The development and evaluation of small molecule-peptide conjugates as probes of prokaryotic ribosome exit tunnel-nascent peptide interaction

Abstract

As the knowledge base grows in regard to peptide synthesis and ribosomal actions, one facet of this is the extent and selectivity of interactions between the ribosomal exit tunnel and the nascent peptide. Small molecule probing techniques have provided insight from binding locations of ribosomal targeting therapeutics as well as potential downstream conformational changes to regions of dynamic flexibility with more solvent exposure aiding in the determination of complex higher-order structures. Crystallography provided a means to explore atomic-level interactions. This not only offered insight into potential questions and controversies posed by previous biochemical data, but also aided in the exploration and design of translation inhibiting antibiotics. However a means of more broadly probing the exit tunnel interactions without relying on mRNA and artificially stalling translation remained elusive. The methodology presented within this document provides an approach to introduce a broad range of customizable peptide probes to discrete regions of the exit tunnel in a manner independent on translation. Through the use of ribosomal large subunit targeting molecules (either clinically relevant or widely used mechanistic probes) as both delivery platforms as well as anchors, a means of presenting peptide sequences of various lengths and amino acid composition became possible. Covalent modification of various anchor types offered the possibility of interaction scanning windows within the peptidyl transferase center and well into the ribosomal exit tunnel. The successful presentation and evaluation of these small molecule-peptide conjugates provides an opportunity to identify regions of interaction within the exit tunnel while potentially displaying preference to amino acid types within these windows. Through the use of four major anchor types, two clearly rose to be viable while a third is continuing to show promise. Probe-dependent interactions within the ribosome have been shown both biochemically and crystallographically strongly indicating that the methodology is not only viable, but a very attractive route of exploration for a region once thought to have little, if any, interaction with nascent peptides. Biochemically, probes 14a-14h all show an orientation that projects the peptide moiety toward the peptidyl transferase center. This is indicated through small molecule probing of the Escherichia coli ribosome and probe-dependent protection of U1963 of the large subunit 23S rRNA. Crystalographic data collected of probe 14c within the Thermos thermophilus further confirmed this by providing atomic level detail of the peptide realigning over the macrolactone ring to project back toward the PTC. This marks the first use of a translation-independent peptide probe resulting in substantial conformational change of multiple residues lining the exit tunnel. Subsequent generations improved upon the design by seeking to project the peptide portion in a specific manner to expand the probing window. Probes 19-28 sought to exploit a strong pi stack interaction existing within the SecM peptide sequence. Within the SecM peptide sequence, tryptophan-155 engages in a pi stack with adenine-751, a 23S nucleotide that lines the wall of the prokaryotic ribosome exit tunnel further down the tunnel away from the PTC and macrolide binding site. Successful engagement of this interaction would serve as a directional handle to encourage threading of the probe peptide selectively down the tunnel. The collective observations taken from all probe classes as well as experimental opportunities therein spurred a new conjugate series. Seeking to improve cell membrane activity in a step to improve whole cell activity of the conjugate probes, the peptide moiety was redesigned to incorporate membrane-active peptidomimetics that resulted in large increases in whole-cell antibiotic activity.