title: Design and Synthesis of Novel Glycolipid Therapeutics and Drug Delivery Systems Targeting Mycobacterium tuberculosis
Creators: name: Farr, Dylan C
Publisher: name: Griffith University
Year: 2021
Subjects: subject: Tuberculosis
subject: TB
subject: Mycobacterium tuberculosis
subject: Novel antimicrobial agents
subject: Novel antitubercular agents
subject: Treatment regimens
subject: Carbohydrate-based chemistry

description: The development of novel medicinal agents is becoming increasingly important in solving several serious healthcare issues. Tuberculosis (TB) is among one of the world’s most serious health threats, especially in developing countries, and although treatment is available, current methods take 6 – 24 months. Prolonged treatment times often result in noncompliance and subsequently result in selection for drug resistance. As there are several multidrug resistant strains of Mycobacterium tuberculosis current treatment methods are proving inadequate, and it is becoming more and more essential to develop novel drugs to control the incidence and spread of TB; alongside other pathogenic bacterial species of clinical importance. Three major approaches towards the creation of novel antitubercular and antimicrobial agents and treatment regimens through the use of carbohydrate-based chemistry were explored within this project; (i) structure-based drug design and synthesis of novel competitive inhibitors targeting mycolic acid synthesis of M. tuberculosis (Chapter 1 &amp; 2); (ii) modification of aminoglycosides (AGs) through amphiphilic substitution, aimed to restore activity towards resistant bacteria or create agents with novel mechanisms of action (Chapter 3 &amp; 4); (iii) the development of novel liposomal drug carriers to selectively deliver therapeutics towards severe intracellular respiratory infections (Chapter 5 &amp; 6). Mycolic acids are essential for the growth and survival of M. tuberculosis, making mycolic acid inhibition a highly promising drug target for treating TB. Recently, a novel pathway of fatty acid synthesis has been identified as a promising drug target. FadD32, a fatty acyl-AMP ligase (FAAL) involved in the progression of TB represents a promising target for novel drug design. A family of sulfonylacetamide compounds have shown promising preliminary results in treating TB, however these failed within in vivo experiments due to their poor pharmacokinetic properties. Further development of these lead compounds with a rational structure-based drug design approach, led to the design and synthesis of compounds directly targeting the FadD32 biosynthetic pathway. This synthesised library of compounds were evaluated for their antitubercular inhibitory effect and several lead structures were identified. The most potent derivatives synthesised within this study displayed an MIC90 of 13 – 16 μg/mL towards M. tuberculosis (H37Rv) within a normoxic resazurin reduction microplate assay. Structural modification of the AG scaffold has historically remained a successful approach towards creating novel antibiotics. This approach led to the discovery of amikacin, a potent AG that is used for the treatment of TB today. Recently, a new class of antibiotics derived from the AG scaffold has produced several promising lead compounds that have revived antimicrobial activities against drug resistant bacterial species of clinical importance. This new class of antimicrobial agents are derived from conjugation of hydrophobic groups to AGs, creating semi-synthetic amphiphilic aminoglycosides (AAGs). AAGs have shown promising preliminary results against several drug resistant bacterial strains, including mycobacteria by evading common mechanisms of resistance and exerting novel mechanisms of action. A variety of novel and structurally diverse AAGs have been synthesised within this study providing lead structures with promising activity for the development of antimicrobial agents. A series of AAGs synthesised from the scaffolds of amikacin, kanamycin and neomycin were synthesised by conjugation of several hydrophobic groups to the AG scaffold. Derivatives were prepared by utilizing either the active functionality from the sulfonylacetamides conjugated via amide or triazole linkers; or by an alternative prodrug-based strategy utilizing long alkyl chains conjugated via ester linkages. Due to AGs broad-spectrum antibacterial activities, these compounds were evaluated for their antimicrobial inhibitory effect towards M. tuberculosis and S. aureus. Several structures were identified to display potent antitubercular activities. One of the kanamycin-based prodrugs was found to exert an increased antitubercular activity in comparison to the parent scaffold (MIC90 3 μg/mL), while amikacin derivatives maintained significant inhibitory effect (MIC90 3 – 6 μg/mL). The most active AAG utilizing the active functionality of the sulfonylacetamides was an amide-linked conjugate of amikacin (MIC90 22 μg/mL). Intracellular infections from bacterial pathogens such as M. tuberculosis are the causative agents of several serious diseases worldwide. Although the mononuclear phagocyte system primarily consists of macrophages, and constitutes the primary line of defence against infections, several bacterial pathogens have evolved the ability to survive and reproduce within intracellular host organelles. Intracellular infections are difficult to eradicate through traditional chemotherapeutic approaches due to the poor intracellular penetration of antibiotics. Moreover, the resulting low intracellular concentration of antibiotics is often ineffective against intracellular pathogens and furthermore promotes the development of drug resistance. Consequently, novel approaches to improve therapeutic effectiveness must be considered to advance treatment towards persistent intracellular bacterial infections. Nanoparticle-based drug delivery systems are one such approach; enabling improved antibiotic bioavailability and targeted site-specific drug delivery, offering the ability to overcome the less than desirable effects most antibiotics display towards intracellular pathogens. Liposomal drug delivery vectors are the most established and investigated nanocarriers, offering nontoxic biological delivery systems that can enhance the antimicrobial efficiency and therapeutic index of various chemotherapeutics. Liposomes provide unique delivery systems for antibiotics, such as AGs, as they can enhance efficiency whilst decreasing toxicity. Liposomal encapsulated AGs have demonstrated enhanced bactericidal activity towards intracellular infections in vitro and in vivo. Liposomal encapsulated amikacin has demonstrated its effectiveness in clinical trials and is currently FDA approved for the treatment of Mycobacterium avium complex. Within this study, amphiphilic-based prodrug derivatives of amikacin and mannosylated targeting ligands were utilized to formulate novel liposomal preparations for the treatment of pulmonary infections. Amphiphilic derivatization of amikacin allowed passive drug loading of the active therapeutic within the lipid bilayer, providing improved drug loading capacities compared to conventional approaches of loading amikacin within the aqueous core. Additionally, incorporation of the therapeutic AAG within the bilayer resulted in vesicles with improved membrane stability and more desirable physiochemical properties compared to conventional formulations. To test these liposomal formulations as a delivery system against intracellular pathogens in vitro, a murine macrophage cell line expressing mannose receptor (RAW264.7) was infected with S. aureus and bacterial inhibition was determined. All formulations displayed concentration-dependent killing efficiencies, while some were capable of complete intracellular bacterial inhibition at higher concentrations. The lipid derivatized amikacin-based prodrug delivery systems provide a novel liposomal delivery platform for intracellular infections, whilst offering the potential to overcome the shortcomings of current liposomal formulations such as poor stability and drug leakage. Once realized, this application could improve treatment towards serious intracellular pulmonary infections such as M. tuberculosis.