular studies. Epigenetic mechanisms regulate the expression of genetic information. A major epigenetic event is DNA cytosine methylation, which is catalyzed by the DNA methyltransferases (DNMTs). Among different mammalian DNMTs, DNMT1 is the most abundant and active, and plays multiple roles in carcinogenesis, embryonic development, and several other biological functions. Reactivation of silenced tumor suppressor genes by DNMT inhibitors (DNMTi) is provides a relatively new approach to cancer therapy. A couple of irreversible nucleoside DNMT inhibitors have been developed clinically. However, due to their low specificity and high cellular toxicity, there is a clear need for the development of reversible inhibitors. S-adenosyl homocysteine (SAH) is a known strong inhibitor of DNA methyltransferases. Based on the crystal structure of SAH bound to human DNMT1-DNA complex, a series of transition state analogues have been designed to occupy the SAH binding site and the cytosine binding site simultaneously. These analogues have been successfully synthesized from adenosine using a modular approach. Inhibition of DNMT1 by these analogues will be studied in the future

Plasma homocysteine, measurement and clinical application

Raised plasma homocysteine (Hcy) levels have been cited as a major risk factor for several vascular disorders. Yet hyperhomocysteinaemia is easily treated through dietary intervention and vitamin supplementation. Commercial assays have facilitated routine plasma Hcy analysis. However, the problem faced by clinicians is stabilisation of Hcy in whole blood samples prior to delivery to the laboratory. Following blood collection, erythrocytes continue to produce and excrete Hcy increasing plasma concentrations by up to 10% per hour. This thesis describes the investigation of stabilising plasma Hcy in whole blood, allowing wide scale screening for hyperhomocysteinaemia. The most effective method appears to be inhibition of the enzyme responsible for Hcy production, Sadenosylhomocysteine hydrolase (SAHH), using a competitive inhibitor 3- deazaadenosine (3DA). Clinical trials were conducted on a pilot batch of evacuated blood tubes. Samples were stored in EDTA whole blood in the presence and absence of 3DA, at ambient temperatures (20 to 25ºC), and under refrigerated conditions (2 to 8ºC). Only samples that were collected into EDTA plus 3DA tubes and stored refrigerated showed stability over 72 hours (p = 0.2761). For wide scale screening, samples must be stable under ambient conditions. As the structure of SAHH is known a molecular modelling approach was adopted in an attempt to identify other potential inhibitors from screened databases. Interference of SAHH, in an immunochemical method for Hcy, was to be utilised for in vitro screening before any further clinical trials were conducted. The thesis also focuses on Hcy as a marker of vitamin deficiency and explores links between thiol metabolism and the development of cognitive decline eventually leading to dementia. Disruption of single carbon metabolism can lead to an increase in Hcy and a decrease in available methyl groups important in regulation of several metabolic pathways. Increased oxidative stress may also be a causative factor.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Identification of Attractive Drug Targets in Neglected-Disease Pathogens Using an In Silico Approach

In cell-based drug development, researchers attempt to create drugs that kill a pathogen without necessarily understanding the details of how the drugs work. In contrast, target-based drug development entails the search for compounds that act on a specific intracellular target—often a protein known or suspected to be required for survival of the pathogen. The latter approach to drug development has been facilitated greatly by the sequencing of many pathogen genomes and the incorporation of genome data into user-friendly databases. The present paper shows how the database TDRtargets.org can identify proteins that might be considered good drug targets for diseases such as African sleeping sickness, Chagas disease, parasitic worm infections, tuberculosis, and malaria. These proteins may score highly in searches of the database because they are dissimilar to human proteins, are structurally similar to other “druggable” proteins, have functions that are easy to measure, and/or fulfill other criteria. Researchers can use the lists of high-scoring proteins as a basis for deciding which potential drug targets to pursue experimentally

A multi-pronged approach targeting SARS-CoV-2 proteins using ultra-large virtual screening.

