FMK

FMK is a potent, highly specific and irreversible ribosomal s6 kinase (RSK) inhibitor with IC50 value of 15 nM.[1]
RSK is named for ribosomal protein s6, part of the translational machinery which are serine/threonine kinases and are activated by the MAPK/ERK pathway. There are two subfamilies of RSK, p90rsk, also known as MAPK-activated protein kinase-1 (MAPKAP-K1), and p70rsk, also known as S6-H1 Kinase or simply S6 Kinase.90 kDa ribosomal S6 kinases(p90rsk),the first substrates of ERK,is a ubiquitous and versatile mediator of ERK signal transduction which regulates protein synthesis by phosphorylation of polyribosomal proteins and glycogen synthase kinase-3 and phosphorylates the Ras GTP/GDP-exchange factor, leading to feedback inhibition of the Ras-ERK pathway.[2]
FMK is an irreversible inhibitor that covalently modifies the C-terminal kinase domain of RSK. It therefore prevents the activation of the N-terminal kinase domain of RSK by the C-terminal kinase domain, but does not affect the activity of the N-terminal domain, explaining why the active forms of RSK1 and RSK2 are not inhibited by FMK in vitro.[3] FMK,the efficacy and specificity of the irreversible RSK inhibitor in ARVMs and subsequently is used to determine the role of RSK as a direct regulator of NHE1 phosphorylation and sarcolemmal NHE activity in this cell type, in response to  1-adrenergic stimulation.[4] FMK-pa, a propargylamine variant, achieves selective and saturable modification of endogenous RSK1 and RSK2 in mammalian cells. Saturating concentrations of fmk-pa inhibited Ser386 phosphorylation and downstream signaling in response to phorbol ester stimulation, but had no effect on RSK activation by lipopolysaccharide.[5]
Metastasis, the spreading of cancer cells from a primary tumor to secondary sites throughout the body, is driven by altered signaling pathways that induce changes in cell-cell adhesion, the cytoskeleton, integrin function, protease expression, epithelial-to-mesenchymal transition and cell survival.[6] The ribosomal S6 kinase (RSK) family of kinases is a group of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) effectors that can regulate these steps of metastasis by phosphorylating both nuclear and cytoplasmic targets. Thus, developing RSK inhibitors as new antimetastasis drugs is urgent.Recently, RSKs have been shown to mediate many cellular functions critical for cancer progression. RSK may be an important driver in PCa progression in bone which regulates anchorage-independent growth through transcriptional regulation of factors that modulate cell survival, including ING3, CKAP2 and PTK6,resulting in promising potential as a therapeutic target for PCa bone metastasis.[7]
Reference:
1.Michael S. Cohen. et al. Structural Bioinformatics-Based Design of Selective, Irreversible Kinase Inhibitors. Science,2005,308(5726):1318-1321.
2.Frodin M, Gammeltoft S. Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction. Mol Cell Endocrinol. 1999, 151(1-2):65-77.
3.Jenny BAIN.et al. The selectivity of protein kinase inhibitors: a further update. Biochem. J. 2007, 408:297-315.
4.Cuello F.et al. Evidence for direct regulation of myocardial Na+/H+ exchanger isoform 1 phosphorylation and activity by 90-kDa ribosomal S6 kinase (RSK): effects of the novel and specific RSK inhibitor fmk on responses to alpha1-adrenergic stimulation. Mol Pharmacol. 2007, 71(3):799-806.
5.Michael S Cohen, Haralambos Hadjivassiliou1, and Jack Taunton. A clickable inhibitor reveals context-dependent autoactivation of p90 RSK. Nat Chem Biol. 2007,3(3): 156-160.
6.Sulzmaier FJ1, Ramos JW. RSK isoforms in cancer cell invasion and metastasis.Cancer Res. 2013,73(20):6099-105.
7.Yu G, Lee YC1. et al. RSK Promotes Prostate Cancer Progression in Bone through ING3, CKAP2 and PTK6-mediated Cell Survival. Mol Cancer Res.2014,0384.

