Hit evaluation of an α-helical peptide: Ala-scan, truncation and sidechain-to-sidechain macrocyclization of an RNA polymerase Inhibitor.

RNA polymerase (RNAP) remains a relatively underexplored target with only rifampicin and fidaxomicin in clinical use. Hence, the concurrent rise in bacterial resistance rate urges the search for novel RNAP inhibitors with a novel mode of action. In this work, we investigated the impact of several systematic modifications including sidechain-to-sidechain macrocylization in the α-helical content and biological activity of a previously identified inhibitory sigma factor fragment. Ala-scan results, peptide truncation from both the N- and C-terminus and modifications inspired by other RNAP inhibitors revealed novel structure activity relationships but did not yield a superior sequence. Additionally, four insertion points for non-natural amino acids bearing side chains required for macrocylization were explored. Linear precursors showed improved stabilization of the α-helical content compared to the original sequence as demonstrated by circular dichroism (CD) spectroscopy. However, this increase in α-helicity did not translate into improved biological activity. Instead, complete abolishment of RNAP inhibitory activity occurred. We hypothesize three possible reasons for such a discrepancy and offer the basis for further optimization efforts for this peptidic RNAP inhibitor.

2-Aminothiazole Derivatives as Selective Allosteric Modulators of the Protein Kinase CK2. 1. Identification of an Allosteric Binding Site.

CK2 is a ubiquitous Ser/Thr protein kinase involved in the control of various signaling pathways and is known to be constitutively active. In the present study, we identified aryl 2-aminothiazoles as a novel class of CK2 inhibitors, which displayed a non-ATP-competitive mode of action and stabilized an inactive conformation of CK2 in solution. Enzyme kinetics studies, STD NMR, circular dichroism spectroscopy, and native mass spectrometry experiments demonstrated that the compounds bind in an allosteric pocket outside the ATP-binding site. Our data, combined with molecular docking studies, strongly suggested that this new binding site was located at the interface between the αC helix and the flexible glycine-rich loop. A first hit optimization led to compound 7, exhibiting an IC50 of 3.4 µM against purified CK2α in combination with a favorable selectivity profile. Thus, we identified a novel class of CK2 inhibitors targeting an allosteric pocket, offering great potential for further optimization into anticancer drugs.