Morphological profiling for drug discovery in the era of deep learning.

Morphological profiling is a valuable tool in phenotypic drug discovery. The advent of high-throughput automated imaging has enabled the capturing of a wide range of morphological features of cells or organisms in response to perturbations at the single-cell resolution. Concurrently, significant advances in machine learning and deep learning, especially in computer vision, have led to substantial improvements in analyzing large-scale high-content images at high throughput. These efforts have facilitated understanding of compound mechanism of action, drug repurposing, characterization of cell morphodynamics under perturbation, and ultimately contributing to the development of novel therapeutics. In this review, we provide a comprehensive overview of the recent advances in the field of morphological profiling. We summarize the image profiling analysis workflow, survey a broad spectrum of analysis strategies encompassing feature engineering- and deep learning-based approaches, and introduce publicly available benchmark datasets. We place a particular emphasis on the application of deep learning in this pipeline, covering cell segmentation, image representation learning, and multimodal learning. Additionally, we illuminate the application of morphological profiling in phenotypic drug discovery and highlight potential challenges and opportunities in this field.

Gatorbulin-1, a distinct cyclodepsipeptide chemotype, targets a seventh tubulin pharmacological site.

Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/ß-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/ß-tubulin-GB1 complex.

Biosynthesis of Lyngbyastatins 1 and 3, Cytotoxic Depsipeptides from an Okeania sp. Marine Cyanobacterium.

Lyngbyastatins (Lbns) 1 (1) and 3 (2) belong to a group of cyclic depsipeptides that inhibit cancer cell proliferation. These compounds have been isolated from different marine cyanobacterial collections, while further development of these compounds relies on their lengthy total synthesis. Biosynthetic studies of these compounds can provide viable strategies to access these compounds and develop new analogs. In this study, we report the identification and characterization of one Lbn biosynthetic gene cluster (BGC) from the marine cyanobacterium Okeania sp. VPG18-21. We initially identified 1 and 2 in the organic extract by mass spectrometry and performed the targeted isolation of these compounds, which feature a (2S,3R)-3-amino-2-methylpentanoic acid (MAP) and a (2S,3R)-3-amino-2-methylhexanoic acid (Amha) moiety, respectively. Parallel metagenomic sequencing of VPG18-21 led to the identification of a putative Lbn BGC that encodes six megaenzymes (LbnA-F), including one polyketide synthase (PKS, LbnE), four nonribosomal peptide synthetases (NRPSs, LbnB-D and -F), and one PKS-NRPS hybrid (LbnA). Bioinformatic analysis of these enzymes suggested that the BGC produces 1 and 2. Furthermore, our biochemical studies of three recombinant adenylation domains uncovered their substrate specificities, supporting the identity of the BGC. Finally, we identified near-complete Lbn-like BGCs in the genomes of two other marine cyanobacteria.

Isolation and Biological Activity of Iezoside and Iezoside B, SERCA Inhibitors from Floridian Marine Cyanobacteria.

Marine cyanobacteria are a rich source of bioactive natural products. Here, we report the isolation and structure elucidation of the previously reported iezoside (1) and its C-31 O-demethyl analogue, iezoside B (2), from a cyanobacterial assemblage collected at Loggerhead Key in the Dry Tortugas, Florida. The two compounds have a unique skeleton comprised of a peptide, a polyketide and a modified sugar unit. The compounds were tested for cytotoxicity and effects on intracellular calcium. Both compounds exhibited cytotoxic activity with an IC50 of 1.5 and 3.0 µΜ, respectively, against A549 lung carcinoma epithelial cells and 1.0 and 2.4 µΜ against HeLa cervical cancer cells, respectively. In the same cell lines, compounds 1 and 2 show an increase in cytosolic calcium with approximate EC50 values of 0.3 and 0.6 µΜ in A549 cells and 0.1 and 0.5 µΜ, respectively, in HeLa cells, near the IC50 for cell viability, suggesting that the increase in cytosolic calcium is functionally related to the cytotoxicity of the compounds and consistent with their activity as SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) inhibitors. The structure-activity relationship provides evidence that structural changes in the sugar unit may be tolerated, and the activity is tunable. This finding has implications for future analogue synthesis and target interaction studies.

