p21-activated kinase 4 counteracts PKA-dependent lipolysis by phosphorylating FABP4 and HSL.

Adipose tissue lipolysis is mediated by cAMP-protein kinase A (PKA)-dependent intracellular signalling. Here, we show that PKA targets p21-activated kinase 4 (PAK4), leading to its protein degradation. Adipose tissue-specific overexpression of PAK4 in mice attenuates lipolysis and exacerbates diet-induced obesity. Conversely, adipose tissue-specific knockout of Pak4 or the administration of a PAK4 inhibitor in mice ameliorates diet-induced obesity and insulin resistance while enhancing lipolysis. Pak4 knockout also increases energy expenditure and adipose tissue browning activity. Mechanistically, PAK4 directly phosphorylates fatty acid-binding protein 4 (FABP4) at T126 and hormone-sensitive lipase (HSL) at S565, impairing their interaction and thereby inhibiting lipolysis. Levels of PAK4 and the phosphorylation of FABP4-T126 and HSL-S565 are enhanced in the visceral fat of individuals with obesity compared to their lean counterparts. In summary, we have uncovered an important role for FABP4 phosphorylation in regulating adipose tissue lipolysis, and PAK4 inhibition may offer a therapeutic strategy for the treatment of obesity.

Pan-Src kinase inhibitor treatment attenuates diabetic kidney injury via inhibition of Fyn kinase-mediated endoplasmic reticulum stress.

Src family kinases (SFKs) have been implicated in the pathogenesis of kidney fibrosis. However, the specific mechanism by which SFKs contribute to the progression of diabetic kidney disease (DKD) remains unclear. Our preliminary transcriptome analysis suggested that SFK expression was increased in diabetic kidneys and that the expression of Fyn (a member of the SFKs), along with genes related to unfolded protein responses from the endoplasmic reticulum (ER) stress signaling pathway, was upregulated in the tubules of human diabetic kidneys. Thus, we examined whether SFK-induced ER stress is associated with DKD progression. Mouse proximal tubular (mProx24) cells were transfected with Fyn or Lyn siRNA and exposed to high glucose and palmitate (HG-Pal). Streptozotocin-induced diabetic rats were treated with KF-1607, a novel pan-Src kinase inhibitor (SKI) with low toxicity. The effect of KF-1607 was compared to that of losartan, a standard treatment for patients with DKD. Among the SFK family members, the Fyn and Lyn kinases were upregulated under diabetic stress. HG-Pal induced p70S6 kinase and JNK/CHOP signaling and promoted tubular injury. Fyn knockdown but not Lyn knockdown inhibited this detrimental signaling pathway. In addition, diabetic rats treated with KF-1607 showed improved kidney function and decreased ER stress, inflammation, and fibrosis compared with those treated with losartan. Collectively, these findings indicate that Fyn kinase is a specific member of the SFKs implicated in ER stress activation leading to proximal tubular injury in the diabetic milieu and that pan-SKI treatment attenuates kidney injury in diabetic rats. These data highlight Fyn kinase as a viable target for the development of therapeutic agents for DKD.

p21-activated kinase 4 inhibition protects against liver ischemia/reperfusion injury: Role of nuclear factor erythroid 2-related factor 2 phosphorylation.

BACKGROUND AND AIMS: p21-activated kinase 4 (PAK4), an oncogenic protein, has emerged as a promising target for anticancer drug development. Its role in oxidative stress conditions, however, remains elusive. We investigated the effects of PAK4 signaling on hepatic ischemia/reperfusion (I/R) injury. APPROACH AND RESULTS: Hepatocyte- and myeloid-specific Pak4 knockout (KO) mice and their littermate controls were subjected to a partial hepatic I/R (HIR) injury. We manipulated the catalytic activity of PAK4, either through genetic engineering (gene knockout, overexpression of wild-type [WT] or dominant-negative kinase) or pharmacological inhibitor, coupled with a readout of nuclear factor erythroid 2-related factor 2 (Nrf2) activity, to test the potential function of PAK4 on HIR injury. PAK4 expression was markedly up-regulated in liver during HIR injury in mice and humans. Deletion of PAK4 in hepatocytes, but not in myeloid cells, ameliorated liver damages, as demonstrated in the decrease in hepatocellular necrosis and inflammatory responses. Conversely, the forced expression of WT PAK4 aggravated the pathological changes. PAK4 directly phosphorylated Nrf2 at T369, and it led to its nuclear export and proteasomal degradation, all of which impaired antioxidant responses in hepatocytes. Nrf2 silencing in liver abolished the protective effects of PAK4 deficiency. A PAK4 inhibitor protected mice from HIR injury. CONCLUSIONS: PAK4 phosphorylates Nrf2 and suppresses its transcriptional activity. Genetic or pharmacological suppression of PAK4 alleviates HIR injury. Thus, PAK4 inhibition may represent a promising intervention against I/R-induced liver injury.

X-ray Crystal Structure-Guided Design and Optimization of 7H-Pyrrolo[2,3-d]pyrimidine-5-carbonitrile Scaffold as a Potent and Orally Active Monopolar Spindle 1 Inhibitor.

