Saudi Cultural Missions Theses & Dissertations
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Item Restricted Assessing isothermal technology for detection of antimicrobial pathogens(Saudi Digital Library, 2024) Abdulrahman Khalid, Ayfan; David, WhileyThis thesis aims to provide solutions for antibiotic resistance by developing rapid point-of-care tests for key bacteria and associated resistances that have been flagged by the World Health Organization (WHO) and Centre for Disease prevention and Control (CDC). The studies initially focused on detection of New Delhi Metallo (NDM), and Verona Integrated Metallo (VIM) Beta Lactamase genes among carbapenemase-producing organisms Gram negative bacteria. The project then expanded to detect Neisseria gonorrhoeae (by targeting the gonococcal porA pseudogene) and associated resistances to ciprofloxacin (by the detection of a single nucleotide polymorphism [SNP] within the gonococcal gyrA gene) and ceftriaxone (by targeting the gonococcal penA 60.001 gene). The technology used for detection was iso-thermal recombinase polymerase amplification (RPA) and the detection was coupled with a lateral flow detection (LFD) system. Recognising the complexity for SNP detection by RPA (e.g., its ability to accommodate several mismatches in assay targets), this thesis explored several approaches to tackle this, including adding additional artificial mismatches in oligonucleotides, as well as using blocker primer and probe approaches. Assays performances compared well to traditional methods of detection (e.g., polymerase chain reaction; PCR), and time needed to perform/read results was less than thirty minutes compared to ninety minutes using PCR. Unlike PCR, RPA can be performed at a relatively low temperature, and assays can be successfully conducted by holding the reaction tubes in the palm of a hand. In addition to the wet-lab experiments, extensive in-silico sequence-based analyses were conducted to help further accelerate the development of point of care (POC) assays; these included improving our understanding of gonococcal gyrA diversity to inform target selection (using publicly available databases; National Centre for Biotechnology Information (NCBI), Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR), Pathogen Watch) as well as to identify new potential N. gonorrhoeae diagnostic gene targets (noting that, for example, the gonococcal porA pseudogene can lead to false-negative results for certain N. gonorrhoeae strains). Overall, this thesis provides further potential POC solutions for antibiotic resistance; specifically, the ability to detect gram negative bacteria-harbouring blaNDM and blaVIM genes, and N. gonorrhoeae gyrA and penA 60.001 resistance targets. These advances will provide valuable information to help clinicians and healthcare teams to tailor antibiotic therapy in a timely manner, with limited skills and training needed to perform and read results.9 0Item Restricted Optimization Of Enantiopure Tetrahydro-Β-Carbolines as Potent Antimalarials & Exploration of Salicylic Acid Analogs for Combating Multidrug-Resistant Neisseria Gonorrhoeae(2023-05-13) Almolhim, Hanan; Carlier, PaulThe emergence of drug resistance towards existing drugs is a constant challenge in the fight against many diseases including Malaria and gonorrhoeae. To evade resistance, new targets must be engaged, and to do that, new structural classes of anti-infective must be prepared and evaluated. During the course of my PhD journey, I had the opportunity to investigate and optimize the antimalarial candidate (Å})-2-3b, and salicylic acid (4-1a) as an anti-gonorrhea treatment. Malaria is a life-threatening mosquito-borne disease. In 2021, there were 247 million cases of malaria and the estimated number of malaria deaths stood at 619,000. Because of the rapid development of resistance to all current antimalarials, discovery of antimalarials with unexploited mechanisms of action is critical to reduce malaria mortality. In the Carlier group, our initial approach focused on discovery of inhibitors of the methylerythritol phosphate (MEP) pathway for isoprenoid precursor biosynthesis, since this pathway is essential for Plasmodium falciparum and absent in human. Application of the isopentenyl pyrophosphate (IPP) chemical rescue screen to the compounds of the Malaria Box, a collection of 400 antimalaria candidates with unknown mechanisms of action, identified tetrahydro-β-carboline 2-1 (MMV008138) as an inhibitor of the MEP pathway. Chapter 2 of this work discusses similarity searching of the Novartis portion of the hit set (5K compounds), from the original 20K compound hit set of the Malaria Box, and identifying tetrahydro-β-carboline GNF-Pf-5009, designated as (Å})-2-3b. Preparation of pure enantiomers, by resolution, demonstrated the pharmacological superiority of (R)-2-3b over (S)-2-3b, which was ound to have good asexual blood stage (ABS) inhibition potency against malarial parasites P. falciparum, and low general cytotoxicity. However, (R)-2-3b was found not orally efficacious in a P. berghei mouse model of malaria. We concluded that the lack of oral efficacy of (R)-2-3b was due to its poor drug-like qualities, in particular its high molecular weight and low solubility. Chapter 3 of this work explores modifications of (R)-2-3b ((R)-3-5Aa) that were expected to improve its properties. We show that the new compounds (R)-3-5Gm and (R)-3-5Gk not only are more potent in vitro than (R)-2-3b ((R)-3-5Aa), but also have molecular weights < 500 g/mol. Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhea. Due to the increased rates of infection as well as the prevalence of multidrug-resistant N. gonorrhoeae strains worldwide, the World Health Organization (WHO) and the U.S. Centers for Disease Control and Prevention (CDC) list N. gonorrhoeae at the highest possible threat level to public health. Dual therapy of azithromycin (AZM) and ceftriaxone has been the standard-of-care for treatment of gonococcal infections. However, due to increasing resistance to azithromycin (>33% in some regions) the CDC removed AZM from the treatment regimen for gonorrhea in 2020. Therefore, ceftriaxone remains the only recommended antibiotic for treatment of gonococcal infections. However, increasing resistance to this treatment option has been reported, consequently there is an urgent need to identify novel therapeutics against N. gonorrhoeae. Drug repurposing is a popular strategy that explores new therapeutic opportunities for approved drugs with available information on their pharmacokinetic data, dosages, and toxicity. Salicylic acid is a highly privileged chemical scaffold. Also, the use of salicylic acid to treat sexually transmitted diseases (including gonorrhea) was reported as early as the 19th century. Recently, Dr. Mohamed N. Seleem reported that salicylic acid (4-1a) exhibited modest activity against N. gonorrhoeae strains including the AZM-resistant strain (CDC-181). Chapter 4 of this work illustrates how the anti-gonococcal activity in this scaffold is easily lost by inopportune substitution. However, we found that substituted naphthyl analogs (4-3b,o,p) have superior activity to salicylic acid itself. In addition, the three analogs showed high selectivity, compared to AZM, against N. gonorrhoeae over the vaginal microbiota.15 0