Chlorinated Enyne Fatty Acid Amides from a Marine Cyanobacterium: Discovery of Taveuniamides L-M and Pharmacological Characterization of Taveuniamide F as a GPCR Antagonist with CNR1 Selectivity.

NMR and MS/MS-based metabolomics of a cyanobacterial extract from Piti Bomb Holes, Guam, indicated the presence of unique enyne-containing halogenated fatty acid amides. We isolated three new compounds of this class, taveuniamides L-N (1-3), along with the previously reported taveuniamide F (4), which was the most abundant analog. The planar structures of the new compounds were established using 1D and 2D NMR as well as mass spectrometry. We established the configuration of this chemical class to be R at C-8 via Mosher's analysis of 4 after reduction of the carboxamide group. Our biological investigations with 4 revealed that the compound binds to the cannabinoid receptor CNR1, acting as an antagonist/inverse agonist in the canonical G-protein signaling pathways. In selectivity profiling against 168 GPCR targets using the ß-arrestin functional assay, we found that 4 antagonizes GPR119, NPSR1b, CCR9, CHRM4, GPR120, HTR2A, and GPR103, in addition to CNR1. Interestingly, 4 showed a 6.8-fold selectivity for CNR1 over CNR2. The binding mode of 4 to CNR1 was investigated using docking and molecular dynamics simulations with both natural and unnatural stereoisomers, revealing important CNR1 residues for the interaction and also providing a possible reasoning for the observed CNR1/CNR2 selectivity.

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.

Discovery, Synthesis, and Biological Evaluation of Anaenamides C and D from a New Marine Cyanobacterium, Hormoscilla sp.

Our ongoing efforts to explore the chemical space associated with marine cyanobacteria from coral reefs of Guam have yielded two new members of the anaenamide family of natural products, anaenamides C (3) and D (4). These compounds were isolated from a novel Hormoscilla sp. (VPG16-58). Our phylogenetic profiling (16S rDNA) of this cyanobacterium indicated that VPG16-58 is taxonomically distinct from the previously reported producer of the anaephenes, VPG16-59 (Hormoscilla sp.), and other previously documented species of the genus Hormoscilla. The planar structures of 3 and 4 were determined via spectroscopic methods, and absolute configurations of the α-hydroxy acids were assigned by enantioselective HPLC analysis. To address the requirement for sufficient material for testing, we first adapted our published linear synthetic approach for 1 and 2 to generate anaenoic acid (7), which served as a point for diversification, providing the primary amides 3 and 4 from synthetic intermediates 5 and 6, respectively. The compounds were then tested for effects on HCT116 colon cancer cell viability and in an ARE-luciferase reporter gene assay for Nrf2 modulation using HEK293 human embryonic kidney cells. Our findings indicate that, in contrast to cytotoxic methyl esters 1 and 2, the primary amides 3 and 4 activate the Nrf2 pathway at noncytotoxic concentrations. Overall, our data suggest that the anaenamide scaffold is tunable to produce differential biological outcomes.

Anti-Inflammatory Dysidazirine Carboxylic Acid from the Marine Cyanobacterium Caldora sp. Collected from the Reefs of Fort Lauderdale, Florida.

