Discovery and Anti-Inflammatory Activity of a Cyanobacterial Fatty Acid Targeting the Keap1/Nrf2 Pathway.

The monounsaturated fatty acid 7(E)-9-keto-hexadec-7-enoic acid (1) and three structurally related analogues with different oxidation states and degrees of unsaturation (2-4) were discovered from a marine benthic cyanobacterial mat collected from Delta Shoal, Florida Keys. Their structures were elucidated using NMR spectroscopy and mass spectrometry. The structure of 1 contained an α,ß-unsaturated carbonyl system, a key motif required for the activation of the Keap1/Nrf2-ARE pathway that is involved in the activation of antioxidant and phase II detoxification enzymes. Compounds 1-4 were screened in ARE-luciferase reporter gene assay using stably transfected HEK293 cells, and only 1 significantly induced Nrf2 activity at 32 and 10 µM, whereas 2-4 were inactive. As there is crosstalk between inflammation and oxidative stress, subsequent biological studies were focused on 1 to investigate its anti-inflammatory potential. Compound 1 induced Nqo1, a well-known target gene of Nrf2, and suppressed iNos transcript levels, which translated into reduced levels of nitric oxide in LPS-activated mouse macrophage RAW264.7 cells, a more relevant model for inflammation. RNA sequencing was performed to capture the effects of 1 on a global level and identified additional canonical pathways and upstream regulators involved in inflammation and immune response, particularly those related to multiple sclerosis. A targeted survey of marine cyanobacterial samples from other geographic locations, including Guam, suggested the widespread occurrence of 1. Furthermore, the previous isolation of 1 from marine diatoms and green algae implied a potentially important ecological role across marine algal eukaryotes and prokaryotes. The previous isolation from sea lettuce raises the possibility of dietary intervention to attenuate inflammation and related disease progression.

Targeting eukaryotic proteases for natural products-based drug development.

Covering: up to April 2020 Proteases are involved in the regulation of many physiological processes. Their overexpression and dysregulated activity are linked to diseases such as hypertension, diabetes, viral infections, blood clotting disorders, respiratory diseases, and cancer. Therefore, they represent an important class of therapeutic targets. Several protease inhibitors have reached the market and >60% of them are directly related to natural products, even when excluding synthetic natural product mimics. Historically, natural products have been a valuable and validated source of therapeutic agents, as over half of the marketed drugs across targets and diseases are inspired by natural product structures. In the past two decades the number of new protease inhibitors discovered from nature has sharply increased. Additionally, the availability of 3D structural information for proteases has permitted structure-based design and accelerated the synthesis of optimized lead structures with improved potency and selectivity profiles, resulting in some of the most-potent-in-class inhibitors. These discoveries were oftentimes maximized by in-depth biological assessments of lead inhibitors, linking them to a relevant disease state. This review will discuss some of the current and emerging drug targets and their involvement in various disease processes, highlighting selected success stories behind several FDA-approved protease inhibitors that have natural products scaffolds as well as recent selected pharmacologically well-characterized inhibitors derived from marine or terrestrial sources.

Structural Diversity and Anticancer Activity of Marine-Derived Elastase Inhibitors: Key Features and Mechanisms Mediating the Antimetastatic Effects in Invasive Breast Cancer.

Three new 3-amino-6-hydroxy-2-piperidone (Ahp)-containing cyclic depsipeptides, named loggerpeptins A-C (1-3), along with molassamide (4), were discovered from a marine cyanobacterium, extending the structural diversity of this prevalent scaffold of cyanobacterial serine protease inhibitors. Molassamide, which contains a 2-amino-butenoic (Abu) unit in the cyclic core, was the most potent and selective analogue against human neutrophil elastase (HNE). Given the growing evidence supporting the role of HNE in breast cancer progression and metastasis, we assessed the cellular effects of compounds 3 and 4 in the context of targeting invasive breast cancer. Both compounds inhibited cleavage of the elastase substrate CD40 in biochemical assays; however, only 4 exhibited significant cellular activity. As CD40 and other receptor proteolytic processing culminates in NFκB activation, we assessed the effects of 4 on the expression of target genes, including ICAM-1. ICAM-1 is also a direct target of elastase and, in our studies, compound 4 attenuated both elastase-induced ICAM-1 gene expression and ICAM-1 proteolytic processing by elastase, revealing a potential dual effect on migration through modulation of gene expression and proteolytic processing. Molassamide also specifically inhibited the elastase-mediated migration of highly invasive triple-negative breast cancer cells.

Grassystatins D-F, Potent Aspartic Protease Inhibitors from Marine Cyanobacteria as Potential Antimetastatic Agents Targeting Invasive Breast Cancer.

