Protecting groups (PGs) in peptide synthesis have inspired advanced design principles that incorporate orthogonality for selective C- and N-terminus and side-chain deprotections. The conventionally acid-stable 9-fluorenylmethoxycarbonyl (Fmoc) group is one of the most widely used N-protection groups in solid- and solution-phase synthesis. Despite the versatility of Fmoc, deprotection by the removal of the Fmoc group to unmask primary amines requires the use of a basic secondary amine nucleophile, but this stratagem poses challenges in sensitive molecules that bear reactive electrophilic groups. An expansion of PG versatility, a tunable orthogonality, in the late-stage synthesis of peptides would add flexibility to the synthetic design and implementation. Here, we report a novel Fmoc deprotection method using hydrogenolysis under mildly acidic conditions for the synthesis of Z-Arg-Lys-acyloxymethyl ketone (Z-R-K-AOMK). This new method is not only valuable for Fmoc deprotection in the synthesis of complex peptides that contain highly reactive electrophiles, or other similar sensitive functional groups, that are incompatible with traditional Fmoc deprotection conditions but also tolerant of N-Boc groups present in the substrate.

Gallinamide A, originally isolated with a modest antimalarial activity, was subsequently reisolated and characterized as a potent, selective, and irreversible inhibitor of the human cysteine protease cathepsin L. Molecular docking identified potential modifications to improve binding, which were synthesized as a suite of analogs. Resultingly, this current study produced the most potent gallinamide analog yet tested against cathepsin L (10, K_i = 0.0937 ± 0.01 nM and k_inact/K_i = 8 730 000). From a protein structure and substrate preference perspective, cruzain, an essential Trypanosoma cruzi cysteine protease, is highly homologous. Our investigations revealed that gallinamide and its analogs potently inhibit cruzain and are exquisitely toxic toward T. cruzi in the intracellular amastigote stage. The most active compound, 5, had an IC₅₀ = 5.1 ± 1.4 nM, but was relatively inactive to both the epimastigote (insect stage) and the host cell, and thus represents a new candidate for the treatment of Chagas disease.

Antibody-drug conjugates (ADCs) represent a new dimension of anticancer chemotherapeutics, with warheads to date generally involving either antitubulin or DNA-directed agents to achieve low-to sub-nanomolar potency. However, other potent cytotoxins working by different pharmacological mechanisms are under investigation, such as α,β-epoxyketone based proteasome inhibitors. These proteasome active agents are an emerging class of anticancer drug that possesses ultra-potent cytotoxicity to some cancer cell lines. The carmaphycins are representatives of this latter class that we isolated and characterized from a marine cyanobacterium, and these as well as several synthetic analogues exhibit this level of potency. In the current work, we investigated the use of these highly potent cytotoxic compounds as warheads in the design of novel ADCs. We designed and synthesized a library of carmaphycin B analogues that contain amine handles, enabling their attachment to an antibody linker. The basicity of these incorporated amine handles was shown to strongly affect their cytotoxic properties. Linear amines resulted in the greatest reduction in cytotoxicity whereas less basic aromatic amines retained potent activity as demonstrated by a 4-sulfonylaniline derivative. These investigations resulted in identifying the P2 residue in the carmaphycins as the most suitable site for linker attachment point, and hence, we synthesized a highly potent analogue of carmaphycin B that contained a 4-sulfonylaniline handle as an attachment point for the linker antibody.

The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB's low specificity for Tv20S compared to the human proteasome and demonstrate CP-17's higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.

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 are fundamental to successful parasitism, including that of the schistosome flatworm parasite, which causes the disease schistosomiasis in 200 million people worldwide. The proteasome is receiving attention as a potential drug target for treatment of a variety of infectious parasitic diseases, but it has been understudied in the schistosome. Adult Schistosoma mansoni were incubated with 1 μM concentrations of the proteasome inhibitors bortezomib, carfilzomib, and MG132. After 24 h, bortezomib and carfilzomib decreased worm motility by more than 85% and endogenous proteasome activity by >75%, and after 72 h, they increased caspase activity by >4.5-fold. The association between the engagement of the proteasome target and the phenotypic and biochemical effects recorded encouraged the chromatographic enrichment of the S. mansoni proteasome (Sm20S). Activity assays with fluorogenic proteasome substrates revealed that Sm20S contains caspase-type (β1), trypsin-type (β2), and chymotrypsin-type (β5) activities. Sm20S was screened with 11 peptide epoxyketone inhibitors derived from the marine natural product carmaphycin B. Analogue 17 was 27.4-fold less cytotoxic to HepG2 cells than carmaphycin B and showed equal potency for the β5 subunits of Sm20S, human constitutive proteasome, and human immunoproteasome. However, this analogue was 13.2-fold more potent at targeting Sm20S β2 than it was at targeting the equivalent subunits of the human enzymes. Furthermore, 1 μM 17 decreased both worm motility and endogenous Sm20S activity by more than 90% after 24 h. We provide direct evidence of the proteasome's importance to schistosome viability and identify a lead for which future studies will aim to improve the potency, selectivity, and safety.