Caspase-9 inhibitor Z-LEHD-FMK enhances the yield of in vitro produced buffalo (Bubalus bubalis) pre-implantation embryos and alters cellular stress response.[Pubmed:26850530]

Res Vet Sci. 2016 Feb;104:4-9.

The present investigation was done to study the effect of caspase-9 inhibitor Z-LEHD-FMK, on in vitro produced buffalo embryos. Z-LEHD-FMK is a cell-permeable, competitive and irreversible inhibitor of enzyme caspase-9, which helps in cell survival. Buffalo ovaries were collected from slaughterhouse and the oocytes were subjected to in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). The culture medium was supplemented with Z-LEHD-FMK at different concentrations i.e. 0 muM (control), 10 muM, 20 muM, 30 muM and 50 muM during IVM and IVC respectively. After day-2 post-insemination, the cleavage rate was significantly higher (74.20 +/- 5.87% at P<0.05) in the group treated with 20 muM of Z-LEHD-FMK than at any other concentration. Same trend was observed in the blastocyst production rate which was higher at 20 muM (27.42 +/- 2.94% at P<0.05). The blastocysts obtained at day-8 of the culture at different concentrations were subjected to TUNEL assay, to determine the level of apoptosis during the culture medium supplied with 20 muM Z-LEHD-FMK which showed apoptotic index significantly lower (1.88 +/- 0.87 at P<0.05). There was a non-significant increase in total cell number in all Z-LEHD-FMK treated blastocysts. The quantitative gene expression of CHOP and HSP10 genes showed significant increase (P<0.05) in the group treated with 50 muM Z-LEHD-FMK, while, HSP40 showed significant increase (P<0.05) at 30 muM and 50 muM Z-LEHD-FMK concentrations. From the afore mentioned results we conclude that, Z-LEHD-FMK at 20 muM increased the cleavage and blastocyst rate of buffalo pre-implantation embryos also affecting the rate of apoptosis and cellular stress at various concentrations.

Effects of a broad-spectrum caspase inhibitor, Z-VAD(OMe)-FMK, on viral hemorrhagic septicemia virus (VHSV) infection-mediated apoptosis and viral replication.[Pubmed:26899629]

Fish Shellfish Immunol. 2016 Apr;51:41-45.

In the development of inactivated or attenuated viral vaccines for cultured fish, viral titers harvested from the cultured cells would be the most important factor for the determination of vaccine's cost effectiveness. In this study, we hypothesized that the lengthening of cell survival time by the inhibition of apoptosis can lead to an increase of the final titer of viral hemorrhagic septicemia virus (VHSV). To test the hypothesis, we investigated the effects of a broad-spectrum caspase inhibitor, Z-VAD(OMe)-FMK, on VHSV infection-mediated apoptosis in Epithelioma papulosum cyprini (EPC) cells and on the VHSV titers. VHSV infection induced the DNA laddering in EPC cells, and the progression of DNA fragmentation was in proportion to the CPE extension. The progression of DNA fragmentation in EPC cells infected with VHSV was clearly inhibited by exposure to Z-VAD(OMe)-FMK, and the inhibition was intensified according to the increase of the inhibitor concentration. These results confirmed the previous reports that the death of host cells by VHSV infection is through apoptosis. Cells infected with a recombinant VHSV, rVHSV-DeltaNV-eGFP, that was generated from our previous study by replacement of the NV gene ORF with the enhanced green fluorescent protein (eGFP) gene ORF, showed earlier and more distinct DNA fragmentations compared to the cells infected with wild-type VHSV, suggesting the inhibitory role of the NV protein in VHSV-mediated apoptosis that was previously reported. The final viral titers in the supernatant isolated from Z-VAD(OMe)-FMK treated cells after showing an extensive CPE were significantly higher than the viral titers from cells infected with virus alone, indicating that the delay of apoptosis by Z-VAD(OMe)-FMK extended the survival time of EPC cells, which lengthen the time for VHSV replication in the cells. In conclusion, Z-VAD(OMe)-FMK-mediated inhibition of apoptosis significantly increased the final titers of both wild-type VHSV and rVHSV-DeltaNV-eGFP, indicating that apoptosis inhibition can be a way to get higher titers of VHSV.