Three new modified peptides named grassystatins D-F (1-3) were discovered from a marine cyanobacterium from Guam. Their structures were elucidated using NMR spectroscopy and mass spectrometry. The hallmark structural feature in the peptides is a statine unit, which contributes to their aspartic protease inhibitory activity preferentially targeting cathepsins D and E. Grassystatin F (3) was the most potent analogue, with IC50 values of 50 and 0.5 nM against cathepsins D and E, respectively. The acidic tumor microenvironment is known to increase the activation of some of the lysosomal proteases associated with tumor metastasis such as cathepsins. Because cathepsin D is a biomarker in aggressive forms of breast cancer and linked to poor prognosis, the effects of cathepsin D inhibition by 1 and 3 on the downstream cellular substrates cystatin C and PAI-1 were investigated. Furthermore, the functional relevance of targeting cathepsin D substrates was evaluated by examining the effect of 1 and 3 on the migration of MDA-MD-231 cells. Grassystatin F (3) inhibited the cleavage of cystatin C and PAI-1, the activities of their downstream targets cysteine cathepsins and tPA, and the migration of the highly aggressive triple negative breast cancer cells, phenocopying the effect of siRNA-mediated knockdown of cathepsin D.

Kempopeptin C, a Novel Marine-Derived Serine Protease Inhibitor Targeting Invasive Breast Cancer.

Kempopeptin C, a novel chlorinated analogue of kempopeptin B, was discovered from a marine cyanobacterium collected from Kemp Channel in Florida. The structure was elucidated using NMR spectroscopy and mass spectrometry (MS). The presence of the basic Lys residue adjacent to the N-terminus of the 3-amino-6-hydroxy-2-piperidone (Ahp) moiety contributed to its selectivity towards trypsin and related proteases. The antiproteolytic activity of kempopeptin C was evaluated against trypsin, plasmin and matriptase and found to inhibit these enzymes with IC50 values of 0.19, 0.36 and 0.28 µM, respectively. Due to the significance of these proteases in cancer progression and metastasis, as well as their functional redundancy with respect to targeting overlapping substrates, we examined the effect of kempopeptin C on the downstream cellular substrates of matriptase: CDCP1 and desmoglein-2 (Dsg-2). Kempopeptin C was shown to inhibit the cleavage of both substrates in vitro. Additionally, kempopeptin C reduced the cleavage of CDCP1 in MDA-MB-231 cells up to 10 µM. The functional relevance of targeting matriptase and related proteases was investigated by assessing the effect of kempopeptin C on the migration of breast cancer cells. Kempopeptin C inhibited the migration of the invasive MDA-MB-231 cells by 37 and 60% at 10 and 20 µM, respectively.

Tasiamide F, a potent inhibitor of cathepsins D and E from a marine cyanobacterium.

In search of novel protease inhibitors with therapeutic potential, our efforts exploring the marine cyanobacterium Lyngbya sp. have led to the discovery of tasiamide F (1), which is an analogue of tasiamide B (2). The structure was elucidated using a combination of NMR spectroscopy and mass spectrometry. The key structural feature in 1 is the presence of the Phe-derived statine core, which contributes to its aspartic protease inhibitory activity. The antiproteolytic activity of 1 and 2 was evaluated in vitro against cathepsins D and E, and BACE1. Tasiamide F (1) displayed IC50 values of 57nM, 23nM, and 0.69µM, respectively, indicating greater selectivity for cathepsins over BACE1 compared with tasiamide B (2). Molecular docking experiments were carried out for compounds 1 and 2 against cathepsins D and E to rationalize their activity towards these proteases. The dysregulated activities of cathepsins D and E have been implicated in cancer and modulation of immune responses, respectively, and these proteases represent potential therapeutic targets.

Empowering competence: A program-wide active learning framework for a pharmacy program.

OBJECTIVE: This study aimed to develop and implement a program-wide active learning framework to guide active learning and assessment efforts in an entry-to-practice competency-based Doctor of Pharmacy program. METHODS: The development of the framework involved 3 stages: creation of a framework aligned with the program's guiding principles, provision of training and support to faculty and students, and evaluation of the students' and academic staff satisfaction using an online survey over 2 academic years (2022-2023). Data from this survey were analyzed descriptively. RESULTS: An active learning framework that was aligned with the program's guiding principles while allowing flexibility for individual teaching styles was developed. It consisted of 4 stages: preclass preparation, in-class work, prelaboratory preparation, and in-laboratory activities (emphasizing knowledge acquisition and competency development). Academic staff surveys reported higher satisfaction of staff in year 2 than year 3 of the program, with indications of further training on specific modalities. Students' satisfaction improved from year 2 to 3, particularly, in areas related to class objectives, learning environment, and feedback. CONCLUSION: The transformation of a curriculum that includes the evolution of the teaching and learning strategy is a complex, long-term project that deserves continuing attention. Having frameworks in place helps the management, instructors, and students to understand the global direction, stay focused, and support the implementation of competency-based education and student-centered learning.

