Discovery and validation of 2-styryl substituted benzoxazin-4-ones as a novel scaffold for rhomboid protease inhibitors

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Abstract

Rhomboids are intramembrane serine proteases with diverse physiological functions in organisms ranging from archaea to humans. Crystal structure analysis has provided a detailed understanding of the catalytic mechanism, and rhomboids have been implicated in various disease contexts. Unfortunately, the design of specific rhomboid inhibitors has lagged behind, and previously described small molecule inhibitors displayed insufficient potency and/or selectivity. Using a computer-aided approach, we focused on the discovery of novel scaffolds with reduced liabilities and the possibility for broad structural variations. Docking studies with the E. coli rhomboid GlpG indicated that 2-styryl substituted benzoxazinones might comprise novel rhomboid inhibitors. Protease in vitro assays confirmed activity of 2-styryl substituted benzoxazinones against GlpG but not against the soluble serine protease α-chymotrypsin. Furthermore, mass spectrometry analysis demonstrated covalent modification of the catalytic residue Ser201, corroborating the predicted mechanism of inhibition and the formation of an acyl enzyme intermediate. In conclusion, 2-styryl substituted benzoxazinones are a novel rhomboid inhibitor scaffold with ample opportunity for optimization.

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Results and discussion

Rhomboids are intramembrane serine proteases present in prokaryotic, archaeal and eukaryotic organisms.1 In 2001, the first rhomboid was discovered in Drosophila and shown to perform a critical proteolysis step in EGF-receptor signaling.2, 3 Since then, rhomboids have been implicated in a wide range of biological processes including bacterial quorum sensing4, mitochondrial dynamics and integrity5, 6, and protein quality control.7 In addition, rhomboids have been identified as putative drug

Acknowledgements

We thank Professors Matthew Freeman (University of Oxford) and Ya Ha (Yale School of Medicine) for bacterial expression constructs. P.G. was supported by a scholarship of the iGRASPseed graduate school of the Heinrich-Heine-University Duesseldorf. K.S. was a recipient of the Purkyne Fellowship of the Academy of Sciences of the Czech Republic and also acknowledges support from the Ministry of Education, Youth and Sports of the Czech Republic (projects no. LK11206 and LO1302), Marie Curie Career

Conflicts of interest

None.

References (49)

  • T. Teshima et al.

    A new class of heterocyclic serine protease inhibitors. Inhibition of human leukocyte elastase, porcine pancreatic elastase, cathepsin G, and bovine chymotrypsin A alpha with substituted benzoxazinones, quinazolines, and anthranilates

    J Biol Chem

    (1982)
  • A.L. Hopkins et al.

    Ligand efficiency: a useful metric for lead selection

    Drug Discovery Today

    (2004)
  • K.R. Vinothkumar et al.

    Structure of rhomboid protease in complex with beta-lactam inhibitors defines the S2' cavity

    Structure

    (2013)
  • K. Strisovsky et al.

    Sequence-specific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates

    Mol Cell

    (2009)
  • A. Ticha et al.

    Sensitive versatile fluorogenic transmembrane peptide substrates for rhomboid intramembrane proteases

    J Biol Chem

    (2017)
  • E.G. DelMar et al.

    A sensitive new substrate for chymotrypsin

    Anal Biochem

    (1979)
  • J. Yang et al.

    Benzoxazin-4-ones as novel, easily accessible inhibitors for rhomboid proteases

    Bioorg Med Chem Lett

    (2018)
  • M. Freeman

    The rhomboid-like superfamily: molecular mechanisms and biological roles

    Annu Rev Cell Dev Biol

    (2014)
  • K.M. Clemmer et al.

    Functional characterization of Escherichia coli GlpG and additional rhomboid proteins using an aarA mutant of Providencia stuartii

    J Bacteriol

    (2006)
  • G.A. McQuibban et al.

    Mitochondrial membrane remodelling regulated by a conserved rhomboid protease

    Nature

    (2003)
  • W. Song et al.

    Rhomboid domain containing 1 promotes colorectal cancer growth through activation of the EGFR signalling pathway

    Nat Commun

    (2015)
  • K. Walder et al.

    The mitochondrial rhomboid protease PSARL is a new candidate gene for type 2 diabetes

    Diabetologia

    (2005)
  • F. Parussini et al.

    Intramembrane proteolysis of Toxoplasma apical membrane antigen 1 facilitates host-cell invasion but is dispensable for replication

    Proc Natl Acad Sci U S A

    (2012)
  • J.M. Santos et al.

    Intramembrane cleavage of AMA1 triggers Toxoplasma to switch from an invasive to a replicative mode

    Science

    (2011)
  • Cited by (0)

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