General and Modular Strategy for Designing Potent, Selective, and Pharmacologically Compliant Inhibitors of Rhomboid Proteases

Abstract

SummaryRhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude. Such peptidyl ketoamides show selectivity for rhomboids, leaving most human serine hydrolases unaffected. Crystal structures show that these compounds bind the active site of rhomboid covalently and in a substrate-like manner, and kinetic analysis reveals their reversible, slow-binding, non-competitive mechanism. Since ketoamides are clinically used pharmacophores, our findings uncover a straightforward modular way for the design of specific inhibitors of rhomboid proteases, which can be widely applicable in cell biology and drug discovery.

Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease

“…Both endogenous and ectopically expressed YqgP cleaved endogenous MgtE, yielding distinct cleavage products (Fig A). The cleavage was abrogated by a rhomboid‐specific peptidyl ketoamide inhibitor (Ticha et al , ) at low nanomolar levels, confirming that it was a rhomboid‐specific event (Fig B). Judging by the apparent molecular size of the N‐terminal cleavage fragment compared with the in vitro ‐translated reference fragments (Lemberg & Martoglio, ) of MgtE, we concluded that the cleavage by YqgP occurred within the extracytoplasmic loop between TMH1 and TMH2 of MgtE.…”

“…Bacillus subtilis genome encodes two rhomboid protease genes, ydcA and yqgP [also known as gluP (Mesak et al , )]. No proteolytic activity has been detected for YdcA so far (Urban et al , ; Lemberg et al , ), while YqgP is a commonly used model rhomboid protease cleaving a number of synthetic substrates (Lemberg et al , ; Urban & Wolfe, ; Ticha et al , ,b). YqgP has homologs in a number of Gram‐positive bacteria, including Bacilli, but also Staphylococci, Listeriae and others (http://www.ebi.ac.uk/interpro/protein/P54493/similar-proteins), and thus represents an attractive system to explore the cell biology and functions of bacterial rhomboid proteases.…”

“…Strain lacking YqgP (Δ yqgP , BTM78, Table EV2) was used as a control. Proteins were detected by immunoblotting with chemiluminescence detection. The cleavage is efficiently inhibited by 1 μM STS736, a specific peptidyl ketoamide rhomboid inhibitor (Ticha et al , ). To map the cleavage site region within endogenous MgtE (second lane from the left, from strain BTM501), MgtE‐derived reference fragments encoding first 300, 315 and 330 amino acids, as well as full‐length MgtE (black arrow), were in vitro ‐transcribed and in vitro‐ translated. Mobility of the N‐terminal cleavage product (red arrow) of MgtE on SDS–PAGE was compared to the mobilities of the translated reference fragments. Diagrammatic display of the mapping shows that YqgP cleaves MgtE in a periplasmic region between transmembrane helices 1 and 2 (red arrow). Data information: In all panels, endogenous full‐length MgtE is indicated by a black arrow, and N‐terminal cleavage product by red arrows.…”

“…Bottom panel: manganese toxicity is prevented by further adding 5 mM magnesium (MgSO 4 ) in otherwise identical conditions. Inhibition of YqgP by a rhomboid‐specific peptidyl ketoamide inhibitor (STS736, i.e. compound 9 from Ticha et al , ) abolishes the YqgP‐induced fitness of B. subtilis under manganese stress, in a dose‐dependent manner, both with endogenous YqgP (top panel) and overexpressed YqgP (bottom panel). Media and growth conditions were identical to those used in panel (B). Overexpression of heterologous MgtE inhibits growth of yqgP ‐deficient strain (BTM610, Table EV2) in rich LB medium supplemented with 75 μM MnSO 4 .…”

“… Schematic of YqgP domain architecture. Transmembrane helices are shown as grey boxes, and the position of the catalytic dyad of S288 and H339 within the transmembrane rhomboid core is displayed. Detection (left panel) and quantification (right panel) of steady‐state conversion of endogenous MgtE and model chimeric substrate derived from Providencia stuartii TatA (Stevenson et al , ; Ticha et al , ,b) (schemes in the middle) by ectopically expressed YqgP and its N‐ and C‐terminally truncated variants (strains BS184–187, Table EV2) in living Bacillus subtilis grown in rich LB medium. Black arrows indicate full‐length substrates, and red arrows indicate cleavage products. Similar analysis of the same strains but grown in minimal M9 medium (with 10 μM MgSO 4 ) supplemented with increasing concentrations of MnCl 2 .…”