L’acarien Varroa destructor est l’un des principaux responsables de l’effondrement des colonies d’abeilles domestique Apis mellifera. L’arsenal thérapeutique disponible pour lutter contre ce parasite ubiquiste apparait insuffisant à ce jour. Dans le cadre de cette thèse, la démarche de drug design, généralement utilisée en santé humaine, a été appliquée pour le développement de nouveaux médicaments vétérinaire à usage varroacide. Les travaux de cette thèse se sont focalisés sur deux cibles du système nerveux : l’acétylcholinestérase et les récepteurs à l’octopamine. Ces deux cibles ont déjà montré leur intérêt varroacide, notamment au travers des médicaments contenant du coumaphos ou de l’amitraze. Concernant l’acétylcholinestérase, un criblage fait avec le modèle 3D de l’enzyme, construit par homologie de séquences, a permis d’identifier deux composés de la chimiothèque du CERMN. Nous avons également exploré le potentiel varroacide d’acaricides de la famille des carbamates, démontrant l’intérêt du pirimicarbe comme varroacide. Concernant l’octopamine, l’étude de quatre dérivés de l’amitraze a montré l’intérêt de l’un d’entre eux. Un criblage par similarité de structure avec ce dérivé a mis en exergue une molécule issue de la chimiothèque du CERMN. Toutes les molécules pointées par ces travaux de thèse montrent de bons résultats lors de tests in vitro ou in vivo. Toutefois, afin de minimiser le risque pour l’abeille et maximiser l’efficacité anti-varroa, ces leads doivent être optimisés avant d’être ajouté à l’arsenal des médicaments varroacides. / The mite Varroa destructor is one of the main contributors to the collapse of honey bee colonies Apis mellifera. The therapeutic arsenal available against this ubiquitous parasite appears insufficient to date. In this thesis, the drug design approach, generally used in human health, was applied to the development of new varroacide veterinary drugs.The works of this thesis focused on two nervous system targets: acetylcholinesterase and octopamine receptors. These two targets have already shown their varroacide interest, especially through drugs containing coumaphos or amitraz. Regarding acetylcholinesterase, a screening made on the 3D model of the enzyme, built by sequence homology, allowed to identify two compounds from the CERMN compound library. We also explored the varroacide potential of carbamate acaricides, demonstrating the interest of pirimicarb as a varroacide. Regarding octopamine, the study of four derivatives of amitraz has shown the interest of one of them. Structural similarity screening with this derivative highlighted one hit from the CERMN compound library. All molecules pointed out by these thesis works show good results during in vitro or in vivo tests. However, to minimize the risk to honey bees and maximize their anti-varroa efficiency, these leads need to be optimized before being added to the arsenal of varroacide drugs.

Malaria is caused by the parasite Plasmodium falciparum and still plagues many parts of the world. To date, efforts to control the spread of the parasites have been largely ineffective. Due to development of resistance by the parasites to current therapeutics there is an urgent need for new classes of therapeutics. The vitamin B6 biosynthetic pathway consists of a PLP synthase which produces pyridoxal 5'-phosphate (PLP) within the parasite. The absence of this pathway in humans makes it attractive for selective targeting using small chemical molecules. The PLP synthase condenses D-ribose 5-phosphate (R5P) and DL-glyceraldehyde 3-phosphate (G3P) with ammonia to form PLP. Two proteins make up this PLP synthase – PfPdx1 and PfPdx2. Computational modelling of Pf Pdx1, and mapping of the R5P-binding site pharmacophore facilitated the identification of several ligands with predicted favourable binding interactions. Confirmatory testing of these on the purified Pf Pdx1 in vitro revealed D-erythrose 4-phosphate (E4P) and an analogue 4-phospho-D-erythronhydrazide (4PEHz) were capable of dose-dependently inhibiting the enzyme. The acyclic tetrose scaffold of E4P, with both aldehyde and phosphate group moieties, was thought to affect R5P imine bond formation in Pf Pdx1, possibly allowing the molecule to enter the R5P-binding site of Pf Pdx1. This hypothesis was supported by molecular docking simulations, and suggested that 4PEHz could similarly enter the R5P-binding site. 4PEHz was detrimental to the proliferation of cultured P. falciparum intraerythrocytic parasites and had an inhibitory concentration (IC50) of 10 µM. The selectivity of 4PEHz in targeting Pf Pdx1 was investigated using transgenic cell lines over-expressing Pf Pdx1 and Pf Pdx2, revealing that complementation of PLP biosynthesis rescued the parasites from the detrimental effects of 4PEHz. Functional transcriptomic and proteomic characterisation of 4PEHz-treated parasites revealed that the expression of Pf Pdx2 increased during 4PEHz treatment, moreover showed that other PLP-related processes were affected. These results supported that Pf Pdx1 is targeted by 4PEHz, and affected PLP biosynthesis de novo. Results from this study allude to alternative regulation of de novo PLP biosynthesis within the parasites by E4P. Moreover, contributions from this work showed that the de novo vitamin B6 pathway of P. falciparum is chemically targetable, and a potential strategy for the development of newer antimalarials. