Development of novel anti-infective drugs targeting microbial proteins

Abstract

The thesis involves targeting bacterial and fungal proteins to cure, or prevent life-threatening microbial infections. Streptococcus pneumoniae is a leading etiological agent in septicemia, pneumonia, bacteremia, meningitis, otitis media and conjunctivitis. b-lactam antibiotics and conjugate vaccines are the mainstay of treating or preventing pneumococcal infections. However, there has been a rapid emergence of multi-drug resistant strains in pneumococci in the past two decades, which are increasingly untreatable with the existing last line of antibiotics. Therefore, there is as urgent need to look for alternative strategies. Targeting pneumococcal virulence factors could potentially be one effective strategy to combat S. pneumoniae. One such factor is the pneumococcal surface antigen A (PsaA), which is a substrate-binding protein (SBP) that functions as a virulence factor, as well as a high-affinity Mn2+-binding protein, facilitating acquisition of Mn2+ and transporting it across the bacterial cell membrane. Mn2+ has an important role in protecting S. pneumoniae from reactive oxygen species, and hence against oxidative stress. Consequently, PsaA is essential for bacterial survival and an important virulence factor, which renders it a promising target for antibiotic drug development. Prior to designing novel inhibitors targeting PsaA, molecular dynamics simulations were performed to understand the conformational flexibility of PsaA and to investigate the molecular movements not captured by the static crystal structures of apo- (open state) and metal-bound (closed state) PsaA. These studies supported the observations of crystal structures and indicated that C-terminal domain of PsaA undergoes an extensive hinge-bending motion in the apo- (open state) PsaA compared to the metal-bound (closed state) PsaA, while, the N-terminal domain remains nearly static in both open and closed states of PsaA. Apart from the conformational flexibility studies of PsaA, novel fragment-like small molecule inhibitors targeting PsaA were also determined using virtual screening and conventional fragment-based drug design approaches. This gave two potent and ligand-efficient hits, 15h and 58f, which could potentially be pursued further for hit-to-lead development. Another major human pathogen is Cryptococcus neoformans, a yeast that can cause fungal meningitis and encephalitis in immune compromised humans, especially as secondary infection in AIDS patients. It is responsible for an estimated 700,000 deaths from cryptococcal meningitis each year. The purine metabolic pathway of C. neoformans has been extensively studied as potential therapeutic target, as it is responsible for the biosynthesis of guanosine triphosphate (GTP) and is thereby essential for cell survival and replication. Inosine monophosphate dehydrogenase (IMPDH) is a key enzyme in this pathway, involved in the first steps of the GTP biosynthesis and therefore, an ideal therapeutic drug target to control C. neoformans persistence. Key differences between fungal and human IMPDHrs have been identified by molecular dynamic simulations, which can assist the design and optimization of fungal-specific IMPDH inhibitors. A high-throughput screening has identified two hit compounds, MCC_005985 and MCC_006189, with activity against C. neoformans IMPDH. In silico structure-activity relationship (SAR) studies have been performed with the two hit structures giving valuable information for further optimization of the hit compounds and their optimization into lead compounds.