Application of enzyme substrate analogues: from inhibitors with anti-mycobacterial properties to synthetic genetic polymers

Part 1: Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis (M. tuberculosis).It is regarded as the leading cause of death from a single infectious agent, even above HIV/AIDS. Although the global tuberculosis incidence has declined marginally over the past two decades, it still affects 10 million people each year and remains out of control in several parts of Africa and Asia. The main threat of TB is the increasing burden of multidrug-resistant (MDR) and extensively drug-resistant cases (XDR). More than 20% of the global TB cases are estimated to be resistant to at least one major tuberculosis drug. MDR-TB (resistance to the most powerful first-line drugs isoniazid and rifampicin) requires switching to second-line medications, which are less effective with more dangerous side effects and higher costs. XDR-TB (MDR-TB plus resistance to fluoroquinolones and second-line injectables) indicates failure of the standardized second-line regimens and thus resorting to insufficient and ineffective drugs to construct a proper treatment regimen. TB-endemic countries have seen selection of more drug resistant strains reaching to incurable or total drug resistance. Four countries: Italy, Iran, India and South Africa, have reported cases of totally drug resistant TB (defined as strains that do not respond to all first-line and second-line drugs). Drug-resistant TB is associated with high morbidity and mortality and is very expensive to treat. Acquired resistance i.e. resistance developing during treatment, represents the major cause of resistance cases. It is driven in part by poor compliance associated with the prolonged treatment course and the severe side-effects. A pipeline of drugs is now underway. However, many drugs are in pre-clinical or clinical development. Of the drugs that have entered into clinical use are bedaquiline and delamanid. However, these drugs are considered as last-resort antibiotics due to incomplete information about safety. Also, acquired resistance to both drugs has been documented. The general goal of this PhD project is to further support the drug discovery and development pipeline for identifying anti-mycobacterial drugs. The effective option is to develop a drug with a novel mode of action and showing selective targeting for the Mycobacteriumversus humans. Thymidylate synthase X (ThyX) represents such a target. This enzyme catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine-5'-monophosphate (dTMP) using methylene tetrahydrofolate (CH2THF) as the methylene source (CH2) and the nicotinamide adenine dinucleotide phosphate/flavin adenine dinucleotide (NADPH/FAD) system as the source of hydride (H-). As this step represents the only de novo source of dTMP for many microorganisms, it has been extensively studied for the development of anti-bacterial drugs. Humans and other eukaryotic organisms carry the genes for another class of thymidylate synthases (TS) known as thymidylate synthase A (ThyA). This enzyme uses CH2THF as the carbon and hydride source for production of dTMP from dUMP. The complex mechanistic differences between both TS such as type and number of reaction intermediates, substrate activation or methylene transfer step, should facilitate the design of selective inhibitors of ThyX. This is further supported by the absence of any sequential or structural homology between the two TS. Genomic studies of the Mycobacterium species revealed that it carries genes for both ThyA and ThyX, raising the question of the true biological role of both enzymes. However, several studies identified ThyX as the essential gene for M. tuberculosis. In this PhD thesis, we aim to identify selective M. tuberculosisThyX inhibitors by the development of a biochemical assay amenable for high-throughput screening (HTS) (Chapter II). The assay monitors the NADPH to NADP+ oxidation step with an absorbance-based readout. We first describe the assay development by adjustment of assay conditions (CH2THF, dUMP, NADPH and ThyX concentrations) followed by assay validation, using dUMP substrate analogues. IC50values comparable to the well-established radioactive assay (monitors product formation using [5-3H]-dUMP]) were obtained. We then focus on the factors essential for feasibility of HTS. These include selection of 50 µM folinic acid to represent the 100% inhibition control, stability of the assay (both signal and enzyme activity) up to 60 minutes