SYNTHETIC STUDIES TOWARDS THE NATURAL PRODUCT LEOPOLIC ACID A

The need to develop new fungicides remains a major driving force as fungal plant pathogens continue to develop resistance against existing fungicides at great speed, and also because new disease situations continually arise. Natural products have been and still are large reservoirs of new biologically active substances for crop protection development, by serving as lead structures for the discovery of active molecules, often featuring novel and unique modes of action. Many pesticides have been developed from naturally occurring lead compounds. An outstanding example is the class of fungicides that has been generated from strobilurins, antifungal metabolites of myxobacteria. Aim of the PhD work was the study of new naturally derived antifungal compounds and the development of synthetic sequences which might, in principle, have value in the preparation of the natural products themselves as well as in synthesizing various analogues. Among a number of possible candidates we have selected a natural products, Leopolic acid A as synthetic target, as it has been reported to be endowed with antifungal activity. During the PhD period the synthesis of this natural product and structurally related analogues has been carried out. Antifungal and antimicrobial activities of the compounds have been evaluated, and preliminary SAR have emerged

Synthesis and evaluation of M.tb Glycosyltransferase (MshA) inhibitors

Includes bibliographical referencesThe emergence of multiple drug resistant (MDR) and extremely drug resistant (XDR) strains of Mycobacterium tuberculosis (M.tb) against the known anti-tuberculosis drug regimens has prompted the need to search for new anti-tubercular drugs. In this study we report the design and synthesis of a series of thiazolidinethione derivatives and substrate mimics, aimed at targeting the mycothiol biosynthetic pathway which is specific to mycobacteria. The strategy involved design of molecules that are expected to compete for the UDP-GlcNAc binding site of the glycosyltransferase (MshA) of M.tb. The bioactivity of the designed molecules against M.tb in cell free and whole cell assays serves as a basis for further inhibitor optimisation. Amongst the thiazolidinethione derivatives screened, compounds (Z)-5-(2,4-dichlorobenzylidene)-2-thioxothiazolidin-4-one (MJ3A) and 2-((Z)-5-(4-hydroxy-3-methoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (MJ7B) were found to be the most potent compounds with a MIC 50 of 10 μg/mL. In addition, substrate mimics were synthesized and screened for anti-tuberculosis activity. Substrate mimics displayed moderate activity, with exception of substrate mimic (4 -34) which displayed th e highest potency. Tunicamycin which is a known glycosyltransferase inhibitor displayed the highest potency against M.tb H37Rv whole cells by inhibiting cell growth with a MIC 50 of 5 μg/mL . Tunicamycin inhibits the transfer of GlcNAc-1-P from UDP-GlcNAc to polyprenyl monophosphates in a variety of organisms including Gram positive bacteria

Analytical tools for the physicochemical profiling of drug candidates to predict absorption/distribution

The measurement of physicochemical properties at an early phase of drug discovery and development is crucial to reduce attrition rates due to poor biopharmaceutical properties. Among these properties, ionization, lipophilicity, solubility and permeability are mandatory to predict the pharmacokinetic behavior of NCEs (new chemical entities). Due to the high number of NCEs, the analytical tools used to measure these properties are automated and progressively adapted to high-throughput technologies. The present review is dedicated to experimental methods applied in the early drug discovery process for the determination of solubility, ionization constants, lipophilicity and permeability of small molecules. The principles and experimental conditions of the different methods are described, and important enhancements in terms of throughput are highlighted. Figure Scheme of the Drug Research Proces

Bioprospecting of marine microorganisms for the discovery of antibacterial compounds - Isolation, structure elucidation and bioactivity assessment of marine microbial natural products

