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Marine cyanobacteria (blue-green algae) have been shown to possess an enormous capacity to produce structurally diverse natural products that exhibit a broad spectrum of potent biological activities, including cytotoxic, antifungal, antiparasitic, antiviral, and antibacterial activities. Using mass-spectrometry-guided fractionation together with molecular networking, cyanobacterial field collections from American Samoa and Palmyra Atoll yielded three new cyclic peptides, tutuilamides A-C. Their structures were established by spectroscopic techniques including 1D and 2D NMR, HR-MS, and chemical derivatization. Structure elucidation was facilitated by employing advanced NMR techniques including nonuniform sampling in combination with the 1,1-ADEQUATE experiment. These cyclic peptides are characterized by the presence of several unusual residues including 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid, together with a novel vinyl chloride-containing residue. Tutuilamides A-C show potent elastase inhibitory activity together with moderate potency in H-460 lung cancer cell cytotoxicity assays. The binding mode to elastase was analyzed by X-ray crystallography revealing a reversible binding mode similar to the natural product lyngbyastatin 7. The presence of an additional hydrogen bond with the amino acid backbone of the flexible side chain of tutuilamide A, compared to lyngbyastatin 7, facilitates its stabilization in the elastase binding pocket and possibly explains its enhanced inhibitory potency.

Proteases within the C1B hydrolase family are encoded by many organisms. We subjected a putative C1B-like cysteine protease secreted by the human gut commensal Parabacteroides distasonis to mass spectrometry-based substrate profiling to find preferred peptide substrates. The P. distasonis protease, which we termed Pd_dinase, has a sequential diaminopeptidase activity with strong specificity for N-terminal glycine residues. Using the substrate sequence information, we verified the importance of the P2 glycine residue with a panel of fluorogenic substrates and calculated kcat and KM for the dipeptide glycine-arginine-AMC. A potent and irreversible dipeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, glycine-arginine- AOMK, was then synthesized and demonstrated that the Pd_dinase active site requires a free N-terminal amine for potent and rapid inhibition. We next determined the homohexameric Pd_dinase structure in complex with glycine-arginine- AOMK and uncovered unexpected active site features that govern the strict substrate preferences and differentiate this protease from members of the C1B and broader papain-like C1 protease families. We finally showed that Pd_dinase hydrolyzes several human antimicrobial peptides and therefore posit that this P. distasonis enzyme may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.

Cysteine proteases are among the most abundant hydrolytic enzymes produced by bacteria, and this diverse family of proteins have significant biological roles in bacterial viability and environmental interactions. Members of the clostripain-like (C11) family of cysteine proteases from commensal gut bacterial strains have recently been shown to mediate immune responses by inducing neutrophil phagocytosis and activating bacterial pathogenic toxins. Development of substrates, inhibitors, and probes that target C11 proteases from enteric bacteria will help to establish the role of these proteins at the interface of the host and microbiome in health and disease. We employed a mass spectrometry-based substrate profiling method to identify an optimal peptide substrate of PmC11, a C11 protease secreted by the commensal bacterium Parabacteroides merdae. Using this substrate sequence information, we synthesized a panel of fluorogenic substrates to calculate kcat and KM and to evaluate the importance of the P2 amino acid for substrate turnover. A potent and irreversible tetrapeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, Ac-VLTK-AOMK, was then synthesized. We determined the crystal structure of PmC11 in complex with this inhibitor and uncovered key active-site interactions that govern PmC11 substrate recognition and specificity. This is the first C11 protease structure in complex with a substrate mimetic and is also the highest resolution crystal structure of a C11 protease to date at 1.12 Å resolution. Importantly, subjecting human epithelial cell lysates to PmC11 hydrolysis in combination with subtiligase-based N-terminal labeling and tandem mass spectrometry proteomics complemented the stringent substrate specificity observed in the in vitro substrate profiling experiment. The combination of chemical biological, biophysical, and biochemical techniques presented here to elucidate and characterize PmC11 substrate selectivity can be expanded to other proteases and the development of chemical tools to study these essential proteins in biologically relevant samples, such as the highly complex distal gut microbiome.