Depression is a major psychological disorder that affects a person's mental and physical abilities. The National Institute of Mental Health (NIMH) classified it as a serious medical illness. It causes huge economic, as well as financial impact on the people, and it is also becoming a major public health issue. Antidepressant drugs are prescribed to mitigate the suffering caused by this disorder. Different generations of antidepressants have been developed with dissimilar mechanisms of action. According to the Center for Disease Control, the usage of antidepressants has skyrocketed by 400 percent increase over 2005- 2008 survey period. This dramatic rise in usage indicates that these are the most prescribed drugs in the US. Even with the FDA mandated "black box" warning of increased suicidal thoughts upon use of selected antidepressants, these drugs are still being used at a higher rate. <br>All classes of antidepressants are plagued by side effects with mainly sexual dysfunction common among them. To avoid the adverse effects, an emphasis is to discover novel structural drug scaffolds that can be further developed as a new generation of antidepressants. The importance of this research is to discover structurally novel antidepressants by performing in silico virtual screening (VS) of chemical databases using the serotonin transporter (SERT). In the absence of a SERT crystal structure, a homology model was developed. The homology model was utilized to develop the first structure-based pharmacophore for the extracellular facing secondary ligand binding pocket. The pharmacophore captured the necessary drug-SERT interaction pattern for SERT inhibitory action. This pharmacophore was employed as one of the filters for VS of candidate ligands. The ten compounds identified were purchased and tested pharmacologically. Out of the ten hits, three structurally novel ligands were identified as lead compounds. Two of these compounds exhibited selectivity towards SERT; the remaining lead compound was selective towards the dopamine transporter and displayed cocaine inhibition. The two SERT selective compounds will provide new opportunities in the development of novel therapeutics to treat depression. <br>For dopamine transporter (DAT), the study was based on recently developed structurally diverse photo probes. In an effort to better understand the binding profile similarities among these different scaffolds, the photo probes were docked into DAT. The finger print analysis of the interaction pattern of docked poses was performed to identify the inhibitor-binding sites. <br>For mitogen activated protein kinase 5 (MEK5), given the lack of structural information, a homology model of MEK5 was developed to guide the rational design of inhibitors. Docking of known MEK5 inhibitors into the homology model was performed to understand the inhibitory interaction profile. Several series of analogues were designed utilizing the generated interaction profile. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry / PhD / Dissertation

Vitamin B1 is an indispensable co-factor for various enzymes inter alia in the Krebs cycle, pentose phosphate pathway, nucleotide and amino acid synthesis. Due to its importance in metabolism, proteins involved in the synthesis of vitamin B1 have been identified as potential drug targets. Thiamine monophosphate kinase (ThiL), catalyses the last reaction in the pathway, the ATP dependent phosphorylation of thiamine monophosphate (TMP) producing thiamine pyrophosphate (TPP) the active and co-factor form of vitamin B1. In this study, thiamine monophosphate kinase from Mycobacterium tuberculosis (MtbThiL, ~36 kDa) was produced as an N-terminally His6-tagged fusion protein, purified by affinity and size exclusion chromatography, and crystallised. Hexagonal MtbThiL crystals belonged to space group P6122. Molecular replacement revealed a symmetric homodimer with a single monomer occupying the asymmetric unit. Analysis of the structure showed that each subunit of MtbThiL has an ATP and TMP binding site and is structurally related to other ThiL enzymes. Ten lead compounds were identified from compound databases as potential ThiL inhibitors, and oxythiamine was chosen for further study. The binding affinities of oxythiamine and TMP to MtbThiL were determined by isothermal titration calorimetry and a pyruvate kinase-lactate dehydrogenase enzyme assay, which revealed that the binding affinity for oxythiamine by MtbThiL is lower than the substrate TMP. / Dissertation (MSc)--University of Pretoria, 2020. / Biochemistry / MSc / Unrestricted