Dysidazirine carboxylic acid (1) was isolated from the lipophilic extract of a collection of the benthic marine cyanobacterium Caldora sp. from reefs near Fort Lauderdale, Florida. The planar structure of this new compound was determined by spectroscopic methods and comparisons between HRMS and NMR data with its reported methyl ester. The absolute configuration of the single chiral center was determined by the conversion of 1 to the methyl ester and the comparison of its specific rotation data with the two known methyl ester isomers, 2 and 3. Molecular sequencing with 16S rDNA indicated that this cyanobacterium differs from Caldora penicillata (Oscillatoriales) and represents a previously undocumented and novel Caldora species. Dysidazirine (2) showed weak cytotoxicity against HCT116 colorectal cancer cells (IC50 9.1 µM), while dysidazirine carboxylic acid (1) was non-cytotoxic. Similar cell viability patterns were observed in RAW264.7 cells with dysidazirine only (2), displaying cytotoxicity at the highest concentration tested (50 µM). The non-cytotoxic dysidazirine carboxylic acid (1) demonstrated anti-inflammatory activity in RAW264.7 cells stimulated with LPS. After 24 h, 1 inhibited the production of NO by almost 50% at 50 µM, without inducing cytotoxicity. Compound 1 rapidly decreased gene expression of the pro-inflammatory gene iNOS after 3 h post-LPS treatment and in a dose-dependent manner (IC50 ~1 µM); the downregulation of iNOS persisted at least until 12 h.

Genomic and Targeted Approaches Unveil the Cell Membrane as a Major Target of the Antifungal Cytotoxin Amantelide A.

Amantelide A, a polyhydroxylated macrolide isolated from a marine cyanobacterium, displays broad-spectrum activity against mammalian cells, bacterial pathogens, and marine fungi. We conducted comprehensive mechanistic studies to identify the molecular targets and pathways affected by amantelide A. Our investigations relied on chemical structure similarities with compounds of known mechanisms, yeast knockout mutants, yeast chemogenomic profiling, and direct biochemical and biophysical methods. We established that amantelide A exerts its antifungal action by binding to ergosterol-containing membranes followed by pore formation and cell death, a mechanism partially shared with polyene antifungals. Binding assays demonstrated that amantelide A also binds to membranes containing epicholesterol or mammalian cholesterol, thus suggesting that the cytotoxicity to mammalian cells might be due to its affinity to cholesterol-containing membranes. However, membrane interactions were not completely dependent on sterols. Yeast chemogenomic profiling suggested additional direct or indirect effects on actin. Accordingly, we performed actin polymerization assays, which suggested that amantelide A also promotes actin polymerization in cell-free systems. However, the C-33 acetoxy derivative amantelide B showed a similar effect on actin dynamics in vitro but no significant activity against yeast. Overall, these studies suggest that the membrane effects are the most functionally relevant for amantelide A mechanism of action.

Fungal Epithiodiketopiperazines Carrying α,ß-Polysulfide Bridges from Penicillium steckii YE, and Their Chemical Interconversion.

Some fungal epithiodiketopiperazine alkaloids display α,ß-polysulfide bridges alongside diverse structural variations. However, the logic of their chemical diversity has rarely been explored. Here, we report the identification of three new (2, 3, 8) and five known (1, 4-7) epithiodiketopiperazines of this subtype from a marine-derived Penicillium sp. The structure elucidation was supported by multiple spectroscopic analyses. Importantly, we observed multiple nonenzymatic interconversions of these analogues in aqueous solutions and organic solvents. Furthermore, the same biosynthetic origin of these compounds was supported by one mined gene cluster. The dominant analogue (1) demonstrated selective cytotoxicity to androgen-sensitive prostate cancer cells and HIF-depleted colorectal cells and mild antiaging activities, linking the bioactivity to oxidative stress. These results provide crucial insight into the formation of fungal epithiodiketopiperazines through chemical interconversions.

Function-Oriented and Modular (+/-)-cis-Pseudoguaianolide Synthesis: Discovery of New Nrf2 Activators and NF-κB Inhibitors.

Described herein is a function-oriented synthesis route and biological evaluation of pseudoguaianolide analogues. The 10-step synthetic route developed retains the topological complexity of the natural product, installs functional handles for late-stage diversification, and forges the key bioactive Michael acceptors early in the synthesis. The analogues were found to be low-micromolar Nrf2 activators and micromolar NF-κB inhibitors and dependent on the local environment of the Michael acceptor moieties.

