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Key Points Juvenile zebrafish tolerate widespread coagulopathy due to complete ablation of antithrombin III, but develop lethal thrombosis as adults. In vivo structure/function analysis of antithrombin III in zebrafish reveals limited roles for heparin-binding and anti-IXa/Xa activity.

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Chemical investigation of the marine-sponge-derived fungus Penicillium adametzioides AS-53 resulted in the identification of two new bisthiodiketopiperazine derivatives, adametizines A (1) and B (2), from cultivation in a liquid potato-dextrose broth (PDB) culture medium, whereas two new acorane sesquiterpenes, adametacorenols A (3) and B (4), were isolated from a rice solid culture medium. The structures of these compounds were elucidated on the basis of spectroscopic analysis. The absolute configuration of compound 1 was determined by X-ray crystallographic analysis, and that of 3 was determined by modified Mosher's method. Compound 1 exhibited lethality against brine shrimp (Artemia salina) with an LD50 value of 4.8 μM and inhibitory activities against Staphyloccocus aureus, Aeromonas hydrophilia, Vibrio spp. V. harveyi and V. parahaemolyticus, and Gaeumannomyces graminis with minimum inhibitory concentration values of 8, 8, 32, 8, and 16 μg/mL, respectively. Chlorination at C-7 significantly increased the brine shrimp lethality and antimicrobial activity of the bisthiodiketopiperazines.

A natural product chemistry-based approach was applied to discover small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1). A Petrosaspongia mycofijiensis marine sponge extract yielded mycothiazole (1), a solid tumor selective compound with no known mechanism for its cell linedependent cytotoxic activity. Compound 1 inhibited hypoxic HIF-1 signaling in tumor cells (IC 50 1 nM) that correlated with the suppression of hypoxia-stimulated tumor angiogenesis in vitro. However, 1 exhibited pronounced neurotoxicity in vitro. Mechanistic studies revealed that 1 selectively suppresses mitochondrial respiration at Complex I (NADH-ubiquinone oxidoreductase). Unlike rotenone, MPP + , annonaceous acetogenins, piericidin A, and other Complex I inhibitors, mycothiazole is a mixed polyketide/peptide-derived compound with a central thiazole moiety. The exquisite potency and structural novelty of 1 suggest that it may serve as a valuable molecular probe for mitochondrial biology and HIF-mediated hypoxic signaling.

The marine-derived macrolides latrunculins A (1) and B, from the Red Sea sponge Negombata magnifica, have been found to reversibly bind actin monomers, forming a 1:1 complex with G-actin and disrupting its polymerization. The microfilament protein actin is responsible for several essential functions within the cell such as cytokinesis and cell migration. One of the main binding pharmacophores of 1 to G-actin was identified as the C-17 lactol hydroxyl moiety that binds arginine 210 NH. Latrunculin A-17-O-carbamates 2-6 were prepared by reaction with the corresponding isocyanates. Latrunculin A (1) and carbamates 4-6 displayed potent anti-invasive activity against the human highly metastatic human prostate cancer PC-3M cells in a Matrigel™ assay at a concentration range of 50 nM-1 µM. Latrunculin A (1, 500 nM) decreased the disaggregation and cell migration of PC-3M-CT+ spheroids by three-fold. Carbamates 4 and 5 were two and half and five-fold more active than 1, respectively, in this assay with less actin binding affinity. Latrunculin A (1, IC 50 6.7 µM) and its 17-O-[N-(benzyl)carbamate (6, IC 50 29 µM) suppress hypoxia-induced HIF-1 activation in T47D breast tumor cells.Latrunculin A (1) and B are macrolides reported by Kashman and coworkers from the Red Sea sponge Negombata magnifica Kelly-Borges and Vacelet (Podospongiidae). 1 Latrunculins are reported to decrease intraocular pressure and increase outflow facility without corneal effects in monkeys.2 , 3 Latrunculin B and analogs showed antiangiogenic, antimetastatic, and antimicrobial activities.4 The most important biological effects of latrunculins are their abilities to disrupt microfilament organization and inhibit microfilament-mediated processes without affecting the organization of the microtubular system. 5 The latrunculins bind reversibly to the cytoskeleton actin monomers, forming 1:1 complexes with G-actin and disrupting polymerization. 5 Actin-active agents are attracting more attention in the field of cancer chemotherapy because microfilament and microtubule proteins form versatile dynamic polymers that can define cell polarity, organize cytoplasmic organelles, control cell shape and promote stable cell-cell and cell-matrix adhesions, and generate protrusive forces required for migration. 6-8 These functions usually fail and become abnormal in cancer cells. 6 Based on X-ray crystallography, the binding site of 1 has been located between subdomains II and IV, in the vicinity of the ATP binding cleft of the protein target in the actin monomer. 9, 10 The binding pharmacophores of 1 to G-actin were identified as follows: C-1 carbonyl oxygen through water to glutamate 214 carboxy, C-17 lactol hydroxyl to arginine 210 NH (major binding), C-17 pyran oxygen to tyrosine 69 hydroxy, thiazolidinone NH to aspartate 157 carboxy, and thiazolidinone C-20 carbonyl oxygen to threonine 186 hydroxy. 9, 10 Only the thiazolidinone NH group acts as a hydrogen bonding donor while the rest of the binding functions act as hydrogen bonding acceptors.9 , 10Semi-s...