The unparalleled global effort to combat the continuing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic over the last year has resulted in promising prophylactic measures. However, a need still exists for cheap, effective therapeutics, and targeting multiple points in the viral life cycle could help tackle the current, as well as future, coronaviruses. Here, we leverage our recently developed, ultra-large-scale in silico screening platform, VirtualFlow, to search for inhibitors that target SARS-CoV-2. In this unprecedented structure-based virtual campaign, we screened roughly 1 billion molecules against each of 40 different target sites on 17 different potential viral and host targets. In addition to targeting the active sites of viral enzymes, we also targeted critical auxiliary sites such as functionally important protein-protein interactions

The Therapeutic Targeting Of Folate Receptor Alpha Positive Tumors Via Folate Receptor Selective Novel 5- And 6- Substituted Pyrrolo [2,3-D]pyrimidine Antifolates

Ovarian Cancer is the fifth leading cause of cancer-related death of women in the United States. Epithelial Ovarian Cancer (EOC) constitutes 85-90% of malignancies within the ovary, with an alarming majority of these cases diagnosed at advanced stage. While most patients are initially highly responsive to the current treatment standard, there is a very high probability that they will recur with a drug resistant fatal disease. Currently there is no validated comprehensive model of disease progression for ovarian cancer, although tremendous progress has been made in understanding the origin of this disease and a putative precursor lesion has been identified via molecular profiling. This progress has led to the identification of molecular signatures that not only distinguish high grade from low grade tumors, but it also highlights mutations that are unique to each histological type. Even though we are armed with this information and are well within the age of molecular targeting, the treatment of ovarian cancer has remained the same for over 40 years, when the use of platinum-based therapies and taxanes were introduced. Approximately 90 percent of EOC are folate receptor alpha (FRα) positive with the extent of receptor over-expression corresponding with stage and grade of disease. FRα based therapies are a subject of increasing interest warranted by a growing number malignancies, from various tissue types, showing FRα expression. Due to this increase of receptor expression in advance disease in EOC, designing FRα-targeted agents will enhance the therapeutic window in a population of patients where most treatments fail. The clinically approved antifolates, MTX and PMX, have greatly improved treatment outcomes in a number of malignancies, however they have been implicated in dose-limiting toxicities that are thought to arise from uptake mediated by the ubiquitously express protein, the reduced folate carrier (RFC). This encourages the development of novel antifolates that utilize other available folate transport mechanisms over RFC. In this study we explored the impact of the addition of various structural components on a pyrrolo[2,3-d]pyrimidine scaffold and demonstrated: (1) the significance of a 5 vs 6 position substitution on the scaffold and how it impacts the pharmacology of the compound; (2) that novel 6-substituted pyrrolo[2,3-d]pyrimidines show remarkable potencies that are mediated via FRα membrane transport; and (3) the impact of purine salvage on the efficacy of novel antifolates

The Histone Methyltransferase DOT1L: Discovery of Small-Molecule Inhibitors and its Role in Wnt Signaling.

Covalent post translational modifications of histone proteins are an important mechanism of epigenetic gene regulation that modulate chromatin structure. Methylation of histone lysine residues is one of several chemical marks that establish the “histone code” essential for proper temporal and spatial expression of gene programs required for cell fate determination in development. Dysregulation of histone methylation contributes to the development of numerous human diseases, particularly cancer. The sole histone H3 lysine 79 (H3K79) methyltransferase, DOT1L, is required for leukemogenic transformation in a subset of leukemias bearing translocations of the MLL gene. Human leukemias carrying MLL gene rearrangements aberrantly recruit DOT1L to leukemogenic genes leading to increased H3K79 methylation and their transcriptional activation. There are also reports that DOT1L plays a role in Wnt signaling, a pathway frequently dysregulated in colon cancer. Small molecule inhibitors of DOT1L are highly sought for the development of therapeutics in leukemia and as chemical tools to probe the role of DOT1L in other human diseases. We applied several approaches for the identification of DOT1L inhibitors, virtual screening, de novo design, and biochemical screening. Here we present the biochemical, biophysical, and cellular characterization of different classes of DOT1L inhibitors. Several S-adenosylmethionine (SAM) analogues have been identified as DOT1L inhibitors by virtual screening and we developed a novel pathway for synthesis of additional 5’ modified adenosine analogues. Additionally, we identified UMD-7, which inhibits H3K79 methylation by a unique mechanism of histone binding and phenocopies genetic loss of DOT1L. Employing a chemical biology approach the requirement for H3K79 methylation in Wnt signaling was investigated by inhibiting DOT1L with EPZ004777, a selective and potent SAM competitive inhibitor. Our findings indicate that H3K79 methylation is not essential for maintenance or activation of Wnt pathway target gene expression in colon cancer cell lines. Furthermore, H3K79 methylation is not elevated in human colon carcinoma samples in comparison with normal colon tissue. Therefore, our findings indicate that inhibition of DOT1L histone methyltransferase activity is likely not a viable therapeutic strategy in colon cancer.PhDMolecular and Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/110377/1/gsgibbon_1.pd