Bifunctional Doscadenamides Activate Quorum Sensing in Gram-Negative Bacteria and Synergize with TRAIL to Induce Apoptosis in Cancer Cells.

New cyanobacteria-derived bifunctional analogues of doscadenamide A, a LasR-dependent quorum sensing (QS) activator in Pseudomonas aeruginosa, characterized by dual acylation of the pyrrolinone core structure and the pendant side chain primary amine to form an imide/amide hybrid are reported. The identities of doscadenamides B-J were confirmed through total synthesis and a strategic focused library with different acylation and unsaturation patterns was created. Key molecular interactions for binding with LasR and a functional response through mutation studies coupled with molecular docking were identified. The structure-activity relationships (SARs) were probed in various Gram-negative bacteria, including P. aeruginosa and Vibrio harveyi, indicating that the pyrrolinone-N acyl chain is critical for full agonist activity, while the other acyl chain is dispensable or can result in antagonist activity, depending on the bacterial system. Since homoserine lactone (HSL) quorum sensing activators have been shown to act in synergy with TRAIL to induce apoptosis in cancer cells, selected doscadenamides were tested in orthogonal eukaryotic screening systems. The most potent QS agonists, doscadenamides S10-S12, along with doscadenamides F and S4 with partial or complete saturation of the acyl side chains, exhibited the most pronounced synergistic effects with TRAIL in triple negative MDA-MB-231 breast cancer cells. The overall correlation of the SAR with respect to prokaryotic and eukaryotic targets may hint at coevolutionary processes and intriguing host-bacteria relationships. The doscadenamide scaffold represents a non-HSL template for combination therapy with TRAIL pathway stimulators.

Heterologous production of cyanobacterial compounds.

Cyanobacteria produce a plethora of compounds with unique chemical structures and diverse biological activities. Importantly, the increasing availability of cyanobacterial genome sequences and the rapid development of bioinformatics tools have unraveled the tremendous potential of cyanobacteria in producing new natural products. However, the discovery of these compounds based on cyanobacterial genomes has progressed slowly as the majority of their corresponding biosynthetic gene clusters (BGCs) are silent. In addition, cyanobacterial strains are often slow-growing, difficult for genetic engineering, or cannot be cultivated yet, limiting the use of host genetic engineering approaches for discovery. On the other hand, genetically tractable hosts such as Escherichia coli, Actinobacteria, and yeast have been developed for the heterologous expression of cyanobacterial BGCs. More recently, there have been increased interests in developing model cyanobacterial strains as heterologous production platforms. Herein, we present recent advances in the heterologous production of cyanobacterial compounds in both cyanobacterial and noncyanobacterial hosts. Emerging strategies for BGC assembly, host engineering, and optimization of BGC expression are included for fostering the broader applications of synthetic biology tools in the discovery of new cyanobacterial natural products.

Largazole Inhibits Ocular Angiogenesis by Modulating the Expression of VEGFR2 and p21.

Ocular angiogenic diseases, characterized by abnormal blood vessel formation in the eye, are the leading cause of blindness. Although Anti-VEGF therapy is the first-line treatment in the market, a substantial number of patients are refractory to it or may develop resistance over time. As uncontrolled proliferation of vascular endothelial cells is one of the characteristic features of pathological neovascularization, we aimed to investigate the role of the class I histone deacetylase (HDAC) inhibitor Largazole, a cyclodepsipeptide from a marine cyanobacterium, in ocular angiogenesis. Our study showed that Largazole strongly inhibits retinal vascular endothelial cell viability, proliferation, and the ability to form tube-like structures. Largazole strongly inhibits the vessel outgrowth from choroidal explants in choroid sprouting assay while it does not affect the quiescent choroidal vasculature. Largazole also inhibits vessel outgrowth from metatarsal bones in metatarsal sprouting assay without affecting pericytes coverage. We further demonstrated a cooperative effect between Largazole and an approved anti-VEGF drug, Alflibercept. Mechanistically, Largazole strongly inhibits the expression of VEGFR2 and leads to an increased expression of cell cycle inhibitor, p21. Taken together, our study provides compelling evidence on the anti-angiogenic role of Largazole that exerts its function through mediating different signaling pathways.