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Biochemistry / Unrestricted

1. SUMMARYMyeloperoxidase (MPO) which belongs to the peroxidase family, is found in mammalian neutrophils. This heme enzyme contributes to the production of (pseudo)halogenous acid such as HOCl which oxidizes proteins, cell membrane, DNA and RNA causing death for the pathogens. It has an antimicrobial effect due to HOCl secreting inside the phagosomes of the neutrophils, whereas it will be released outside neutrophils causing oxidative damages for the host tissues. Proteins, lipids, lipoproteins, DNA and RNA are potential targets of the MPO resulting in several chronic syndromes. Many researchers have discovered the harmful effects of MPO and its products demonstrating its role in many inflammatory chronic diseases such as: Cardiovascular diseases as in atherosclerosis. MPO contribution in atherosclerosis development has been demonstrated. Neurodegenerative diseases also was related to MPO: such as Alzheimer’s disease (AD), multiple sclerosis (MSc) and Parkinson’s disease The enzyme has been also pointed out in other diseases such as renal disease and cancer.For these reasons, MPO as a target of drug discovery has attracted the attention of many researchers. X-ray 3D structures were resolved for this enzyme, biological activity and mechanism of action were investigated in depth, and many medicinal chemists have investigated and screened for new MPO inhibitors. Indeed, this cumulative work including X-ray data, the role of MPO in different pathologies, MPO inhibitory mechanism of action, screening and various chemical entities that inhibit MPO, provided sufficient elements to start a new drug design and drug discovery process on MPO.The aim of the present study was to apply a rational drug design approach to the myeloperoxidase inhibition: from in silico to pharmacological activity. This includes:─ Conducting high throughput virtual screening in order to find new potential hits to inhibit MPO followed by mechanism of inhibition determination. ─ Selecting one hit and then implementing a whole pharmacomodulation process in order to increase the potency of the inhibition greater than the starting hit and to improve the selectivity.Firstly, a rational drug design process was launched to find new hits using high throughput virtual screening. The chosen database for the screening was ASINEX database published in ZINC.X-ray structure of human peroxidase complexed to cyanide and thiocyanate (PDB 1DNW) was selected to conduct High-Throughput Virtual Screening (HTVS). Three successive protocols with different levels of accuracy in the docking and scoring processes were used starting with HTVS, followed by Standard Precision (SP) and finally with Xtra Precision (XP). The quality of the docking process performed was validated by docking a set of 60 chosen molecules of varying chemical structure and known as MPO inhibitors. From the result of the HTVS conducted on 1,350,000 compounds, the 100 best compounds were selected. Among them, 81 molecules were available for purchase from ASINEX, those compounds were tested with a MPO inhibition assay. Thirty-two compounds (39 %) were active, but only 8 compounds were selected, featuring different chemical structures with IC50 values ranging between 0.46 ± 0.07 and 12 ± 3 μM. Among these molecules, two compounds were the best and considered as hits. One has purinedione structure which is similar with the structure of thioxanthine derivatives (F9, IC50=0.46±0.07μM). The second compound has a hexahydropyrimidine structure (A1, IC50 = 0.5 ± 0.1 μM) The most common interactions found among all 8 docked ligands are the ionic bond with Glu102 and a stacking (shifted or not) with pyrrole ring D of the prosthetic group. Hydrogen bonds with Glu102, Thr100, Gln91, Arg239, or the propionate groups of the heme are also found in several docked geometries of the complexes. Interestingly, interactions with Glu102 and pyrrole ring D of the heme were also seen with fluorotryptamine derivatives and also salicylhydroxamic acid (SHA).For measuring MPO-dependent LDL oxidation, the two best compounds