Identification and Co-complex Structure of a New S. pyogenes SpeB Small Molecule Inhibitor

Abstract

The secreted Streptococcus pyogenes cysteine protease SpeB is implicated in host immune system evasion and bacterial virulence. We present a small molecule inhibitor of SpeB 2477 identified from a high-throughput screen based on the hydrolysis of a fluorogenic peptide substrate Ac-AIK-AMC. 2477 inhibits other SpeB-related proteases but not human caspase-3, suggesting that the molecule targets proteases with the papain-like structural fold. A 1.59 Å X-ray crystal structure of 2477 bound to the SpeB active site reveals the mechanism of inhibition and the essential constituents of 2477 necessary for binding. An assessment against a panel of 2477 derivatives confirms our structural findings and shows that a carbamate and nitrile on 2477 are required for SpeB inhibition, as these moieties provide an extensive network of electrostatic and hydrogen-bonding interactions with SpeB active site residues. Surprisingly, despite 2477 having a reduced inhibitory potential against papain, the majority of 2477-related compounds inhibit papain to a much greater and broader extent than SpeB. These findings indicate that SpeB is more stringently selective than papain for this panel of small molecule inhibitors. On the basis of our structural and biochemical characterization, we propose modifications to 2477 for subsequent rounds of inhibitor design that will impart specificity to SpeB over other papain-like proteases, including alterations of the compound to exploit the differences in CA protease active site pocket sizes and electrostatics.

IL-1β is an innate immune sensor of microbial proteolysis

“…Confirming that SpeB is sufficient for the activation of IL-1β, caspase inhibitors YVAD-fmk, IETD-fmk, and DEVD-fmk did not alter IL-1 signaling in these cells, whereas cysteine-protease inhibitors that target SpeB, E64 (14), and N-(benzyloxycarbonyl)-2-amino-acetonitrile (15) did (Fig. 3C).…”

Leaderless secreted peptide signaling molecule alters global gene expression and increases virulence of a human bacterial pathogen

“…The protease degrades various host proteins to contribute to tissue damage and cleaves bacterial cell-surface proteins to promote disease dissemination (14-19, 21, 22). Consistent with its documented significance in infection, interference strategies targeting SpeB or its proteolytic activity confer protection against GAS infection (23)(24)(25)(26).…”

Sulfur(VI) Fluoride Exchange (SuFEx)-Enabled High-Throughput Medicinal Chemistry

“…Our proof-of-concept started with a modest inhibitor (cmpd 1, IC50 = 14 µM) of the cysteine protease SpeB, a virulence factor secreted from the bacterial pathogen Streptococcus pyogenes, previously identified in our HTS campaign (Figure 2a) [21][22] . Although peptidic SpeB inhibitors were reported, such as E64 [23][24] , potent small molecule inhibitors have not been developed against SpeB.…”

“…It should be noted that the reactions between isodifluor-containing molecules with the amines showed an improved yield when PBS (pH 7.4) was added to the solvent (Table S2). The reaction products were directly screened for SpeB inhibition with an established kinetic fluorogenic substrate assay [22][23] . Scatter plots of the screening results are shown in Figure 2c.…”

“…In preliminary SAR studies, introduction of an ( S )-benzyl moiety ( 2 , Figure a) improved the potency to 1.8 μM. The SpeB: 1 cocomplex X-ray structure and the initial SAR campaign (Table S1) suggested that additional surface sites on SpeB were accessible for compound optimization via extension of 2 from the meta positions of both benzyl substituents. An iminosulfur oxydifluoride SuFEx handle was therefore introduced at the meta positions of each benzyl group in 2 , generating 3 and 4 , respectively.…”