Nature-Inspired Gallinamides Are Potent Antischistosomal Agents: Inhibition of the Cathepsin B1 Protease Target and Binding Mode Analysis.

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.

Secondary Metabolites Profiling, Antimicrobial and Cytotoxic Properties of Commiphora gileadensis L. Leaves, Seeds, Callus, and Cell Suspension Extracts.

Commiphora gileadensis L. is an important endangered medicinal plant that belongs to the family Burseraceae. In this study, C. gileadensis callus culture was established successfully using mature leaves as explants cultured on Murashige and Skoog (MS) media supplemented with 24.50 µM of indole butyric acid (IBA) and 2.22 µM 6-Benzylaminopurine (BAP) (callus induction media). The obtained callus was maintained on MS medium supplemented with 16.11 µM naphthalene acetic acid (NAA) in combination with 6.66 µM BAP, which resulted in a substantial increase in callus fresh and dry weights. The cell suspension culture was established successfully using liquid callus induction media supplemented with 3.0 mg·L-1 proline. Thereafter, the chemical constituents of different C. gileadensis methanolic extracts (callus, cell suspension, leaves, and seeds) were profiled, and their cytotoxic and antimicrobial properties were investigated. The LC-MS GNPS analyses were applied for chemical profiling of the methanolic plant extracts, and several natural products were identified, including flavonols, flavanones, and flavonoids glycosides, with two unusual families that included puromycin, 10-hydroxycamptothecin, and justicidin B. The methanolic extracts have shown selective antimicrobial and cytotoxic properties against different microbes and cancer cell lines. For instance, leaf extract showed the highest zone of inhibition for Staphylococcus aureus, while cell suspension culture was effective against Staphylococcus epidermidis and Staphylococcus aureus. All extracts showed selective activity against A549 cell lines for the cytotoxicity assay, while the leaf extract had a broad cytotoxic effect against all tested cell lines. This study revealed that C. gileadensis callus and cell suspension cultures can be employed to increase the in vitro formation of biologically active compounds that may have cytotoxicity and antibacterial action against different cancer cell lines and bacterial species. Further studies are required to isolate and identify such constituents that corroborate the observed activities.

Discovery of Novel Epoxyketone Peptides as Lipase Inhibitors.

Obesity is the most common nutritional disorder in the developed world and is associated with important comorbidities. Pancreatic lipase (PL) inhibitors play a key role in the metabolism of human fat. A series of novel epoxyketones peptide derivatives were investigated for their pancreatic lipase inhibitory activity. The epoxyketone moiety is a well-known reactive electrophile group that has been used as part of proteasome inhibitors in cancer therapy, and it is widely believed that these are very selective for targeting the proteasome active site. Here we investigated various peptide derivatives with an epoxide warhead for their anti-lipase activity. The assessment of these novel epoxyketones was performed by an in-house method that we developed for rapid screening and identification of lipase inhibitors using GC-FID. Herein, we present a novel anti-lipase pharmacophore based on epoxyketone peptide derivatives that showed potent anti-lipase activity. Many of these derivatives had comparable or more potent activity than the clinically used lipase inhibitors such as orlistat. In addition, the lipase appears to be inhibited by a wide range of epoxyketone analogues regardless of the configuration of the epoxide in the epoxyketone moiety. The presented data in this study shows the first example of the use of epoxyketone peptides as novel lipase inhibitors.

Portobelamides A and B and Caciqueamide, Cytotoxic Peptidic Natural Products from a Caldora sp. Marine Cyanobacterium.

Three new compounds, portobelamides A and B (1 and 2), 3-amino-2-methyl-7-octynoic acid (AMOYA) and hydroxyisovaleric acid (Hiva) containing cyclic depsipeptides, and one long chain lipopeptide caciqueamide (3), were isolated from a field-collection of a Caldora sp. marine cyanobacterium obtained from Panama as part of the Panama International Cooperative Biodiversity Group Program. Their planar structures were elucidated through analysis of 2D NMR and MS data, especially high resolution (HR) MS2/MS3 fragmentation methods. The absolute configurations of compounds 1 and 2 were deduced by traditional hydrolysis, derivative formation, and chromatographic analyses compared with standards. Portobelamide A (1) showed good cytotoxicity against H-460 human lung cancer cells (33% survival at 0.9 µM).

Applying a Chemogeographic Strategy for Natural Product Discovery from the Marine Cyanobacterium Moorena bouillonii.

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.

Tutuilamides A-C: Vinyl-Chloride-Containing Cyclodepsipeptides from Marine Cyanobacteria with Potent Elastase Inhibitory Properties.

