Date of Award

5-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular and Systems Pharmacology

Research Advisor

Richard. E. Lee, Ph.D.

Committee

Taosheng Chen, Ph.D. Glen. E. Palmer, Ph.D. Anang. A. Shelat, Ph.D. D. Parker Suttle, Ph.D.

Keywords

Assay Development, Drug Discovery, Fragment Screening, High Throughput Screening

Abstract

The discovery of antibacterial drugs has been among most significant achievements of mankind in saving millions of lives across the planet from infectious diseases. With rise in resistance to almost all existing chemotypes, the design of next generation novel antibiotics has become much more challenging and difficult. The early 21st century witnessed the advancement of multiple novel chemotypes during golden age of antibiotics however the pace of antibiotic drug discovery has slowed down tremendously, contributing to life threatening antimicrobial discovery void since 1980’s. Therefore the need to develop novel antibiotics with unique mechanism of action to leverage against multi drug resistance pathogens, is paramount. In this direction the Caseinolytic Protease P (ClpP) is an emerging drug discovery target with significant potential for treatment of recalcitrant biofilm forming infections from pathogens such as Methicillin-resistant Staphylococcus aureus (MRSA) This dissertation highlights the ongoing efforts to facilitate the discovery of novel non peptidic ClpP activator compounds and improvement of pharmacological profile of existing ClpP targeting Acyldepsipeptides (ADEPs) series antibiotics. The chapter one discusses the history and synopsis of conventional antibiotics drug discovery screening approaches, and transitions to modern era structure or fragment based screening approaches. The merits and challenges of such approaches of targeting a well conserved bacterial protease (ClpP) are discussed along with dissertation aims toward development of biophysical and biochemical screening approaches. Chapter two discusses optimization of thermal shift assay as primary screening assay for ClpP and its utility toward screening of fragment collections and buffer conditions. Chapter three discussed the development of a site specific Fluorescence Polarization based FP probe based on ADEP scaffold and its utility as a robust high throughput capable primary screening assay for screening of diverse collections ranging from bioactives to fragments. Chapter four discusses development of a label free Surface Plasmon Resonance (SPR) based assay geared toward screening of fragment as well as in house small and large (ADEP analogs) series compounds in addition to determining full kinetics for lead prioritization. Chapter five discusses the results of multiple screening campaigns utilizing combination of above assays to generate multiple hits with superior ligand efficiency and chemical tractability. Chapter six concludes with analysis of the best of compounds among individual series or from screening campaigns and highlights effectiveness of above screening assays toward hit exploration along with outlook on anticipated challenges and future directions.

ORCID

http://orcid.org/0000-0002-1455-5045

DOI

10.21007/etd.cghs.2017.0571

Comments

Embargo expires May 2021.

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