were tested. Compounds A1 and F9 showed good inhibition on MPO-dependent LDL oxidation (62 ± 6, 4.5 ± 0.9, 11 ± 2% and 11 ± 2, 2.6 ± 0.8, 6 ± 4%, respectively).Consequently, in order to determine the mechanism of inhibition transient-state kinetics were further investigated of all the 8 selected compounds.Both new lead compounds (A1 and F9) act as electron donors of both Compound I and Compound II of MPO. The reaction with Compound I was significantly faster (k2 ≫ k3). As a consequence, the enzyme is trapped in the Compound II state. They reversibly inactivated the enzyme blocking the harmful halogenation activity of MPO by transferring it to the MPO peroxidase cycle. In the present study, 8 active and reversible MPO inhibitors were selected. They act as electron donors of the oxidoreductase and efficiently block the halogenation activity with reversible inactivation. Two of the selected compounds have a submicromolar activity and inhibit MPO-dependent LDL oxidation. The high-throughput virtual screening was proved to be a successful tool to find new leads of MPO inhibitors. Conducting HTVS on a large-scale database enabled selection of novel scaffolds of MPO inhibitors never explored before in less time and at less expenses.Finding 8 new different chemical scaffolds through the first step of this drug discovery process led us to choose a new hit, compound A1, which has a hexahydropyrimidine structure, compound F9 was not chosen despite being more active due to its similarity to compounds discovered by AstraZeneca. To conduct pharmacomodulation, a validation of the docking procedure was conducted by comparing the X-ray structures of MPO with 2-(3,5-bistrifluoromethylbenzylamino)-6-oxo-1H-pyrimidine-5- carbohydroxamic acid, HX1, and SHA in the X-ray structures of human MPO in complex with cyanide and thiocyanate (PDB code 1DNW) as well as in complex with HX1 (PDB code 4C1M). Compound A1 was docked into both target structures 1DNW and 4C1M. In both cases, A1 showed almost the same poses.Based on the binding modes of A1, different strategies were developed for the design of derivatives which were mainly focused on the substitution of the aromatic rings A and B, the 2 amino groups and the side chain bridges.Pharmacomodulation was carried out on the hit A1 with different strategies:─ Investigating the role of hydroxyl groups on both aromatic rings─ Shifting the position of the amino groups in the hexahydropyrimidine ring to obtain piperazine derivatives and introduction of fluorine ─ Eliminating of one ring and of an amino group in the hexahydropyrimidine ring leading to piperidine derivatives ─ Opening the hexahydropyrimidine ring while keeping amine function and changing the length of the bridge between this amino group and aromatic ring as well as the impact of substitutions on aromatic rings.─ Hybridization of fluorotryptamine derivatives (effective MPO inhibitors) with hit A1.Based on of the docking experiments, 37 designed compounds were synthesized. The assessment of inhibition of the chlorination activity of MPO was undertaken over the 37 compounds. The hit A1 IC50 = 500 nM. The best compounds inhibiting MPO exhibited the following characteristics:─ One amino group on the bridge between aromatic rings was sufficient for the establishment of binding to Glu102 ─ The presence of three methylene groups between the secondary amine and an aromatic ring improved the inhibition of chlorination and thus decreased the IC50 values. These results showed that the position of the hydroxyl group is important. The distance between the hydrogen bond acceptor (HBA) group of one aromatic ring and the amino group is very important. The docking experiments of bisarylpropylamine derivatives showed ionic and hydrogen bonding interactions between Glu102 and hydroxyl group on aromatic ring linked to the longer side chain.─ Hybridized compounds which carry a fluorotryptamine instead of the phenol ring obtained by hybridization of hit A1 and the potent MPO inhibitors fluorotryptamine derivatives. Actually, compound 38 (which had one aromatic ring and a propyl bridge attached to indole ring) had an IC50 = 54 nM which was 10 times more powerful than the starting hit.The 3 best compounds were tested to examine the transient kinetics. They act as electron donors of the oxidoreductase and efficiently shift MPO from the chlorination cycle to the peroxidase cycle. Due to the similarity of the best compound 38 to serotonin it was tested with the two other best compounds on serotonin transporter (SERT) to examine the selectivity between MPO and SERT.Compound 38 had higher selectivity over MPO but the best selective compound was 28 that contains two aromatic rings carrying one hydroxyl and one fluorine.Electron density maps were conducted to predict