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.

Islet macrophages are associated with islet vascular remodeling and compensatory hyperinsulinemia during diabetes.

ß-Cells respond to peripheral insulin resistance by first increasing circulating insulin during diabetes. Islet remodeling supports this compensation, but its drivers remain poorly understood. Infiltrating macrophages have been implicated in late-stage type 2 diabetes, but relatively little is known on islet resident macrophages, especially during compensatory hyperinsulinemia. We hypothesized that islet resident macrophages would contribute to islet vascular remodeling and hyperinsulinemia during diabetes, the failure of which results in a rapid progression to frank diabetes. We used chemical (clodronate), genetics (CD169-diphtheria toxin receptor mice), or antibody-mediated (colony-stimulating factor 1 receptor α) macrophage ablation methods in diabetic (db/db) and diet-induced models of compensatory hyperinsulinemia to investigate the role of macrophages in islet remodeling. We transplanted islets devoid of macrophages into naïve diabetic mice and assessed the impact on islet vascularization. With the use of the above methods, we showed that macrophage depletion significantly and consistently compromised islet remodeling in terms of size, vascular density, and insulin secretion capacity. Depletion of islet macrophages reduced VEGF-A secretion in both human and mouse islets ex vivo, and this functionally translated to delayed revascularization upon transplantation in vivo. We revealed that islet resident macrophages were associated with islet remodeling and increased insulin secretion during diabetes. This suggests utility in harnessing islet macrophages during this phase to promote islet vascularization, remodeling, and insulin secretion.

Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

Epigenetic mediation through bromodomain and extraterminal (BET) proteins have progressively translated protein imbalance into effective cancer treatment. Perturbation of druggable BET proteins through proteolysis-targeting chimeras (PROTACs) has recently contributed to the discovery of effective therapeutics. Unfortunately, precise and microenvironment-activatable BET protein degradation content with promising tumor selectivity and pharmacological suitability remains elusive. Here, we present an enzyme-derived clicking PROTACs (ENCTACs) capable of orthogonally cross-linking two disparate small-molecule warhead ligands that recognize BET bromodomain-containing protein 4 (BRD4) protein and E3 ligase within tumors only upon hypoxia-induced activation of nitroreductase enzyme. This localized formation of heterobifunctional degraders promotes specific down-regulation of BRD4, which subsequently alters expression of epigenetic targets and, therefore, allows precise modulation of hypoxic signaling in live cells, zebrafish, and living mice with solid tumors. Our activation-feedback system demonstrates compelling superiorities and may enable the PROTAC technology with more flexible practicality and druggable potency for precision medicine in the near future.

Collaborative Regulation of LRG1 by TGF-ß1 and PPAR-ß/δ Modulates Chronic Pressure Overload-Induced Cardiac Fibrosis.

BACKGROUND: Despite its established significance in fibrotic cardiac remodeling, clinical benefits of global inhibition of TGF (transforming growth factor)-ß1 signaling remain controversial. LRG1 (leucine-rich-α2 glycoprotein 1) is known to regulate endothelial TGFß signaling. This study evaluated the role of LRG1 in cardiac fibrosis and its transcriptional regulatory network in cardiac fibroblasts. METHODS: Pressure overload-induced heart failure was established by transverse aortic constriction. Western blot, quantitative reverse transcription polymerase chain reaction, immunofluorescence, and immunohistochemistry were used to evaluate the expression level and pattern of interested targets or pathology during fibrotic cardiac remodeling. Cardiac function was assessed by pressure-volume loop analysis. RESULTS: LRG1 expression was significantly suppressed in left ventricle of mice with transverse aortic constriction-induced fibrotic cardiac remodeling (mean difference, -0.00085 [95% CI, -0.0013 to -0.00043]; P=0.005) and of patients with end-stage ischemic-dilated cardiomyopathy (mean difference, 0.13 [95% CI, 0.012-0.25]; P=0.032). More profound cardiac fibrosis (mean difference, -0.014% [95% CI, -0.029% to -0.00012%]; P=0.048 for interstitial fibrosis; mean difference, -1.3 [95% CI, -2.5 to -0.2]; P=0.016 for perivascular fibrosis), worse cardiac dysfunction (mean difference, -2.5 ms [95% CI, -4.5 to -0.4 ms]; P=0.016 for Tau-g; mean difference, 13% [95% CI, 2%-24%]; P=0.016 for ejection fraction), and hyperactive TGFß signaling in transverse aortic constriction-operated Lrg1-deficient mice (mean difference, -0.27 [95% CI, -0.47 to -0.07]; P<0.001), which could be reversed by cardiac-specific Lrg1 delivery mediated by adeno-associated virus 9. Mechanistically, LRG1 inhibits cardiac fibroblast activation by competing with TGFß1 for receptor binding, while PPAR (peroxisome proliferator-activated receptor)-ß/δ and TGFß1 collaboratively regulate LRG1 expression via SMRT (silencing mediator for retinoid and thyroid hormone receptor). We further demonstrated functional interactions between LRG1 and PPARß/δ in cardiac fibroblast activation. CONCLUSIONS: Our results established a highly complex molecular network involving LRG1, TGFß1, PPARß/δ, and SMRT in regulating cardiac fibroblast activation and cardiac fibrosis. Targeting LRG1 or PPARß/δ represents a promising strategy to control pathological cardiac remodeling in response to chronic pressure overload.