GPCR Pharmacological Profiling of Aaptamine from the Philippine Sponge Stylissa sp. Extends Its Therapeutic Potential for Noncommunicable Diseases.

We report the first isolation of the alkaloid aaptamine from the Philippine marine sponge Stylissa sp. Aaptamine possessed weak antiproliferative activity against HCT116 colon cancer cells and inhibited the proteasome in vitro at 50 µM. These activities may be functionally linked. Due to its known, more potent activity on certain G-protein coupled receptors (GPCRs), including α-adrenergic and δ-opioid receptors, the compound was profiled more broadly at sub-growth inhibitory concentrations against a panel of 168 GPCRs to potentially reveal additional targets and therapeutic opportunities. GPCRs represent the largest class of drug targets. The primary screen at 20 µM using the ß-arrestin functional assay identified the antagonist, agonist, and potentiators of agonist activity of aaptamine. Dose-response analysis validated the α-adrenoreceptor antagonist activity of aaptamine (ADRA2C, IC50 11.9 µM) and revealed the even more potent antagonism of the ß-adrenoreceptor (ADRB2, IC50 0.20 µM) and dopamine receptor D4 (DRD4, IC50 6.9 µM). Additionally, aaptamine showed agonist activity on selected chemokine receptors, by itself (CXCR7, EC50 6.2 µM; CCR1, EC50 11.8 µM) or as a potentiator of agonist activity (CXCR3, EC50 31.8 µM; CCR3, EC50 16.2 µM). These GPCRs play a critical role in the treatment of cardiovascular disease, diabetes, cancer, and neurological disorders. The results of this study may thus provide novel preventive and therapeutic strategies for noncommunicable diseases (NCDs).

Natural products as modulators of eukaryotic protein secretion.

Covering: up to the end of 2019Diverse natural product small molecules have allowed critical insights into processes that govern eukaryotic cells' ability to secrete cytosolically synthesized secretory proteins into their surroundings or to insert newly synthesized integral membrane proteins into the lipid bilayer of the endoplasmic reticulum. In addition, many components of the endoplasmic reticulum, required for protein homeostasis or other processes such as lipid metabolism or maintenance of calcium homeostasis, are being investigated for their potential in modulating human disease conditions such as cancer, neurodegenerative conditions and diabetes. In this review, we cover recent findings up to the end of 2019 on natural products that influence protein secretion or impact ER protein homeostasis, and serve as powerful chemical tools to understand protein flux through the mammalian secretory pathway and as leads for the discovery of new therapeutics.

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.

Dolastatin 15 from a Marine Cyanobacterium Suppresses HIF-1α Mediated Cancer Cell Viability and Vascularization.

Chemical investigation of a benthic marine cyanobacterium yielded the anticancer agent dolastatin 15, originally isolated from a mollusk. Dolastatin 15 is a microtubule-destabilizing agent with analogues undergoing clinical evaluation. Profiling against a panel of isogenic HCT116 colorectal cancer cells showed remarkable differential cytotoxicity against the parental cells over isogenic cells lacking HIF or other key players in the pathway, including oncogenic KRAS and VEGF. Dolastatin 15 displayed an antivascularization effect in human endothelial cells and in zebrafish vhl mutants with activated Hif, thus signifying its clinical potential as a treatment for solid tumors with an angiogenic component. Global transcriptome analysis with RNA sequencing suggested that dolastatin 15 could affect other major cancer pathways that might not directly involve tubulin or HIF. The identification of the true producer of a clinically relevant agent is important for sustainable supply, as is understanding the biosynthesis, and future genetic manipulation of the biosynthetic gene cluster for analogue production.