Caspase inhibitor zVAD-fmk protects against acute pancreatitis-associated lung injury via inhibiting inflammation and apoptosis.[Pubmed:27324074]

Pancreatology. 2016 Sep-Oct;16(5):733-8.

BACKGROUND/OBJECTIVES: Pulmonary apoptosis is an important pathogenic mechanism of acute lung injury induced by many factors. This study aims to investigate whether the caspase inhibitor zVAD-FMK has a protective effect against lung injury in the severe acute pancreatitis model (SAP) in rats. METHODS: Seventy-two Sprague-Dawley rats were randomly divided into Sham, SAP, and SAP + zVAD-FMK groups. The SAP model was established by injection of 5% sodium taurocholate into the pancreatic duct. Animals were sacrificed at 3 h, 6 h, 12 h, and 24 h after operation and then HE staining analysis was performed to assess the lung injury. ELISA was used to detect the activity of myeloperoxidase (MPO) and the concentrations of tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta). Western blotting was used to detect the expression of cleaved caspase-3 in the lung tissues. RESULTS: Rats in SAP group showed obvious lung injury through pathologic examination. Pretreatment with zVAD-FMK significantly inhibited a post-SAP increase in the activation of MPO, TNF-alpha, IL-1beta, and caspase-3, and decreased lung injury induced by SAP as determined by the pathologic score. CONCLUSION: Our results suggest that apoptosis plays an important role in acute pancreatitis-associated lung injury (APALI), and inhibition of caspase activity may represent a new therapeutic approach for the treatment of APALI.

Positive effect of apoptotic inhibitor z-vad-fmk on vitrified-thawed porcine mii stage oocytes.[Pubmed:27393955]

Cryo Letters. 2016 May-Jun;37(3):188-95.

BACKGROUND: The developmental potential of vitrified porcine oocytes is very lower, and apoptosis is considered as one of the key factors involved. OBJECTIVE: To investigate the effects of apoptotic inhibitor Z-VAD-FMK addition into the incubation medium after warming on apoptosis and developmental ability of vitrified porcine MII-stage oocytes. MATERIALS AND METHODS: The activities of several caspases, mitochondrial membrane potential (DeltaPsim) and early apoptotic levels were measured. Parthenogenetic developmental ability and relative expression levels of apoptosis related genes were also detected. RESULTS: Caspase activity and early apoptotic level of the Z-VAD-FMK group were significantly lower than those of the group without Z-VAD-FMK addition, but were much higher than those of fresh group (P < 0.05). The DeltaPsim of Z-VAD-FMK group was 1.19, higher than the vitrified group (0.91) and lower than the fresh group (1.33). The cleavage rate and blastocyst rate after parthenogenetic activation in the Z-VAD-FMK group were much higher than those in the vitrified group, and much lower than those in the fresh group (P < 0.05). Vitrified porcine oocytes exhibited increased expression of pro-apoptotic genes (caspase 3, 8, 9, TNF-alpha) and decreased genes expression levels of anti-apoptotic genes (Bcl-2, CuZnSOD), and the Z-VAD-FMK addition in incubaiton medium significantly decreased the transcripts levels of caspase 3,8,9, Bax, TNF-alpha and increased Bcl-2 and CuZnSOD genes expression. CONCLUSION: The addition of apoptotic inhibitor Z-VAD-FMK into the incubation medium after warming improved the in vitro developmental ability of vitrified porcine oocytes by increasing mitochondrial function, reducing apoptotic level and changing apoptosis-elated gene expression.