the site of oxidation. Results suggested it occurs preferentially at the benzene ring or the indole ring in the best compounds.Determination of redox potentials for the synthesized compounds were tested. Best compounds act as electron donors allowing a one-electron reduction of Compound I.In conclusion, the present study succeeded through rational drug design including structure-based drug design and HTVS to identify new chemical entities for MPO inhibition. Eight compounds were more active than the starting hit A1 with submicromolar inhibition potency. Hybridization and structure based design also gave improvement of selectivity of inhibitors against MPO such as compound 38. Bis-arylalkylamine derivatives are a new group of MPO inhibitors with higher selectivity which could be a new hit for future development. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Tuberculosis (TB) remains the leading cause of death due to a single infectious agent with more than 1.5 million people killed each year. In 2018, the World Health Organization (WHO) estimated that one third of the world’s population was infected with Mycobacterium tuberculosis (Mtb), the pathogen responsible for the disease.In 2000, EthR, a mycobacterial transcriptional repressor, was identified as a key modulator of ethionamide (ETH) bioactivation. ETH is one of the main second-line drugs used to treat drug-resistant strains and it is a prodrug that is activated in Mtb by the mono-oxygenase EthA and then inhibits InhA, an enzyme involved in the mycolic acid biosynthesis. In 2009, it was demonstrated that co-administration of ETH with the drug-like inhibitors of EthR was able to boost ETH activity by a factor three in a mouse-model of TB-infection, thus validating EthR protein as a target for a new therapeutic strategy. The first part of this thesis deals with the validation and deep characterization of the solved EthR-ligand structures based on all analysis of how each ligand bind to the EthR. In this section, based on the study of both co-crystal structures and the physicochemical properties of the ligands, we have rationalized the information currently available and understood the interaction of all EthR inhibitors in order to lead to more effective inhibitor design.More recently, another mycobaterial repressor, denoted EthR2, was identified as a putative target that appears to be functionally comparable to EthR (then the locus has been termed EthA2/EthR2, due to its similarity to the EthA/EthR locus). Furthermore, a spiroisoxazoline family of small-molecules, generically denoted as SMARt, has been identified as effective ligand of EthR2. However, according to the data present in the literature, this spiroisoxazoline family can also bind to the former EthR. In order to investigate this proposition, I have solved these small molecules in complex with EthR and compared their binding interactions to the EthR2 protein as well. The opportunity for the design small-molecules is capable of targeting both repressors, thereby opening the way to a dual-target approach.Finally, the third part of this thesis is devoted to the mycobacterial transcriptional factor MabR (Rv2242). Several studies identified this protein as a regulatory transcription factor of the fatty acid synthase II operon, which is mainly responsible for the mycolic acid biosynthesis in Mtb. I therefore purified to homogeneity and characterized the MabR protein as well as I determined the crystal structure of its C-terminal part. Finally, the functional role of MabR is largely discussed, and the way on how to interfere with its DNA binding ability is commented with respect to our results. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

Herein we report the structure-activity and structure-physicochemical property relationships of a series of class I selective ortho-aminoanilides targeting the “foot-pocket” in HDAC1&2. To balance the structural benefits and the physicochemical disadvantages of these substances, we started with a set of HDACi related to tacedinaline (CI-994) and evaluated their solubility, lipophilicity (log D7.4) and inhibition of selected HDAC isoforms. Subsequently, we selected the most promising “capless” HDACi and transferred its ZBG to our previously published scaffold featuring a peptoid-based cap group. The resulting hit compound 10c (LSH-A54) showed favorable physicochemical properties and is a potent, selective HDAC1/2 inhibitor. The following evaluation of its slow binding properties revealed that LSH-A54 binds tightly to HDAC1 in an induced-fit mechanism. The potent HDAC1/2 inhibitory properties were reflected by attenuated cell migration in a modified wound healing assay and reduced cell viability in a clonogenic survival assay in selected breast cancer cell lines.

