Identification of an Optimized Clinical Development Candidate from Cilagicin, an Antibiotic That Evades Resistance by Dual Polyprenyl Phosphate Binding.

Cilagicin is a dual polyprenyl phosphate binding lipodepsipeptide antibiotic with strong activity against clinically relevant Gram-positive pathogens while evading antibiotic resistance. Cilagicin showed high serum binding that reduced its in vivo efficacy. Cilagicin-BP, which contains a biphenyl moiety in place of the N-terminal myristic acid found on cilagicin, showed reduced serum binding and increased in vivo efficacy but decreased potency against some pathogens. Here, we manipulated the acyl tail and the peptide core of cilagicin to identify an optimized collection of structural features that maintain potent antibiotic activity against a wide range of pathogens in the presence of serum. This led to the identification of the optimized antibiotic dodecacilagicin, which contains an N-terminal dodecanoic acid. Dodecacilagicin exhibits low MICs against clinically relevant pathogens in the presence of serum, retains polyprenyl phosphate binding, and evades resistance development even after long-term antibiotic exposure, making dodecacilagicin an appealing candidate for further therapeutic development.

Tapcin, an In Vivo Active Dual Topoisomerase I/II Inhibitor Discovered by Synthetic Bioinformatic Natural Product (Syn-BNP)-Coupled Metagenomics.

DNA topoisomerases are attractive targets for anticancer agents. Dual topoisomerase I/II inhibitors are particularly appealing due to their reduced rates of resistance. A number of therapeutically relevant topoisomerase inhibitors are bacterial natural products. Mining the untapped chemical diversity encoded by soil microbiomes presents an opportunity to identify additional natural topoisomerase inhibitors. Here we couple metagenome mining, bioinformatic structure prediction algorithms, and chemical synthesis to produce the dual topoisomerase inhibitor tapcin. Tapcin is a mixed p-aminobenzoic acid (PABA)-thiazole with a rare tri-thiazole substructure and picomolar antiproliferative activity. Tapcin reduced colorectal adenocarcinoma HT-29 cell proliferation and tumor volume in mouse hollow fiber and xenograft models, respectively. In both studies it showed similar activity to the clinically used topoisomerase I inhibitor irinotecan. The study suggests that the interrogation of soil microbiomes using synthetic bioinformatic natural product methods has the potential to be a rewarding strategy for identifying potent, biomedically relevant, antiproliferative agents.

DPP8/9 are not Required to Cleave Most Proline-Containing Peptides.

Small molecule inhibitors of the intracellular serine peptidases DPP8 and DPP9 (DPP8/9) activate the NLRP1 and CARD8 inflammasomes, but the key DPP8/9 substrates have not yet been identified. DPP8/9 cleave after proline to remove N-terminal dipeptides from peptides or proteins, and studies using pseudo-peptide reporter substrates have suggested that these enzymes may play key roles in the catabolism of many proline-containing peptides generated by the proteasome. Here, we evaluated the degradation of a wide array of actual peptides in cell lysates, and discovered that DPP8/9 are not in fact involved in the processing of the vast majority of proline-containing peptides. Overall, these results indicate that DPP8/9 have a much more limited substrate scope than previously thought, and likely specifically cleave some critically important, but as yet unknown, intracellular peptide or protein that regulates inflammasome activation.

Optimized M24B Aminopeptidase Inhibitors for CARD8 Inflammasome Activation.

Inflammasomes are innate immune signaling platforms that trigger pyroptotic cell death. NLRP1 and CARD8 are related human inflammasomes that detect similar danger signals, but NLRP1 has a higher activation threshold and triggers a more inflammatory form of pyroptosis. Both sense the accumulation of intracellular peptides with Xaa-Pro N-termini, but Xaa-Pro peptides on their own without a second danger signal only activate the CARD8 inflammasome. We recently reported that a dual inhibitor of the Xaa-Pro-cleaving M24B aminopeptidases PEPD and XPNPEP1 called CQ31 selectively activates the CARD8 inflammasome by inducing the build-up of Xaa-Pro peptides. Here, we performed structure-activity relationship studies on CQ31 to develop the optimized dual PEPD/XPNPEP1 inhibitor CQ80 that more effectively induces CARD8 inflammasome activation. We anticipate that CQ80 will become a valuable tool to study the basic biology and therapeutic potential of selective CARD8 inflammasome activation.

Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation.

