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24 Sep 2013

Monocyclic Beta-Lactams Are Selective, Mechanism-Based Inhibitors of Rhomboid Intramembrane Proteases

Monocyclic Beta-Lactams Are Selective, Mechanism-Based Inhibitors of Rhomboid Intramembrane Proteases

Chem. Biol. 2011, 6, 325-335

O. Pierrat et al.


Discovered a decade ago, Rhomboids are a class of enzymes belonging to the super family of intramembrane proteases which comprises metalloproteases such as site-2 protease (S2P) and aspartyl proteases such as signal peptide peptidase (SPP) and presenilin/g-secretase. They are serine proteases and like all intramembrane proteases, they cleave proteins within their transmembrane domains.

Since the early 2000s, the Freeman group at the MRC Laboratory of Molecular Biology1 has been studying rhomboids (for comprehensive reviews on these unusual enzymes, see ‘Rhomboids: 7 years of a new protease family’2 or ‘Proteolysis within the membrane: Rhomboids revealed’3) and recently, they commissioned Cambridge Research Biochemicals (CRB) to synthesise fluorescent substrate peptides for a screening campaign against a library of potential rhomboid inhibitors.

Peptide FRET pairs

Short peptides are often suitable candidates to study enzyme activity using Fluorescence Resonance Energy Transfer (FRET). Importantly, donor and acceptor have to be introduced in the peptide sequence so that they do not interfere with the recognition motif of the substrate. The Freeman group decided to work with synthetic peptides derived from Drosophila Gurken (PQRKVRMA*HIVFSFPV – cleavage site for AarA marked by an asterisk). A pegylated version of the substrate was synthesised to improve solubility during the assay and various FRET pairs were tested, such as AlexaFluor647/QSY21 and Chromis645A/QSY21.

The most efficient substrate for AarA (E.Coli rhomboid) was identified as Ac-[PEG]4-QR-K(QSY21)-VRMA*HIVFSFP-C(Chromis645A)-amide, a peptide designed following consultation with CRB’s technical experts. The screening campaign started with more than 50000 compounds and identified a new class of rhomboid inhibitors, a series of monocyclic beta-lactams.


Seminars in Cell & Developmental Biology 20 (2009) 231–239

Nature Reviews Molecular Cell Biology (2004) 188–197