<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>

Stroke is the third leading cause of death and disability in the United States. At present, intravenous administration of tissue plasminogen activator (t-PA) is the only thrombolytic therapy approved by the FDA for the treatment of acute ischemic stroke. There are no other effective treatments available so far. The discovery of new drugs and new treatments for stroke to reduce mortality and disability is an urgent medical research priority. In this study, the protective effects and mechanisms of two novel agents Gly14 humanin (HNG) and necrostatin-1 (Nec-1) were examined. HNG, a highly potent neuropeptide against amyloid toxicity, exhibited anti-apoptotic properties on cerebral ischemia injury. HNG reduced infarct volume after ischemia/reperfusion injury with pre-treatment or post-treatment (i.c.v. and i.p.) in a middle cerebral artery occlusion model in mice and decreased neurological deficits induced by ischemia. The protection of HNG was mediated by inhibiting ERK activation and activating PI3K/Akt pathway. Inhibition of the PI3K/Akt pathway blocked the protective effects of HNG. Nec-1 is a specific inhibitor of necroptosis, a newly identified cell death, and was reported to reduce infarct volume even when it was administered at 6 h post-ischemia in a mouse stroke model. Interestingly, this small molecule protected against glutamate-induced oxidative toxicity in a hippocampal HT-22 cell line. It inhibited the translocation of apoptosis-inducing factor from the mitochondria to the nucleus, increased the cellular glutathione level, and decreased free radical formation after glutamate treatment. More importantly, Nec-1 inhibited BNIP3-mediated caspase-independent cell death. Cerebral ischemia/reperfusion injury involves the activation of different pathways that lead to neuronal cell death. Given this multifactorial pathnogenesis, it is possible that a cocktail of neuroprotective agents would be superior to monotherapy. In this study, a cocktail of HNG and Nec-1 was examined in vitro and in vivo. HNG and Nec-1 exerted synergistic neuroprotection on oxygen-glucose deprivation-induced cell death and cerebral ischemia/reperfusion injury. This study provided a new therapeutic strategy for the treatment of stroke by the combination of anti-apoptosis and anti-necroptosis therapy.

The bacterial ribosome is a target for inhibition by numerous antibiotics. Neomycin and paromomycin are aminoglycoside antibiotics that specifically stimulate the misreading of mRNA by binding to the decoding site of 16S rRNA in the 30S ribosomal subunit. Recent work has shown that both antibiotics also inhibit 30S subunit assembly in Escherichia coli and Staphylococcus aureus cells. This work describes the characteristics of an assembly intermediate produced in E.coli cells grown with neomycin or paromomycin. Antibiotic treatment stimulated the accumulation of a 30S assembly precursor with a sedimentation coefficient of 21S. The particle was able to bind radio labeled antibiotics both in vivo and in vitro. Hybridization experiments showed that the 21S precursor particle contained 16S and 17S rRNA. Ten 30S ribosomal proteins were found in the precursor after inhibition by each drug in vivo. In addition, cell free reconstitution assays generated a 21S particle during incubation with either aminoglycoside. Precursor formation was inhibited with increasing drug concentration. This work examines features of a novel antibiotic target for aminoglycoside and will provide information that is needed for the design of more effective antimicrobial agents.

Machine learning (ML) has enjoyed great success in chemistry and drug design, from designing synthetic pathways to drug screening, to biomolecular property predictions, etc.. However, ML model's generalizability and robustness require high-quality training data, which is often difficult to obtain, especially when the training data is acquired from experimental measurements. While one can always discard all data associated with noisy and/or missing values, this often results in discarding invaluable data. This thesis presents and applies mathematical techniques to solve this problem, and applies them to problems in molecular medicinal chemistry. In chapter 1, we indicate that the missing-data problem can be expressed as a matrix completion problem, and we point out how frequently matrix completion problems arise in (bio)chemical problems. Next, we use matrix completion to impute the missing values in protein-NMR data, and use this as a stepping-stone for understanding protein allostery in Chapter 2. This chapter also used several other techniques from statistical data analysis and machine learning, including denoising (from robust principal component analysis), latent feature identification from singular-value decomposition, and residue clustering by a Gaussian mixture model. In chapter 3, Δ-learning was used to predict the free energies of hydration (Δ𝐺). The aim of this study is to correct estimated hydration energies from low-level quantum chemistry calculations using continuum solvation models without significant additional computation. Extensive feature engineering, with 8 different regression algorithms and with Gaussian process regression (38 different kernels) were used to construct the predictive models. The optimal model gives us MAE of 0.6249 kcal/mol and RMSE of 1.0164 kcal/mol. Chapter 4 provides an open-source computational tool Procrustes to find the maximum similarities between metrics. Some examples are also given to show how to use Procrustes for chemical and biological problems. Finally, in Chapters 5 and 6, a database for permeability of the blood-brain barrier (BBB) was curated, and combined with resampling strategies to form predictive models. The resulting models have promising performance and are released along with a computational tool B3clf for its evaluation. / Thesis / Doctor of Science (PhD)

No description available.