NLRP1 and CARD8 are related pattern-recognition receptors (PRRs) that detect intracellular danger signals and form inflammasomes. Both undergo autoproteolysis, generating N-terminal (NT) and C-terminal (CT) fragments. The proteasome-mediated degradation of the NT releases the CT from autoinhibition, but the stimuli that trigger NT degradation have not been fully elucidated. Here, we show that several distinct agents that interfere with protein folding, including aminopeptidase inhibitors, chaperone inhibitors, and inducers of the unfolded protein response, accelerate NT degradation. However, these agents alone do not trigger inflammasome formation because the released CT fragments are physically sequestered by the serine dipeptidase DPP9. We show that DPP9-binding ligands must also be present to disrupt these complexes and allow the CT fragments to oligomerize into inflammasomes. Overall, these results indicate that NLRP1 and CARD8 detect a specific perturbation that induces both protein folding stress and DPP9 ligand accumulation.

M24B aminopeptidase inhibitors selectively activate the CARD8 inflammasome.

Inflammasomes are multiprotein complexes that sense intracellular danger signals and induce pyroptosis. CARD8 and NLRP1 are related inflammasomes that are repressed by the enzymatic activities and protein structures of the dipeptidyl peptidases 8 and 9 (DPP8/9). Potent DPP8/9 inhibitors such as Val-boroPro (VbP) activate both NLRP1 and CARD8, but chemical probes that selectively activate only one have not been identified. Here we report a small molecule called CQ31 that selectively activates CARD8. CQ31 inhibits the M24B aminopeptidases prolidase (PEPD) and Xaa-Pro aminopeptidase 1 (XPNPEP1), leading to the accumulation of proline-containing peptides that inhibit DPP8/9 and thereby activate CARD8. NLRP1 is distinct from CARD8 in that it directly contacts DPP8/9's active site; these proline-containing peptides, unlike VbP, do not disrupt this repressive interaction and thus do not activate NLRP1. We expect that CQ31 will now become a valuable tool to study CARD8 biology.

DPP9's Enzymatic Activity and Not Its Binding to CARD8 Inhibits Inflammasome Activation.

Inflammasomes are multiprotein complexes formed in response to pathogens. NLRP1 and CARD8 are related proteins that form inflammasomes, but the pathogen-associated signal(s) and the molecular mechanisms controlling their activation have not been established. Inhibitors of the serine dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both NLRP1 and CARD8. Interestingly, DPP9 binds directly to NLRP1 and CARD8, and this interaction may contribute to the inhibition of NLRP1. Here, we use activity-based probes, reconstituted inflammasome assays, and mass spectrometry-based proteomics to further investigate the DPP9-CARD8 interaction. We show that the DPP9-CARD8 interaction, unlike the DPP9-NLRP1 interaction, is not disrupted by DPP9 inhibitors or CARD8 mutations that block autoproteolysis. Moreover, wild-type, but not catalytically inactive mutant, DPP9 rescues CARD8-mediated cell death in DPP9 knockout cells. Together, this work reveals that DPP9's catalytic activity and not its binding to CARD8 restrains the CARD8 inflammasome and thus suggests the binding interaction likely serves some other biological purpose.

2-Arylidene Hydrazinecarbodithioates as Potent, Selective Inhibitors of Cystathionine γ-Lyase (CSE).

Hydrogen sulfide is produced from l-cysteine by the action of both cystathionine γ-lyase (CSE) and cystathionine ß-synthase (CBS) and increasingly has been found to play a profound regulatory role in a range of physiological processes. Mounting evidence suggests that upregulation of hydrogen sulfide biosynthesis occurs in several disease states, including rheumatoid arthritis, hypertension, ischemic injury, and sleep-disordered breathing. In addition to being critical tools in our understanding of hydrogen sulfide biology, inhibitors of CSE hold therapeutic potential for the treatment of diseases in which increased levels of this gasotransmitter play a role. We describe the discovery and development of a novel series of potent CSE inhibitors that show increased activity over the benchmark inhibitor and, importantly, display high selectivity for CSE versus CBS.

Oxamidation of Unsaturated O-Alkyl Hydroxamates: Synthesis of the Madangamine Diazatricylic (ABC Rings) Skeleton.

A novel approach to the diazatricyclic madangamine ABC ring system and the synthesis of an advanced, differentially protected intermediate for the synthesis of madangamine D is reported. Central to the success of this approach is the iodine(III)-mediated intramolecular oxamidation of an unsaturated O-methyl hydroxamate, a π-N+-type cyclization which proceeds in high yield and with complete regioselectivity to generate the 2-azabicyclo[3.3.1]nonane (morphan) system encompassing rings A and C.