Infectious diseases have been a problem for humans since the beginning of human existence. The “golden age” of antibiotics started at the end of the 1920s, with the discovery of penicillin by Sir Alexander Fleming. This was followed by the discovery of several life-saving antibiotics. The number of new marketed antibiotics has declined, and most pharmaceutical companies are no longer working in antibiotic development. This in itself would be unproblematic, had it not been for the rapid ability of bacteria to become resistant towards previously debilitating agents. The need for new antibiotics is therefore eminent. Natural products have been important contributors for antibiotic drug discovery and development. Microorganisms have been a particularly proliferative source of antibiotics, providing us with among others the penicillins, aminoglycosides, tetracyclines and polymyxins. Most naturally derived pharmaceuticals, including antibiotics, originate from terrestrial organisms. This is mainly because the terrestrial environment historically has been easier to access compared to the marine environment below the intertidal zone. The marine environment is highly diverse, and there is still a huge biodiversity that is yet to be explored. In this project, Arctic and sub-Arctic marine bacteria and fungi were cultivated and studied for their production of natural products. The cultures were extracted and fractionated, and the fractions were tested for bioactivity, mainly focusing on antibacterial activity. Using bioactivity-guided isolation, compounds were isolated and structurally characterized. Finally, the bioactivity of the isolated compounds was broadly evaluated. In paper I, a known siderophore, serratiochelin A, was isolated from a co-culture of two bacteria, Serratia sp. and Shewanella sp. The compound was not detected in axenic cultures, indicating that cocultivation triggered production. The acid-catalyzed degradation of serratiochelin A into serratiochelin C was also observed. Serratiochelin A had weak activity against Staphylococcus aureus, melanoma cells and non-malignant lung fibroblasts. No activity was observed for the degradation product serratiochelin C, indicating that the oxazoline moiety in the original compound is essential for the bioactivity. In paper II, a marine bacterium Lacinutrix sp. was cultivated and studied for its ability to produce bioactive natural products. Through bioactivity-guided isolation, two new lyso-ornithine lipids were isolated, and their structures elucidated, showing that they only differed by the length of the hydrocarbon tail. Analysis by UHPLC-HR-MS indicate that the purified solutions are mixtures of isomers, but these were not possible to separate by preparative HPLC-MS. The compounds were evaluated for antibacterial activity and antiproliferative activity against human cells. Compound 1 displayed weak activity against Streptococcus agalactiae, while compound 2 had weak activity against melanoma cells. In paper III, a new dimeric naphthopyrone substituted with a sulphate group was isolated in high yields from cultures of an obligate marine fungus in the family Lulworthiaceae. The compound was tested against an extended panel of clinical bacterial isolates and showed potent antibacterial activity against several clinical methicillin-resistant Staphylococcus aureus isolates, with MICs down to 1.56 μg/mL. Acid-catalyzed degradation was also observed. The compound also displayed moderate activities against three human cell lines: melanoma, hepatocellular carcinoma, and non-malignant lung fibroblast. In paper IV, a new chlovalicin variant, chlovalicin B, was isolated from cultures of the obligate marine fungus Digitatispora marina. The fungus has previously been studied for its distribution in the marine environment but has not been extensively studied for its biosynthetic potential. The compound was isolated in low yields, and the structure was elucidated by NMR and HRMS experiments. The compound was assessed for a range of bioactivities and had weak antiproliferative activity against human melanoma cells

Identifying the modes and mechanisms of action of transmission-blocking antimalarials against Plasmodium falciparum

Phenotypic drug screening has transformed the antimalarial development pipeline, facilitating the discovery of compounds targeting unexplored parasite proteins at a drastically accelerated rate. Breaking the cycle of malaria transmission is crucial to eradication and centres around inhibition of the sexual stage gametocytes and gametes. Transmissible sexual stages represent a population bottleneck of the Plasmodium life-cycle which face little selective pressure and are hence a desirable target of drug intervention. Recently discovered in a phenotypic screen, the N-((4- hydroxychroman-4-yl)methyl)-sulphonamide (N-4HCS) compounds potently inhibit P. falciparum male gamete formation (microgametogenesis) in the mosquito. By combining study of the remarkable Plasmodium gametocyte cellular biology with medicinal chemistry and chemical proteomics, here we performed a detailed analysis of N-4HCS compound activity. Adhering to a traditional drug developmental pipeline, we first performed N- 4HCS hit-to-lead development and yielded compounds with activity in the nanomolar concentration range and favourable safety profiles. Furthermore, an N-4HCS photoaffinity probe was utilised in a proteome-wide photoaffinity labelling study, identifying the P. falciparum sexual stage-specific protein, Pfs16, as the compound target. Notably, the Cellular Thermal Shift Assay confirmed label-free specificity of N-4HCS compounds to Pfs16. Flow cytometry and phase-contrast, widefield- fluorescence and electron microscopy revealed compounds exclusively inhibited microgametogenesis, with a cellular phenotype consistent with published reports on the targeted gene disruption of Pfs16. We therefore present the 16kDa parasitophorous vacuole membrane protein, Pfs16, as a desirable antimalarial target and the N-4HCS compounds as potent candidates for future development. Additionally, a live-cell fluorescence microscopy workflow was devised to enable visualisation of microgametogenesis from the onset of activation through to exflagellation. Crucially, our imaging approach was applicable to drug discovery and is accessible to the malaria research community, thereby facilitating future study of transmission-blocking drug candidates. This thesis hence addressed the urgent requirement for novel antimalarial interventions, developing and aiding future discovery of transmission-blocking drugs.Open Acces

The application of the Multi-Component Reaction (MCR) strategy in the design and synthesis of new antiplasmodial and antimycobacterial agents

Includes bibliographical references.Malaria and tuberculosis are ancient diseases that continue to have a profound impact on mankind, 5 millennia after their first documentation. Malaria is endemic in more than 100 countries and about 50% of the world's population is at risk of infection. Sub-Saharan Africa accounts for nearly 91% of malaria-related deaths annually. Tuberculosis on the other hand infects about one third of the word's population and is the second major cause of death in adults worldwide, with about 1.8 million deaths reported annually. The major challenge to the control of these diseases has been the rapid emergence of multi-drug resistant strains to the currently administered drugs, as such, these exert an enormous pressure on health care systems, especially in resource-limited areas. Alleviation of this pressure requires the development of highly efficacious new chemical entities (NCEs) to curb or manage these pathogens. The main aim of this study was to design NCEs based on quinoline-, PA-824-, and tetrazole-scaffolds, which exhibit in vitro antiplasmodial and antimycobacterial activity

IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY

Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled “In silico Methods for Drug Design and Discovery,” which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD