THE APPLICATION OF METABOLIC NETWORK ANALYSIS IN UNDERSTANDING AND TARGETING METABOLISM FOR DRUG DISCOVERY

Metabolic networks provide a vital framework for understanding the cellular metabolism in both physiological and pathophysiological states, which will ultimately facilitate network analysis-based drug discovery. In this thesis, we aim to employ a metabolic network analysis approach to study cancer metabolism (a pathophysiological state) and the metabolism of the bacterial pathogen, S. aureus (a physiological state), in order to understand, predict, and ultimately target cell metabolism for drug discovery. Cancer cells have distinct metabolism that highly depend on glycolysis instead of mitochondrial oxidative phosphorylation alone, even in the presence of oxygen, also called aerobic glycolysis or the Warburg effect, which may offer novel therapeutic opportunities. However, the origin of the Warburg effect is only partially understood. To understand the origin of cancer metabolism, our theoretical collaborator, Prof. Alexei Vazquez, developed a reduced flux balance model of human cell metabolism incorporating the macromolecular crowding (MC) constraint and the maximum glucose uptake constraint. The simulations successfully captured the main characteristics of cancer metabolism (aerobic glycolysis), indicating that MC constraint may be a potential origin of the Warburg effect. Notably, when we experimentally tested the model with mammalian cells from low to high growth rates as a proxy of MC alteration, we find that, consistent with the model, faster growing cells indeed have increased aerobic glycolysis. Moreover, the metabolic network analysis approach has also been shown to be capable of predicting the drug targets against pathogen metabolism when completely reconstructed metabolic networks are available. We deduced common antibiotic targets in Escherichia coli and Staphylococcus aureus by identifying shared tissue-specific or uniformly essential metabolic reactions in their metabolic networks. We then predicted through virtual screening dozens of potential inhibitors for several enzymes of these reactions and demonstrated experimentally that a subset of these inhibited both enzyme activities in vitro and bacterial cell viability. Our results indicate that the metabolic network analysis approach is able to facilitate the understanding of cellular metabolism by identifying potential constraints and predicting as well as ultimately targeting the metabolism of the organisms whose complete metabolic networks are available through the seamless integration of virtual screening with experimental validation

Computational Approaches To Anti-Toxin Therapies And Biomarker Identification

This work describes the fundamental study of two bacterial toxins with computational methods, the rational design of a potent inhibitor using molecular dynamics, as well as the development of two bioinformatic methods for mining genomic data. Clostridium difficile is an opportunistic bacillus which produces two large glucosylating toxins. These toxins, TcdA and TcdB cause severe intestinal damage. As Clostridium difficile harbors considerable antibiotic resistance, one treatment strategy is to prevent the tissue damage that the toxins cause. The catalytic glucosyltransferase domain of TcdA and TcdB was studied using molecular dynamics in the presence of both a protein-protein binding partner and several substrates. These experiments were combined with lead optimization techniques to create a potent irreversible inhibitor which protects 95% of cells in vitro. Dynamics studies on a TcdB cysteine protease domain were performed to an allosteric communication pathway. Comparative analysis of the static and dynamic properties of the TcdA and TcdB glucosyltransferase domains were carried out to determine the basis for the differential lethality of these toxins. Large scale biological data is readily available in the post-genomic era, but it can be difficult to effectively use that data. Two bioinformatics methods were developed to process whole-genome data. Software was developed to return all genes containing a motif in single genome. This provides a list of genes which may be within the same regulatory network or targeted by a specific DNA binding factor. A second bioinformatic method was created to link the data from genome-wide association studies (GWAS) to specific genes. GWAS studies are frequently subjected to statistical analysis, but mutations are rarely investigated structurally. HyDn-SNP-S allows a researcher to find mutations in a gene that correlate to a GWAS studied phenotype. Across human DNA polymerases, this resulted in strongly predictive haplotypes for breast and prostate cancer. Molecular dynamics applied to DNA Polymerase Lambda suggested a structural explanation for the decrease in polymerase fidelity with that mutant. When applied to Histone Deacetylases, mutations were found that alter substrate binding, and post-translational modification

Plasmodium yoelii acetyl-coa carboxylase : detection and characterisation of the recombinant biotinoyl domain.

Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.Human malaria, caused by four species of the intracellular protozoan parasite Plasmodium, is a major health and economic burden in the tropics where the disease is endemic. The biotindependent enzyme acetyl-CoA carboxylase catalyses the commitment step in de novo fatty acid biosynthesis in several organisms. Acetyl-CoA carboxylase is a target for anti-parasitic drug development due to its relevance in membrane biogenesis. This study describes the detection of acetyl-CoA carboxylase and the partial characterisation of the biotinoyl domain of the enzyme of the mouse malaria parasite, Plasmodium yoelii. Acetyl-CoA carboxylase mRNA was detected by RT-PCR performed on total RNA isolated from P. yoelii 17XL-infected mouse erythrocytes using primers designed from PY01695 ORF of the Plasmodb-published MALPY00458 gene of P. yoelii 17XNL. The RT-PCR was confirmed by sequencing and comparative analysis of the sequenced RT-PCR cDNA products. Northern blot analysis performed on total RNA using probes designed from a 1 kb region of the gene showed that the transcript was greater than the predicted 8.7 kb ORF. An immunogenic peptide corresponding to the P. yoelii theoretical acetyl-CoA carboxylase sequence was selected using epitope prediction and multiple sequence alignment algorithms. The immunogenic peptide was coupled to rabbit albumin carrier for immunisation in chickens and the affinity purified antibody titre was approximately 25 mg. The anti-peptide antibodies detected a 330 kD protein in P. yoelii lysate blot, which corresponds to the predicted size of the enzyme. The enzyme was also detected in situ by immunofluorescence microscopy using the anti-peptide antibodies. A 1 kb region of the P. yoelii acetyl-CoA carboxylase gene containing the biotinoyl domain was cloned and expressed in E. coli as 66 kD GST-tag and 45 kD His-tag protein. Both recombinant biotinoyl proteins were shown to contain bound biotin using peroxidaseconjugated avidin-biotin detection system. This suggested in vivo biotinylation of the recombinant P. yoelii biotinoyl protein, possibly by the E. coli biotin protein ligase. The Proscan™ and the NetPhos 2.0™ algorithms were used to predict protein kinase phosphorylation sites on the biotin carboxylase and the carboxyltransferase domains of the enzyme. The three-dimensional structure of the biotinoyl and the biotin carboxylase domains were predicted using the SWISS-MODEL™ homology modelling algorithm. Homology modelling revealed a similarity in the 3D conformation of the predicted P. yoelii biotinoyl domain and the E. coli biotinoyl protein with negligible root mean square deviation. The model also revealed the possibility of inhibiting P. yoelii and falciparum acetyl-CoA carboxylases with soraphen A based on the similarity in conformation with S. cerevisiae biotin carboxylase and the stereochemical properties of the residues predicted to interact with soraphen A. This study demonstrated that malaria parasite expresses acetyl-CoA carboxylase and, combined with data on other enzymes involved in fatty acid metabolism suggests that the parasite synthesizes fatty acids de novo. This enzyme could be a target for rational drug design

Enzymatic Ring Formation in Polyether Tetronate Antibiotic Biosynthesis

The formation of rings in carbon backbones is essential for the biological activity of many natural products. The polyether tetronate antibiotics tetronasin, tetronomycin, and tetromadurin (SF2487/A80577) are notable for their ring diversity, each possessing a tetronate, cyclohexane, tetrahydropyran, and at least one tetrahydrofuran ring. The antibiotic activity and complexity of these polyether tetronates has led to research on their biosynthesis from actinomycete bacteria. Despite this, the mechanism of stereospecific cyclohexane and tetrahydropyran formation has remained mysterious. Although no formal [4+2] cycloaddition (Diels-Alder reaction) is predicted in the biosynthesis of these compounds, the biosynthetic gene clusters of all three were found to contain a pair of genes that encode homologues of two different [4+2] cyclases previously identified in complex spirotetronate pathways. Specific gene deletions demonstrated that both classes of [4+2] cyclase homologue are essential for polyether tetronate biosynthesis. In the tetronasin producer, Streptomyces longisporoflavus, deletion of the [4+2] cyclase enzyme homologue gene tsn11 resulted in production of a new metabolite that was characterised as an open form of tetronasin lacking both cyclohexane and tetrahydropyran rings. Incubating this metabolite with purified Tsn11 resulted in the production of an unknown intermediate labelled T-22. The structure of T-22 was determined using NMR to contain an unexpected oxadecalin moiety but still lack the tetrahydropyran ring, implicating Tsn11 as catalysing an apparent inverse-electron-demand hetero-Diels-Alder reaction. Remarkably, incubating T-22 with purified Tsn15, the other [4+2] cyclase homologue, formed the tetrahydropyran ring and fragmented the oxadecalin moiety to a cyclohexane ring, producing tetronasin. To gain structural insight into the novel activity of Tsn15 it was successfully crystallised. The structure of Tsn15 was then solved at 1.8 Å using SAD phasing; and Brazilian collaborators solved a Tsn15-ligand structure at 1.7 Å. Tsn15 shares the same eight-stranded β-barrel fold as its [4+2] cyclase homologues. The two main mechanisms considered here for the Tsn15-catalysed conversion of T-22 into tetronasin were a general acid/ base or a pericyclic mechanism. Site-directed mutagenesis of the Tsn15 active site indicated that none of the acid/base amino acid side chains were essential for activity, favouring instead the pericyclic mechanism.Woolf Fisher Trust Cambridge Commonwealth European & International Trus

Washington University Senior Undergraduate Research Digest (WUURD), Spring 2018

From the Washington University Office of Undergraduate Research Digest (WUURD), Vol. 13, 05-01-2018. Published by the Office of Undergraduate Research. Joy Zalis Kiefer, Director of Undergraduate Research and Associate Dean in the College of Arts & Scienc

Molecular dynamics and docking simulations as a proof of high flexibility in E. coli FabH and its relevance for accurate inhibitor modeling

Bacterial β-ketoacyl-acyl carrier protein synthase III (FabH) has become an attractive target for the development of new antibacterial agents which can overcome the increased resistance of these pathogens to antibiotics in clinical use. Despite several efforts have been dedicated to find inhibitors for this enzyme, it is not a straightforward task, mainly due its high flexibility which makes difficult the structure-based design of FabH inhibitors. Here, we present for the first time a Molecular Dynamics (MD) study of the E. colil FabH enzyme to explore its conformational space. We compare the flexibility of this enzyme for the unliganded protein and an enzyme-inhibitor complex and find a correspondence between our modeling results and the experimental evidence previously reported for this enzyme. Furthermore, through a 100 ns MD simulation of the unliganded enzyme we extract useful information related to the concerted motions that take place along the principal components of displacement. We also establish a relation between the presence of water molecules in the oxyanion hole with the conformational stability of structural important loops. Representative conformations of the binding pocket along the whole trajectory of the unliganded protein are selected through cluster analysis and we find that they contain a conformational diversity which is not provided by the X-ray structures of ecFabH. As a proof of this last hypothesis, we use a set of 10 FabH inhibitors and show that they cannot be correctly modeled in any available X-ray structure, while by using our set of conformations extracted from the MD simulations, this task can be accomplish. Finally, we show the ability of short MD simulations for the refinement of the docking binding poses and for MM-PBSA calculations to predict stable protein-inhibitor complexes in this enzyme.status: publishe

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

From a multitarget antidiabetic glycosyl isoflavone towards new molecular entities against diabetes and Alzheimer’s disease : generation of lead series and target assessment

Type 2 diabetes and Alzheimer’s disease are closely related amyloid diseases globally affecting millions of people. However, the pathophysiological mechanisms connecting both diseases still require further investigation. In this work, we compile the existing evidence in the literature to allow the establishment of etiological links needed for drug discovery against diabetes-induced dementia. Furthermore, we provide an extensive revision of bioactive lead molecules that encourage further studies, particularly focusing on polyphenol sugar conjugates endowed with antidiabetic and neuroprotective activities. The state-of-the-art synthetic approaches for the generation of these types of molecules are also covered, thus setting the organic chemistry background for the original research work here developed. The use of carbohydrate-based molecules in drug research and development has multiple recognized benefits. In addition to enhanced solubility, bioavailability, and antidiabetic effects as previously reported, in this work we show, for the first time, that C-glucosylation is able to reverse the membrane dipole potential decrease induced by planar lipophilic polyphenols, elsewhere described as Pan-Assay Interference Compounds. This is a relevant discovery for drug development, particularly in the context of this thesis due to the polyphenolic nature of the compounds here presented. One of these compounds, 8-β-D-glucosylgenistein, was investigated in a diet-induced obese mouse of type 2 diabetes and found to exert a multitarget antidiabetic mechanism of action that goes beyond prior conjectures. Indeed, this antihyperglycemic glucosyl isoflavone reduces the renal threshold for glucose reabsorption, ameliorates diabetes-associated non-alcoholic fatty liver disease and hypercholesterolemia, normalizes insulin-degrading enzyme expression, and increases glucosestimulated insulin secretion. However, the detected inability of this polyphenol to permeate the blood brain barrier and to exert neuroprotective effects encouraged the pursuit of new scaffolds with therapeutic potential against diabetes-induced dementia. The role of amyloid β in the neurodegenerative processes occurring in Alzheimer’s disease and diabetes-induced dementia is, nowadays, unquestionable. Yet, targeted therapies aimed at inhibiting amyloid secretion or aggregation have, so far, failed clinical trials. In the past decade, the role of the cellular prion protein (PrPC) – a high-affinity ligand of amyloid β oligomers (Aβo) – has, in fact, been regarded as the limiting step in the cascade of events leading to neurodegeneration. Fyn kinase is one of the key players in this cascade, which culminates with the formation of neurofibrillary tangles composed by hyperphosphorylated tau, eventually leading to cell death. In this perspective, we have identified innovative N-methylpiperazinyl flavones and their glucosyl derivatives as Aβo-binders and non-toxic disruptors of Aβo-PrPC interactions. Furthermore, easily accessed glucosyl polyphenols with improved pharmacokinetic properties were also investigated and revealed to inhibit Aβ-induced Fyn activation with concomitant decrease in tau phosphorylation. Fyn kinase inhibition is considered a novel therapeutic strategy for Alzheimer’s disease, and these compounds are the first to accomplish this goal, with proven downstream effects. These molecules thus share the potential for further development against Alzheimer’s disease and diabetes-induced dementia. The work presented in this thesis elucidates the therapeutic relevance of natural and nature-inspired C-glucosyl polyphenols in the studied biological context, and highlights the usefulness of carbohydrate-based molecules for medicinal chemistry applications