Here, we present remarkable epoxyketone-based proteasome inhibitors with low nanomolar in vitro potency for blood-stage Plasmodium falciparum and low cytotoxicity for human cells. Our best compound has more than 2,000-fold greater selectivity for erythrocytic-stage P. falciparum over HepG2 and H460 cells, which is largely driven by the accommodation of the parasite proteasome for a D-amino acid in the P3 position and the preference for a difluorobenzyl group in the P1 position. We isolated the proteasome from P. falciparum cell extracts and determined that the best compound is 171-fold more potent at inhibiting the β5 subunit of P. falciparum proteasome when compared to the same subunit of the human constitutive proteasome. These compounds also significantly reduce parasitemia in a P. berghei mouse infection model and prolong survival of animals by an average of 6 days. The current epoxyketone inhibitors are ideal starting compounds for orally bioavailable anti-malarial drugs.

The tropical marine cyanobacterium Moorena bouillonii occupies a large geographic range across the Indian and Western Tropical Pacific Oceans and is a prolific producer of structurally unique and biologically active natural products. An ensemble of computational approaches, including the creation of the ORCA (Objective Relational Comparative Analysis) pipeline for flexible MS1 feature detection and multivariate analyses, were used to analyze various M. bouillonii samples. The observed chemogeographic patterns suggested the production of regionally specific natural products by M. bouillonii. Analyzing the drivers of these chemogeographic patterns allowed for the identification, targeted isolation, and structure elucidation of a regionally specific natural product, doscadenamide A (1). Analyses of MS2 fragmentation patterns further revealed this natural product to be part of an extensive family of herein annotated, proposed natural structural analogs (doscadenamides B-J, 2-10); the ensemble of structures reflect a combinatorial biosynthesis using nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) components. Compound 1 displayed synergistic in vitro cancer cell cytotoxicity when administered with lipopolysaccharide (LPS). These discoveries illustrate the utility in leveraging chemogeographic patterns for prioritizing natural product discovery efforts.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1 and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

Trichomoniasis is a sexually transmitted disease with hundreds of millions of annual cases worldwide. Approved treatment options are limited to two related nitro-heterocyclic compounds, yet resistance to these drugs is an increasing concern. New antimicrobials against the causative agent, Trichomonas vaginalis, are urgently needed. We show here that clinically approved anticancer drugs that inhibit the proteasome, a large protease complex with a critical role in degrading intracellular proteins in eukaryotes, have submicromolar activity against the parasite in vitro and on-target activity against the enriched T. vaginalis proteasome in cell-free assays. Proteomic analysis confirmed that the parasite has all seven α and seven β subunits of the eukaryotic proteasome although they have only modest sequence identities, ranging from 28 to 52%, relative to the respective human proteasome subunits. A screen of proteasome inhibitors derived from a marine natural product, carmaphycin, revealed one derivative, carmaphycin-17, with greater activity against T. vaginalis than the reference drug metronidazole, the ability to overcome metronidazole resistance, and reduced human cytotoxicity compared to that of the anticancer proteasome inhibitors. The increased selectivity of carmaphycin-17 for T. vaginalis was related to its >5-fold greater potency against the β1 and β5 catalytic subunits of the T. vaginalis proteasome than against the human proteasome subunits. In a murine model of vaginal trichomonad infection, proteasome inhibitors eliminated or significantly reduced parasite burden upon topical treatment without any apparent adverse effects. Together, these findings validate the proteasome of T. vaginalis as a therapeutic target for development of a novel class of trichomonacidal agents.