Improved Mass Spectrometry-Based Approaches to Screening and Structural Analysis in Drug Development

Abstract

Mass spectrometry (MS)-based analysis is highly versatile and provides powerful tools for addressing a plentitude of analytical challenges. For example, MS allows for high-throughput screening via direct analysis of microfluidic droplets, label-free quantification of enzyme-catalyzed reactions, and probing of protein structures and stability by coupling with hydrogen/deuterium exchange (HDX). This dissertation focuses on expanding the role of MS in drug development beginning with the development of a droplet microfluidic-MS workflow for whole cell screening of the catalytic efficiency of a saxitoxin biosynthetic enzyme for directed evolution towards non-native substrates in chemoenzymatic synthesis (Chapter 2). Specifically, variants of the polyketide-like synthase, saxitoxin biosynthetic module A (SxtA) from the cyanobacterium Cylinderspermopsis raciborskii T3, capable of transforming α-amino acids to α-aminoketones, are screened for activity with the non-native substrate tryptophan. Direct infusion of droplets containing whole cell lysate with overexpressed enzyme and substrates, increased screening throughput to 362 samples/h from 4 samples/h with a previous liquid chromatography (LC)-MS workflow. A structure-activity relationship (SAR) study of cathepsin B-mediated release of model amine-containing payloads from cleavable linkers employed in antibody drug-conjugate (ADC) design is discussed in Chapter 3. While there has been significant development on linker optimization for efficient payload release, no studies to date have investigated amine substrate (i.e., drug) dependent release rates as currently available payload options are extremely limited. Our results indicate that, in addition to aniline substrates, aryl amines and substituted ortho pyridyl methyl amines cleave at similar rates, thus eliminating the spacer requirement for efficient release and enabling access to more options for payload chemistries. In addition to the established tetrapeptide Gly-Gly-Phe-Gly, various capping groups were conjugated to the N-terminus of the dipeptide valine-citrulline (Val-Cit) and screened for efficient cathepsin-mediated release with results indicating a strong preference for capped peptides. Additional purified peptidases besides cathepsin B as well as rat liver lysosomes were included to assess substrate performance using a scoring system to determine relative rates. Our findings demonstrated that the substrates screened were enzyme agnostic. Because the structural integrity of monoclonal antibodies (mAbs) in ADCs can be compromised by payload conjugation, characterizing changes in protein conformation and dynamics as well as stability is crucial. HDX-MS determines protein conformational changes through changes in deuterium uptake rates in peptic peptides generated following time-dependent exposure to deuterated solvents. Localization of deuterium content to individual amino acids is desired for optimum structural resolution. To accomplish such localization, electron-based tandem MS activation techniques have been shown to allow fragmentation of deuterated peptides without deuterium scrambling. However, high charge state (greater than or equal to 2+) precursor ions are required, which is problematic with pepsin digestions. Supercharging reagents such as m-nitrobenzyl alcohol have been shown to increase the average peptic peptide charge state for smaller standard proteins, thus allowing for more efficient fragmentation. In Chapter 4, this supercharging approach is demonstrated for antibody pepsin digests where additional peptides were identified only upon supercharging. Overall, the improved MS-based approaches for characterization and screening presented in this dissertation have implications for enabling more efficient next generation drug design.