Magister Pharmaceuticae - MPharm / Malaria disease poses substantial health risks to many nations, especially in Africa, where it primarily affects pregnant women, children, and immunocompromised patients. However, current antimalarial drugs have limitations such as low safety profile and particularly widespread treatment failure due to the increasing resistance of Plasmodium falciparum, the major causative organism to artemisinin-based therapy (ACT) and other chemotherapeutics. In the light of this, there is a pressing need for new antimalarial drugs with novel mechanisms of action and satisfactory pharmacokinetic properties, which has led to the current study. Furthermore, current antimalarial drugs target specific stages of the Plasmodium life cycle. For instance, chloroquine targets the erythrocytic stage while primaquine targets the liver stage. However, these therapies cannot achieve complete elimination of the parasite once the life cycle has been established in the body. Hence, the goal of this study is to combat resistance by finding novel compounds that can bind to a multiple-staged protein in Plasmodium falciparum. Based on this consideration, falstatin was chosen as the protein target for this study because it was observed to play a crucial role in the degradation of haemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. Hence, the protein, falstatin can be targeted to inhibit cell growth and cause plasmodial cell death in merozoites as well as schizonts of Plasmodium falciparum. Therefore, it is intended that compounds that bind to falstatin could serve as novel antimalarials that target multiple stages of the Plasmodium life cycle. Consequently, this study explored the structure-based virtual screening approach to identify compounds that could bind to the protein target, falstatin in Plasmodium falciparum. An extensive literature review identified falstatin as the multi-staged drug target for this study, while homology modelling was used to generate the three-dimensional structure of falstatin. Molecular docking was conducted to predict the binding energy of compiled antiplasmodial compounds to falstatin while druglikeness analysis was used to prioritize compounds according to their ADMET (absorption, distribution, metabolism, excretion and toxicity) properties. The top-ranked compound, based on a novel ligand scoring function, was then subjected to molecular dynamics (MD). Following this step, rescoring analysis was performed on the top 5 compounds using the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) scoring function to gain insight into their component binding energies. Thereafter, a pharmacophore hypothesis was developed based on the 5 top-ranking compounds in order to screen other compound libraries in the future. From the results, TCMDC 131646, TCMDC-124274, TCMDC-138266, TCMDC 123844 and TCMDC 131234 possessed good binding energies and satisfactory ADMET properties showing high ligand scores of 77.1, 75.4, 75.4, 75.4 and 73.1 respectively (on a total scale of 100). Also, the study revealed that the top-ranked compound, TCMDC 131646 had a binding energy of -6.15 KJ/mol, contained no toxicophore and conformed to Lipinski, Egan and Muegge rules of druglikeness. Findings from the MD simulation demonstrated that TCMDC 131646 strongly interacted with the protein, falstatin. Morealso, the study revealed that TCMDC 131646 is structurally diverse from chloroquine, artemisinin, artemether and lumefantrine, indicating that it may possess a distinct mechanism of action. The rescoring analysis of TCMDC-131646, TCMDC 124274, TCMDC-138266, TCMDC 123844 and TCMDC 131234 predicted negative binding energies ≤ -4.662 KJ/mol for the top compounds, further indicating that these compounds are likely to bind strongly with falstatin. Additionally, the developed pharmacophore hypothesis contained -H-N-C=O and N-H moieties which strongly suggested that the presence of electron-withdrawing groups could be vital for the inhibition of falstatin at the active site. Overall, TCMDC 131646 was predicted to be a drug-like and safe compound that could inhibit falstatin in Plasmodium falciparum. Chemical-disease co-occurrence analysis in literature revealed that this compound showed in-vitro antiplasmodial activity at an IC50 of 0.226μM and has also shown in vitro activity for neuralgia, hyperalgesia and arthritis. The research recommends TCMDC 131646 as a potential antimalarial hit compound that could yield novel analogues by hit expansion. However, confirmatory in-vitro and in-vivo studies are required to substantiate these predictions

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第20482号 / エネ博第351号 / 新制||エネ||70(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井 孝, 教授 木下 正弘, 教授 片平 正人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