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.

Design and Synthesis of New Sulfonamides-Based Flt3 Inhibitors.

BACKGROUND: Flt3 is an oncogenic kinase involved in different leukemias. It is most prominently associated with acute myeloid leukemia (AML). Flt3-specific inhibitors have shown promising results in interfering with AML. METHODS: The crystallographic structures of two inhibitors complexed within Flt3, namely, quizartinib and F6M, were used to guide the synthesis of new sulfonamide-based Flt3 inhibitors. RESULTS: One of the prepared compounds showed low micromolar anti-Flt3 bioactivity, and interestingly, low micromolar bioactivity against the related oncogenic kinase VEGFR2. CONCLUSION: Sulfonamides were successfully used as privileged scaffolds for the synthesis of novel Flt3 inhibitors of micromolar potencies.

Design of Gallinamide A Analogs as Potent Inhibitors of the Cysteine Proteases Human Cathepsin L and Trypanosoma cruzi Cruzain.

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, Ki = 0.0937 ± 0.01 nM and kinact/Ki = 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.

20S Proteasome as a Drug Target in Trichomonas vaginalis.

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 ß 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 ß1 and ß5 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.

The Proteasome as a Drug Target in the Metazoan Pathogen, Schistosoma mansoni.

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 (ß1), trypsin-type (ß2), and chymotrypsin-type (ß5) 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 ß5 subunits of Sm20S, human constitutive proteasome, and human immunoproteasome. However, this analogue was 13.2-fold more potent at targeting Sm20S ß2 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.

Exploration of the carmaphycins as payloads in antibody drug conjugate anticancer agents.

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,ß-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.

Discovery and Synthesis of Caracolamide A, an Ion Channel Modulating Dichlorovinylidene Containing Phenethylamide from a Panamanian Marine Cyanobacterium cf. Symploca Species.

A recent untargeted metabolomics investigation into the chemical profile of 10 organic extracts from cf. Symploca spp. revealed several interesting chemical leads for further natural product drug discovery. Subsequent target-directed isolation efforts with one of these, a Panamanian marine cyanobacterium cf. Symploca sp., yielded a phenethylamide metabolite that terminates in a relatively rare gem-dichlorovinylidene moiety, caracolamide A (1), along with a known isotactic polymethoxy-1-alkene (2). Detailed NMR and HRESIMS analyses were used to determine the structures of these molecules, and compound 1 was confirmed by a three-step synthesis. Pure compound 1 was shown to have in vitro calcium influx and calcium channel oscillation modulatory activity when tested as low as 10 pM using cultured murine cortical neurons, but was not cytotoxic to NCI-H460 human non-small-cell lung cancer cells in vitro (IC50 > 10 µM).

Development of a Potent Inhibitor of the Plasmodium Proteasome with Reduced Mammalian Toxicity.

Naturally derived chemical compounds are the foundation of much of our pharmacopeia, especially in antiproliferative and anti-infective drug classes. Here, we report that a naturally derived molecule called carmaphycin B is a potent inhibitor against both the asexual and sexual blood stages of malaria infection. Using a combination of in silico molecular docking and in vitro directed evolution in a well-characterized drug-sensitive yeast model, we determined that these compounds target the ß5 subunit of the proteasome. These studies were validated using in vitro inhibition assays with proteasomes isolated from Plasmodium falciparum. As carmaphycin B is toxic to mammalian cells, we synthesized a series of chemical analogs that reduce host cell toxicity while maintaining blood-stage and gametocytocidal antimalarial activity and proteasome inhibition. This study describes a promising new class of antimalarial compound based on the carmaphycin B scaffold, as well as several chemical structural features that serve to enhance antimalarial specificity.

Dudawalamides A-D, Antiparasitic Cyclic Depsipeptides from the Marine Cyanobacterium Moorea producens.

A family of 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya)-containing cyclic depsipeptides, named dudawalamides A-D (1-4), was isolated from a Papua New Guinean field collection of the cyanobacterium Moorea producens using bioassay-guided and spectroscopic approaches. The planar structures of dudawalamides A-D were determined by a combination of 1D and 2D NMR experiments and MS analysis, whereas the absolute configurations were determined by X-ray crystallography, modified Marfey's analysis, chiral-phase GCMS, and chiral-phase HPLC. Dudawalamides A-D possess a broad spectrum of antiparasitic activity with minimal mammalian cell cytotoxicity. Comparative analysis of the Dhoya-containing class of lipopeptides reveals intriguing structure-activity relationship features of these NRPS-PKS-derived metabolites and their derivatives.