Chemical Biology Approaches to Drug Design: Activated Cysteine-Like Protein Ligation, SARS-CoV-2 Drug Repurposing and Hypoxia Activated Prodrug Triggers
Abstract
Chemical biology creates a unique opportunity to reexamine and alter biological processes by means of chemical manipulations. To better understand biological systems, chemical functionalities can be used to generate selective probes that can detect, alter and measure biological reactions as well as other influencing factors and mechanisms. In order to incorporate desirable chemical functionalities into biological systems, peptide/protein synthesis is used as well as various ligation techniques. These ligation methods allow for incorporation of large fluorophores, tags, reactive species, as well as altering the terminal ends of proteins that are essential for their biological breakdown, resulting in longer half-lives. In chapter two, a new ligation method is explored that improves upon expressed protein ligation. This method employs a small molecule that selectively activates the c-terminal end of a protein, making a once unreactive carbonyl more electrophilic and labile towards substitution. This chapter shows the ease in which a small peptide or a large protein can be easily altered at its c-terminus without the need of an intein to undergo protein splicing. This method opens the field of drug discovery to altering proteins at their c-terminus and adding desirable functionalities to peptide therapeutics to treat and better understand biological systems.
Chapter three focuses on the repurposing of a drug to treat severe acute respiratory syndrome coronavirus 2, (SARs-CoV-2). Sometimes drug discovery is re-evaluating biologically active compounds in new conditions. The FDA is a huge hurdle to overcome when getting drugs approved for human use and in situations where time is of the essence, sometimes an effective alternative can be re-discovered. To repurpose a drug the structure and function of the virus has to be understood. Mapping a virus’ genome and establishing a crystal structure is essential to identifying possible drug candidates. Because of SARs-CoV-2’s sequence similarity and quick characterization of its crystal structure, many previous compounds are identifiable as possible candidates. These candidates are chosen by being previously approved by the FDA as well as their structure and fitting in the enzymes active site. our studies on main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is described. The turmoil created by SARS-CoV-2 prompted us to study main protease (Mpro) which is one of the key enzymes in life cycle of SARS-CoV-2. Our studies began by docking selected FDA/EMA (Food Drug Administration/European Medical Agency) drugs to active site of Mpro. Next, the promising drugs were purchased and their IC50 values were determined. Next, three most promising drugs were tested in live-virus microneutralization assay. Bepridil (an antiginal medicine) showed complete eradication of SARS-CoV-2 replication in low-micromolar concentrations showing promise for animal studies.
In chapter four, a new drug is designed for the treatment of tumor cells. This drug is designed to be a biologically activated prodrug that is triggered in response to hypoxic conditions. Tumors generate a microenvironment around themselves typified by low oxygen concentrations. These environments make tumor cells resistant to chemotherapies and radiation due to poor blood flow/vasculature and lack of reactive oxygen species, respectively. These Hypoxia activated prodrugs (HAPs) circumvent these obstacles by targeting the environments that have previously prevented therapies. The selectivity of these compounds comes from their nitro containing triggers. These Nitro compounds are selectively reduced enzymatically by 1 electron transfers with the ability to be back-oxidized to their parent compounds in the presence of oxygen. This back-oxidation assures that the therapeutic effector will only be released in low oxygen environments and not in normoxic, healthy cells. The purpose of this study was not only to develop an effective therapeutic but to also challenge the previous rationale in designing these types of prodrug triggers. Nitro aromatic reduction is well established in the sense of electrochemical reduction potential and mechanism, but these compounds have shown to behave much differently in biological environments and their rate of reduction shows to be influenced by many other factors unrelated to the trigger’s overall reduction potential. Aqueous, enzymatic and in vivo reduction must be considered when designing these compounds. We tested various trigger moieties in aqueous conditions, then selected the best candidates to synthesize our fluorophore analogs. Our fluorescent compounds were then subjected to chemical reduction, deuterium kinetics isotope reduction, and reduction by two different enzymes.
Citation
Kratch, Kaci Caitlin (2021). Chemical Biology Approaches to Drug Design: Activated Cysteine-Like Protein Ligation, SARS-CoV-2 Drug Repurposing and Hypoxia Activated Prodrug Triggers. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /196055.