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Defense: Empirical determination of the individual permeabilities of thousands of geometrically diverse cyclic hexa- and heptapeptides via multiplex PAMPA and MSMS sequencing

Speaker Name: 
Chad Townsend
Speaker Title: 
PhD Candidate
Speaker Organization: 
Biomolecular Engineering & Bioinformatics PhD
Start Time: 
Wednesday, December 9, 2020 - 1:00pm
End Time: 
Wednesday, December 9, 2020 - 2:00pm
Location: 
Zoom - https://ucsc.zoom.us/j/8568582235?pwd=aUZmbWIxNXlCTHFveHpnMVM5Q1Yydz09 - Passcode: 781881

Abstract: Much of modern pharmaceutical development has occurred within or nearby the chemical space delineated by Lipinski’s rules of five (< 500 molecular weight, < 5 hydrogen bond donors, < 5 octanol/water partition coefficient, < 10 hydrogen bond acceptors) due to the practical advantages in pharmacokinetic properties evinced by such small molecules, especially cell permeation. This leaves intracellular interactions which occur over a larger surface area (i.e. not involving native small molecules) “undruggable”. Cyclic peptides of up to 1200 molecular weight have demonstrated the ability to inhibit a wide variety of such undruggable intracellular interactions. However, engineering cell permeability into cyclic peptides remains a major barrier to their therapeutic utility.

Here I present my work using tandem mass spectrometry and the PAMPA artificial membrane permeability assay to empirically evaluate the individual permeabilities of thousands of geometrically diverse cyclic hexa- and heptapeptides. Analysis of the 823 permeable hexamers and 1330 permeable heptamers identified by matched pairs has yielded insights into the structural impact on permeability of the various backbone elements probed. In addition to general insights into the design of passively cell permeable cyclic peptides this set of known permeable backbone geometries can be used as a resource to design screening libraries biased toward permeable hits – an especially valuable strategy for DNA/mRNA encoded libraries which often generate impermeable leads and cannot select for cell permeability due to the encoding molecule attached to each compound. My exploration of passive permeability “motifs” found them sufficiently impactful to bias combinatorically generated screening libraries without restricting them to a single backbone geometry.

I also present a computational workflow used to match individual PAMPA permeabilities originating from mixtures of isobaric compounds to sequence identities through tandem mass spectrometry. The need to sequence thousands of peptides at speed motivated development of CycLS, a non-de novo cyclic peptide sequencing program using a database-matching approach. I increased PAMPA throughput to similar levels by removing data analysis as the bottleneck. Matching sequences to permeabilities by their retention times over identical LC conditions allowed me to assign individual permeabilities to 1063 cyclic hexamers and 2023 cyclic heptamers. These tools can easily be adapted for other mass spectrometry quantified assays and peptide classes.

Event Type: 
Advancement/Defense
Advisor: 
Scott Lokey
Graduate Program: 
Biomolecular Engineering & Bioinformatics PhD