Variable-Sized bis(4-spiro-fused-ß-lactam)-Based Unsaturated Macrocycles: Synthesis and Characterization.

The synthesis with structural identifications including NMR and HRMS spectral data along with single-crystal X-ray diffraction analysis (for 20b, 23b, 25b-27b) of a family of 14 new syn/anti bis-4-spiro-ß-lactam-based unsaturated macrocycles (19a,b-27a,b), obtained by multistep synthesis including (i) diimine formation, (ii) Staudinger [2 + 2] ketene-imine cycloaddition, and (iii) ring-closing metathesis (RCM), is reported.

Largazole is a Brain-Penetrant Class I HDAC Inhibitor with Extended Applicability to Glioblastoma and CNS Diseases.

Largazole is a potent class I selective histone deacetylase inhibitor prodrug with anticancer activity against solid tumors in preclinical models. Largazole possesses in vitro activity against glioblastoma multiforme (GBM) cells and sufficiently crosses the blood-brain barrier based on measurement of the active species, largazole thiol, to achieve therapeutically relevant concentrations in the mouse brain. The effective dose resulted in pronounced functional responses on the transcript level based on RNA sequencing and quantitative polymerase chain reaction after reverse transcription (RT-qPCR), revealing desirable expression changes of genes related to neuroprotection, including Bdnf and Pax6 upregulation, extending the applicability of largazole to the treatment of brain cancer and neurodegenerative disorders. The largazole-induced modulation of Pax6 unifies both activities, since Pax6 expression suppresses GBM proliferation and invasion and inversely correlates with GBM tumor grade, while it is also implicated in neurogenesis, neuronal plasticity, and cognitive ability. Our results suggest that largazole could be repurposed for diseases of the brain.

Discovery, Synthesis, Pharmacological Profiling, and Biological Characterization of Brintonamides A-E, Novel Dual Protease and GPCR Modulators from a Marine Cyanobacterium.

Five novel modified linear peptides named brintonamides A-E (1-5) were discovered from a marine cyanobacterial sample collected from Brinton Channel, Florida Keys. The total synthesis of 1-5 in addition to two other structurally related analogues (6 and 7) was achieved, which provided more material to allow rigorous biological evaluation and SAR studies. Compounds were subjected to cancer-focused phenotypic cell viability and migration assays and orthogonal target-based pharmacological screening platforms to identify their protease and GPCR modulatory activity profiles. The cancer related serine protease kallikrein 7 (KLK7) was inhibited to similar extents with an IC50 near 20 µM by both representative members 1 and 4, which differed in the presence or lack of the N-terminal unit. In contrast to the biochemical protease profiling study, clear SAR was observed in the functional GPCR screens, where five GPCRs in antagonist mode (CCR10, OXTR, SSTR3, TACR2) and agonist mode (CXCR7) were modulated by compounds 1-7 to varying extents. Chemokine receptor type 10 (CCR10) was potently modulated by brintonamide D (4) with an IC50 of 0.44 µM. We performed in silico modeling to understand the structural basis underlying the differences in the antagonistic activity among brintonamides toward CCR10. Because of the significance of KLK7 and CCR10 in cancer progression and metastasis, we demonstrated the ability of brintonamide D (4) at 10 µM to significantly target downstream cellular substrates of KLK7 (Dsg-2 and E-cad) in vitro and to inhibit CCL27-induced CCR10-mediated proliferation and the migration of highly invasive breast cancer cells.

Modulation of Activity Profiles for Largazole-Based HDAC Inhibitors through Alteration of Prodrug Properties.

Largazole is a potent and class I-selective histone deacetylase (HDAC) inhibitor purified from marine cyanobacteria and was demonstrated to possess antitumor activity. Largazole employs a unique prodrug strategy, via a thioester moiety, to liberate the bioactive species largazole thiol. Here we report alternate prodrug strategies to modulate the pharmacokinetic and pharmacodynamics profiles of new largazole-based compounds. The in vitro effects of largazole analogues on cancer cell proliferation and enzymatic activities of purified HDACs were comparable to the natural product. However, in vitro and in vivo histone hyperacetylation in HCT116 cells and implanted tumors, respectively, showed differences, particularly in the onset of action and oral bioavailability. These results indicate that, by employing a different approach to disguise the "warhead" moiety, the functional consequence of these prodrugs can be significantly modulated. Our data corroborate the role of the pharmacokinetic properties of this class of compounds to elicit the desired and timely functional response.