Triple-negative breast cancer (TNBC) is an aggressive breast-cancer subtype associated with poor prognosis and high relapse rates. Monopolar spindle 1 kinase (MPS1) is an apical dual-specificity protein kinase that is over-expressed in TNBC. We herein report a highly selective MPS1 inhibitor based on a 7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile scaffold. Our lead optimization was guided by key X-ray crystal structure analysis. In vivo evaluation of candidate (9) is shown to effectively mitigate human TNBC cell proliferation.

KF-1607, a Novel Pan Src Kinase Inhibitor, Attenuates Obstruction-Induced Tubulointerstitial Fibrosis in Mice.

Src family kinases (SFKs), an important group of non-receptor tyrosine kinases, are suggested to be excessively activated during various types of tissue fibrosis. The present study investigated the effect of KF-1607, an orally active and a newly synthesized Src kinase inhibitor (SKI) with proposed low toxicity, in preventing the progression of renal interstitial fibrosis. Unilateral ureteral obstruction (UUO) surgery was performed in 6-week-old male C57BL/6 mice to induce renal interstitial fibrosis. Either KF-1607 (30 mg/kg, oral gavage) or PP2 (2 mg/kg, intraperitoneal injection), a common experimental SKI, was administered to mice for seven days, started one day prior to surgery. UUO injury-induced SFK expression, including Src, Fyn, and Lyn kinase. SFK inhibition by KF-1607 prevented the progression of tubular injury in UUO mice, as indicated by decreases in albuminuria, urinary KIM-1 excretion, and kidney NGAL protein expression. Renal tubulointerstitial fibrosis was attenuated in response to KF-1607, as shown by decreases in α-SMA, collagen I and IV protein expression, along with reduced Masson's trichrome and collagen-I staining in kidneys. KF-1607 also inhibited inflammation in the UUO kidney, as exhibited by reductions in F4/80 positive-staining and protein expression of p-NFκB and ICAM. Importantly, the observed effects of KF-1607 were similar to those of PP2. A new pan Src kinase inhibitor, KF-1607, is a potential pharmaceutical agent to prevent the progression of renal interstitial fibrosis.

Discovery of a potent dual ALK and EGFR T790M inhibitor.

The mutational activations of anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR) are validated oncogenic events and the targets of approved drugs to treat non-small cell lung cancer (NSCLC). Here we report highly potent dual small molecule inhibitors of both ALK and EGFR, particularly the T790M mutant which confers resistance to first generation EGFR inhibitors. Dual ALK/EGFR inhibitors may provide an efficient approach to prevent resistance that arises as a consequence of clinically reported reciprocal activation mechanisms. Our lead compound 7c displayed remarkable inhibitory activities against both ALK and EGFR in enzymatic and cellular assays. We demonstrate that 7c is capable of recapitulating the signaling effects and antiproliferative activity of combined treatment with the approved ALK inhibitor ceritinib and T790M EGFR inhibitor osimertinib against patient-derived non-small cell lung cancer cell line, DFCI032 which harbors both EML4-ALK and activated EGFR.

Suppression of the metastatic spread of breast cancer by DN10764 (AZD7762)-mediated inhibition of AXL signaling.

Breast cancer is the most common malignant disease occurring in women and represents a substantial proportion of the global cancer burden. In these patients, metastasis but not the primary tumor is the main cause of breast cancer-related deaths. Here, we report the novel finding that DN10764 (AZD7762, a selective inhibitor of checkpoint kinases 1 and 2) can suppress breast cancer metastasis. In breast cancer cells, DN10764 inhibited cell proliferation and GAS6-mediated AXL signaling, consequently resulting in suppressed migration and invasion. In addition, DN10764 induced caspase 3/7-mediated apoptosis in breast cancer cells and inhibited tube formation of human umbilical vein endothelial cells. Finally, DN10764 significantly suppressed the tumor growth and metastasis of breast cancer cells in in vivo metastasis models. Taken together, these data suggest that therapeutic strategies targeting AXL in combination with systemic therapies could improve responses to anti-cancer therapies and reduce breast cancer recurrence and metastases.

Enantioselective total synthesis of (-)-clavosolide B.

Enantioselective synthesis of 2, a revised structure for (-)-clavosolide B, was accomplished by a convergent approach, where syn-selective aldol, hydroxy-directed cyclopropanation, Mitsunobu inversion, Schmidt-type glycosylation, and macrolactonization reactions were utilized as key reactions. Comparison of 1H and 13C NMR spectra and optical rotation measurement confirmed the relative and absolute stereochemistry of clavosolide B (2).

Total synthesis, structural revision, and absolute configuration of (-)-clavosolide [corrected] A.

[reaction: see text] Enantioselective synthesis of 3, a revised structure for clavosolide A, was completed. Both (1)H and (13)C NMR spectra of the natural and synthetic compounds were identical, and optical rotation measurements identified the absolute configuration of the natural clavosolide A as [corrected] 3.