Deficiency of factor X (F10) in humans is a rare bleeding disorder with a heterogeneous phenotype and limited therapeutic options. Targeted disruption of and other common pathway factors in mice results in embryonic/neonatal lethality with rapid resorption of homozygous mutants, hampering additional studies. Several of these mutants also display yolk sac vascular defects, suggesting a role for thrombin signaling in vessel development. The zebrafish is a vertebrate model that demonstrates conservation of the mammalian hemostatic and vascular systems. We have leveraged these advantages for in-depth study of the role of the coagulation cascade in the developmental regulation of hemostasis and vasculogenesis. In this article, we show that ablation of zebrafish by using genome editing with transcription activator-like effector nucleases results in a major embryonic hemostatic defect. However, widespread hemorrhage and subsequent lethality does not occur until later stages, with absence of any detectable defect in vascular development. We also use zebrafish to confirm 5 novel human variants as causative mutations in affected patients, providing a rapid and reliable in vivo model for testing the severity of variants. These findings as well as the prolonged survival of mutants will enable us to expand our understanding of the molecular mechanisms of hemostasis, including a platform for screening variants of uncertain significance in patients with F10 deficiency and other coagulation disorders. Further study as to how fish tolerate what is an early lethal mutation in mammals could facilitate improvement of diagnostics and therapeutics for affected patients with bleeding disorders.

Cessation of bleeding after trauma is a necessary evolutionary vertebrate adaption for survival. One of the major pathways regulating response to hemorrhage is the coagulation cascade, which ends with the cleavage of fibrinogen to form a stable clot. Patients with low or absent fibrinogen are at risk for bleeding. While much detailed information is known about fibrinogen regulation and function through studies of humans and mammalian models, bleeding risk in patients cannot always be accurately predicted purely based on fibrinogen levels, suggesting an influence of modifying factors and a need for additional genetic models. The zebrafish has orthologs to the three components of fibrinogen (fga, fgb, and fgg), but it hasn’t yet been shown that zebrafish fibrinogen functions to prevent bleeding in vivo. Here we show that zebrafish fibrinogen is incorporated into an induced thrombus, and deficiency results in hemorrhage. An Fgb-eGFP fusion protein is incorporated into a developing thrombus induced by laser injury, but causes bleeding in adult transgenic fish. Antisense morpholino knockdown results in intracranial and intramuscular hemorrhage at 3 days post fertilization. The observed phenotypes are consistent with symptoms exhibited by patients with hypo- and afibrinogenemia. These data demonstrate that zebrafish possess highly conserved orthologs of the fibrinogen chains, which function similarly to mammals through the formation of a fibrin clot.