<p>  </p> <p>Sulfonation is a widespread biological reaction catalyzed by a supergene family of enzymes called sulfotransferases (SULTs). SULTs utilize 3’-phosphoadenosine-5’-phospho-sulfate (PAPS) as the universal sulfonate donor to conjugate with a diverse range of endo- and xenobiotic substrates, including neurotransmitters, hormones, and drugs resulting in altering their biological activity. This reaction serves as a major detoxification pathway as conjugation with a sulfonate group renders substrates more hydrophilic and facilitates excretion. Therefore, this process is responsible for reducing the bioavailability of some drugs. In some cases, sulfo-conjugation causes the bio-activation of pro-mutagens and pro-carcinogens, leading to SULTs being risk factors in some cancers. Despite the biological relevance, understanding of this family of enzymes is still scarce. One SULT member that is the focus of the studies described herein is human sulfotransferase 2B1b (SULT2B1b), which had been identified as a potential drug target in prostate cancer. However, the inconsistency in reported kinetic data obtained using radiolabeled assays and the lack of robust assays have become significant limitations for SULT2B1b-targeted drug discovery studies. A label-free assay was developed to bridge this knowledge gap that directly quantifies SULT2B1b sulfonated products. This novel assay utilized high-throughput technology based on Desorption Electrospray Ionization Mass Spectrometry (DESI-MS). Results obtained from the DESI-MS-based assay were compared with those from a fluorometric, coupled-enzyme assay already developed in the Mesecar lab. Both methods provided consistent kinetic data for the reaction of SULT2B1b. Therefore, this novel assay is promising for the application of drug discovery efforts aiming at identifying SULT2B1b inhibitors. The other SULT member studied and described herein is human sulfotransferase 1A1 (SULT1A1), one of humans' most vital detoxifying and drug-metabolizing SULT isoforms that can also be a potential drug target in some cancers. The detailed kinetic mechanism of SULT1A1 was elucidated using steady-state kinetic, product inhibition, dead-end inhibition, and X-crystallographic studies. to gain insights into the role of this enzyme in detoxification, drug metabolism, and the development of inhibitors.</p>

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Bromodomains (BRDs) are protein modules which bind to acetylated lysines on histones and transcriptional regulating proteins. BRD-containing proteins are involved in a large variety of critical cellular processes and their misregulation, or mutation of the genes encoding for them, has been linked to pathogenesis in humans. The generation of chemical probes (potent, selective and cell permeable small molecules) in cellular experiments to investigate the biological role of the BRDs is thus desirable. A chemical probe for the CREB (cyclic-AMP response element binding protein) binding-protein (CBP) and E1A binding protein (p300) BRDs was developed, starting from a low molecular weight, weak and non-selective dimethylisoxazole benzimidazole compound. Parallel synthesis was used to optimise the initial hit into a weak, but selective CBP inhibitor. Further modification of the two N-1 and C-2 moieties of the benzimidazole scaffold, led to highly potent and selective CBP inhibitors. Structure-guided design was then applied to optimise the selectivity of the series for CBP over the first domain of bromodomain-containing protein 4 BRD4(1). A strategy to reduce the flexibility of the N-1 and C-2 ethylene linker groups through the incorporation of conformational constraints led to inhibitors with increased selectivity. The optimal compound was highly potent for the CBP and p300 BRDs (K_d 21 nM and 32 nM, respectively) and selective over BRD4(1) (40-fold and 27-fold, respectively). On-target cellular activity was observed in a fluorescence recovery after photobleaching (FRAP) assay (0.1 μM), a p53 reporter gene assay (IC₅₀ 1.5 μM) and a Förster resonance energy transfer (FRET) assay (5 μM). A weak indolizine bromodomain-containing protein 9 (BRD9) inhibitor was used as the starting point for the development of a BRD9/BRD7 chemical probe. Analogues were synthesised via [3+2] cycloadditions. An optimised compound was found to be highly potent (68 nM) and selective over BRD4(1) (34-fold). On-target cellular activity was observed in a FRAP assay (5 μM). Efforts were made to improve the cellular activity through the introduction of an ionisable centre to aid solubility. A selection of piperazine analogues were shown to be potent and selective, and these compounds warrant further investigation of their selectivity and cellular activity. Overall, the work has led to the first potent and selective inhibitors of the CBP/p300 and BRD9 BRDs. It also highlights the role of structural analysis in the development of inhibitors that modulate protein-protein interactions.