Discovery of new pyrimidine-5-carbonitrile derivatives as anticancer agents targeting EGFRWT and EGFRT790M.

A new series of pyrimidine-5-carbonitrile derivatives has been designed as ATP mimicking tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR). These compounds were synthesized and evaluated for their in vitro cytotoxic activities against a panel of four human tumor cell lines, namely colorectal carcinoma (HCT-116), hepatocellular carcinoma (HepG-2), breast cancer (MCF-7), and non-small cell lung cancer cells (A549). Five of the synthesized compounds, 11a, 11b, 12b, 15b and 16a, were found to exhibit moderate antiproliferative activity against the tested cell lines and were more active than the EGFR inhibitor erlotinib. In particular, compound 11b showed 4.5- to 8.4-fold erlotinib activity against HCT-116, HepG-2, MCF-7, and A549 cells with IC50 values of 3.37, 3.04, 4.14, and 2.4 µM respectively. Moreover, the most cytotoxic compounds that showed promising IC50 values against the four cancer cell lines were subjected to further investigation for their kinase inhibitory activities against EGFRWT and EGFRT790M using homogeneous time resolved fluorescence (HTRF) assay. Compound 11b was also found to be the most active compound against both EGFRWT and mutant EGFRT790M, exhibiting IC50 values of 0.09 and 4.03 µM, respectively. The cell cycle and apoptosis analyses revealed that compound 11b can arrest the cell cycle at the G2/M phase and induce significant apoptotic effects in HCT-116, HepG-2, and MCF-7 cells. Additionally, compound 11b upregulated the level of caspase-3 by 6.5 fold in HepG-2 when compared with the control. Finally, molecular docking studies were carried out to examine the binding mode of the synthesized compounds against the proposed targets; EGFRWT and EGFRT790M. Additional in silico ADMET studies were performed to explore drug-likeness properties.

Ahp-Cyclodepsipeptides as tunable inhibitors of human neutrophil elastase and kallikrein 7: Total synthesis of tutuilamide A, serine protease selectivity profile and comparison with lyngbyastatin 7.

We describe the total synthesis of tutuilamide A, a potent porcine pancreatic elastase (PPE) inhibitor and a representative member of the 3-amino-6-hydroxy-2-piperidone (Ahp) cyclodepsipeptide family, isolated from marine cyanobacteria. The Ahp unit serves as a pharmacophore and the adjacent 2-amino-2-butenoic acid (Abu) is a main driver of the selectivity among serine proteases. We adapted our previous convergent strategy to generate the macrocycle, common with lyngbyastatin 7 and related elastase inhibitors, and then appended the tutuilamide A-specific side chain bearing a vinyl chloride. Tutuilamide A and lyngbyastatin 7 were evaluated side by side for the inhibition of the disease-relevant human neutrophil elastase (HNE). Tutuilamide A and lyngbyastatin 7 were approximately equipotent against HNE, while tutuilamide A was previously shown to be more active against PPE compared with lyngbyastatin 7, further demonstrating that the side chain provides opportunities to not only modulate potency but also selectivity among proteases of the same function from different organisms. Profiling of tutuilamide A against mainly human serine proteases revealed high selectivity for HNE (IC50 0.73 nM) and pleiotropic activity against kallikrein 7 (KLK7, IC50 5.0 nM), without affecting other kallikreins, similarly to lyngbyastatin 7 (IC50 0.85 nM for HNE and 3.1 nM for KLK7). A comprehensive molecular docking study for elastases and KLK7 afforded deeper insight into the intricate differences between inhibitor interactions with HNE and PPE, accounting for the differential activities for both compounds. The synthesis and molecular studies serve as a proof-of-concept that the macrocyclic scaffold can be diversified to fine-tune the activity of serine protease inhibitors.