at room temperature and assay compatibility with DMSO. Finally, we describe the results of the 40,000-compound screen conducted in collaboration with Cistim. The assay maintained a Z' value > 0.5, indicating high assay quality and robustness. We identified 14 compounds, of which 7 (from different chemical classes) were further tested for inhibition of M. tuberculosisThyA. The 7 compounds were selective to ThyX. The best inhibitor had a moderate IC50of 710 nM and was further evaluated by kinetic experiments to understand its mechanism of inhibition in relation of the current mechanistic findings. Part 2: Nucleic acids (DNA and RNA) are fundamental biopolymers with the capacity for information storage, transfer and expression of genetic information. Their single-stranded form can also behave as functional molecules binding to protein of interest or catalyzing biochemical reactions. This combined with ease of manipulation, through availability of different DNA/RNA-modifying enzymes, creates a high interest for the application of nucleic acids with new activities. Many nucleotide modifications (base, sugar or phosphate unit) can be incorporated into nucleic acids and provide enhancement of stability or improvement in activity. Bases can be modified by introducing functional chemical groups such as fluorescent dyes. Base substitutions in nucleic acids can be used to mask restriction sites allowing creation of stable genetic templates to introduce desired information or function (such inhibition of proteins) in vivo. These synthetic genetic polymers can be used to design proteins by coding for unnatural amino acids. In the second part of this PhD thesis, we evaluate nucleobase analogues for their base pairing, DNA polymerase recognition and selectivity. In Chapter V, we investigate 8-aza-guanine (GN1) and 8-aza 9-deaza-guanine (GN2) linked to the deoxyribose sugar through the N8position (N8-glycosylated purines). In the search for the best base pair combinations, they were tested for pairing with 1)8-substituted N9-glycosylated deoxyinosine [8-amino-deoxyinosine (Hx1), 1-methyl 8-amino-deoxyinosine (Hx2) and 8-oxo-deoxyinosine (Hx3)], or 2)the pyrimidine, 5-methyl deoxyisocytidine (d-isoCMe). We observed that small structural changes such as the N9 in GNXor the 1-methyl group in HxX can have a significant effect on incorporation degree and selectivity. The most efficiently recognized base pair was the GN2:isoCMefollowed by GN1or GN2pairing with Hx1or Hx2. In terms of selectivity, the best base pairs were GN1:isoCMefollowed by GN1:Hx2. The latter purine:purine combination demonstrates the formation of a Hoogsteen base pair at the polymerase level as the 1-methyl group of Hx2blocks the Watson-Crick side of pairing. Furthermore, we demonstrate that GN2can form a self-complementary base pair. The enzymatic biocompatibility of base pairs consisting of N8-linked purine with a Hoogsteen base pair recognition system should encourage the development of base pair moving away from the chemistry of the natural system. Modification of the sugar unit can be used to create nuclease-resistant biopolymers towards development of therapeutics nucleic acids. When combined with base analogues, they can support the formation of an orthogonal genetic system used for establishment of safe genetically-modified organisms. In Chapter VI, we study hexitol nucleic acids (HNA) in combination with the isoguanine (isoG):5-methyl-isocytosine (isoCMe) base pair to answer the question of the influence of sugar chemistry on the base pairing by measures of duplex stability and in vitroand in vivoenzymatic recognition. Thein vitroDNA polymerase incorporation experiments demonstrate that HNA-dependent HNA synthesis (of an isoG:isoCMebase pair) of only two nucleotides is possible. However, with a DNA backbone, incorporation of isoG against 3 d-isoCMenucleotides in the template (or isoCMeagainst isoG template nucleotides) was observed. In agreement with the in vitroand in vivoexperiments, isoG was mainly recognized by dT whereas isoCMewas mainly recognized by dA. The in vitroincorporation experiments demonstrated that HNA had a variable effect on selectivity (polymerase-dependent effect). However, in vivo, the recognition of hexitol nucleosides of isoG and isoCMeby the natural system was considerably lower than the deoxyribose nucleosides. This demonstrates further steps towards achieving an HNA-based orthogonal genetic information system.

Publication year:2019

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