MKT 077

Selective Mitochondrial Uptake of MKT-077 Can Suppress Medullary Thyroid Carcinoma Cell Survival In Vitro and In Vivo.[Pubmed:26485469]

Endocrinol Metab (Seoul). 2015 Dec;30(4):593-603.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the rearranged during transfection (RET) proto-oncogene. Not all patients with progressive MTC respond to current therapy inhibiting RET, demanding additional therapeutic strategies. We recently demonstrated that disrupting mitochondrial metabolism using a mitochondria-targeted agent or by depleting a mitochondrial chaperone effectively suppressed human MTC cells in culture and in mouse xenografts by inducing apoptosis and RET downregulation. These observations led us to hypothesize that mitochondria are potential therapeutic targets for MTC. This study further tests this hypothesis using1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2- ylidenemethyl] pyridinium chloride (MKT-077), a water-soluble rhodocyanine dye analogue, which can selectively accumulate in mitochondria. METHODS: The effects of MKT-077 on cell proliferation, survival, expression of RET and tumor protein 53 (TP53), and mitochondrial activity were determined in the human MTC lines in culture and in mouse xenografts. RESULTS: MKT-077 induced cell cycle arrest in TT and MZ-CRC-1. Intriguingly, MKT-077 also induced RET downregulation and strong cell death responses in TT cells, but not in MZ-CRC-1 cells. This discrepancy was mainly due to the difference between the capacities of these cell lines to retain MKT-077 in mitochondria. The cytotoxicity of MKT-077 in TT cells was mainly attributed to oxidative stress while being independent of TP53. MKT-077 also effectively suppressed tumor growth of TT xenografts. CONCLUSION: MKT-077 can suppress cell survival of certain MTC subtypes by accumulating in mitochondria and interfering with mitochondrial activity although it can also suppress cell proliferation via other mechanisms. These results consistently support the hypothesis that mitochondrial targeting has therapeutic potential for MTC.

Synthesis and initial evaluation of YM-08, a blood-brain barrier permeable derivative of the heat shock protein 70 (Hsp70) inhibitor MKT-077, which reduces tau levels.[Pubmed:23472668]

ACS Chem Neurosci. 2013 Jun 19;4(6):930-9.

The molecular chaperone, heat shock protein 70 (Hsp70), is an emerging drug target for treating neurodegenerative tauopathies. We recently found that one promising Hsp70 inhibitor, MKT-077, reduces tau levels in cellular models. However, MKT-077 does not penetrate the blood-brain barrier (BBB), limiting its use as either a clinical candidate or probe for exploring Hsp70 as a drug target in the central nervous system (CNS). We hypothesized that replacing the cationic pyridinium moiety in MKT-077 with a neutral pyridine might improve its clogP and enhance its BBB penetrance. To test this idea, we designed and synthesized YM-08, a neutral analogue of MKT-077. Like the parent compound, YM-08 bound to Hsp70 in vitro and reduced phosphorylated tau levels in cultured brain slices. Pharmacokinetic evaluation in CD1 mice showed that YM-08 crossed the BBB and maintained a brain/plasma (B/P) value of approximately 0.25 for at least 18 h. Together, these studies suggest that YM-08 is a promising scaffold for the development of Hsp70 inhibitors suitable for use in the CNS.

Uptake rate of cationic mitochondrial inhibitor MKT-077 determines cellular oxygen consumption change in carcinoma cells.[Pubmed:22616013]

PLoS One. 2012;7(5):e37471.

OBJECTIVE: Since tumor radiation response is oxygen-dependent, radiosensitivity can be enhanced by increasing tumor oxygenation. Theoretically, inhibiting cellular oxygen consumption is the most efficient way to increase oxygen levels. The cationic, rhodacyanine dye-analog MKT-077 inhibits mitochondrial respiration and could be an effective metabolic inhibitor. However, the relationship between cellular MKT-077 uptake and metabolic inhibition is unknown. We hypothesized that rat and human mammary carcinoma cells would take up MKT-077, causing a decrease in oxygen metabolism related to drug uptake. METHODS: R3230Ac rat breast adenocarcinoma cells were exposed to MKT-077. Cellular MKT-077 concentration was quantified using spectroscopy, and oxygen consumption was measured using polarographic electrodes. MKT-077 uptake kinetics were modeled by accounting for uptake due to both the concentration and potential gradients across the plasma and mitochondrial membranes. These kinetic parameters were used to model the relationship between MKT-077 uptake and metabolic inhibition. MKT-077-induced changes in oxygen consumption were also characterized in MDA-MB231 human breast carcinoma cells. RESULTS: Cells took up MKT-077 with a time constant of approximately 1 hr, and modeling showed that over 90% of intracellular MKT-077 was bound or sequestered, likely by the mitochondria. The uptake resulted in a rapid decrease in oxygen consumption, with a time constant of approximately 30 minutes. Surprisingly the change in oxygen consumption was proportional to uptake rate, not cellular concentration. MKT-077 proved a potent metabolic inhibitor, with dose-dependent decreases of 45-73% (p = 0.003). CONCLUSIONS: MKT-077 caused an uptake rate-dependent decrease in cellular metabolism, suggesting potential efficacy for increasing tumor oxygen levels and radiosensitivity in vivo.

Analogs of the Allosteric Heat Shock Protein 70 (Hsp70) Inhibitor, MKT-077, as Anti-Cancer Agents.[Pubmed:24312699]

ACS Med Chem Lett. 2013 Nov 14;4(11).

The rhodacyanine, MKT-077, has anti-proliferative activity against cancer cell lines through its ability to inhibit members of the heat shock protein 70 (Hsp70) family of molecular chaperones. However, MKT-077 is rapidly metabolized, which limits its use as either a chemical probe or potential therapeutic. We report the synthesis and characterization of MKT-077 analogs designed for greater stability. The most potent molecules, such as 30 (JG-98), were at least 3-fold more active than MKT-077 against the breast cancer cell lines MDA-MB-231 and MCF-7 (EC50 values of 0.4 +/- 0.03 muM and 0.7 +/- 0.2 muM, respectively). The analogs modestly destabilized the chaperone "clients", Akt1 and Raf1, and induced apoptosis in these cells. Further, the microsomal half-life of JG-98 was improved at least 7-fold (t1/2 = 37 min) compared to MKT-077 (t1/2 < 5 min). Finally, NMR titration experiments suggested that these analogs bind an allosteric site that is known to accommodate MKT-077. These studies advance MKT-077 analogs as chemical probes for studying Hsp70's roles in cancer.

Pharmacological targeting of the Hsp70 chaperone.[Pubmed:19860737]

Curr Top Med Chem. 2009;9(15):1337-51.

The molecular chaperone, heat shock protein 70 (Hsp70), acts at multiple steps in a protein's life cycle, including during the processes of folding, trafficking, remodeling and degradation. To accomplish these various tasks, the activity of Hsp70 is shaped by a host of co-chaperones, which bind to the core chaperone and influence its functions. Genetic studies have strongly linked Hsp70 and its co-chaperones to numerous diseases, including cancer, neurodegeneration and microbial pathogenesis, yet the potential of this chaperone as a therapeutic target remains largely underexplored. Here, we review the current state of Hsp70 as a drug target, with a special emphasis on the important challenges and opportunities imposed by its co-chaperones, protein-protein interactions and allostery.

Rhodacyanine dye MKT-077 inhibits in vitro telomerase assay but has no detectable effects on telomerase activity in vivo.[Pubmed:12154051]

Cancer Res. 2002 Aug 1;62(15):4434-8.

MKT-077, a cationic rhodacyanine dye analogue, causes selective toxicity to cancer cells. Its cellular targets elucidated thus far include oncogenic Ras, F-actin, mortalin (hmot-2)/mthsp70, and telomerase. Here we report that MKT-077 causes growth arrest of cancer cells in culture independent of their Ras, p53, or telomerase status. Telomerase activity is inhibited in vitro by MKT-077 in the telomerase assay used. However, the in vivo toxicity seen in telomerase-positive cancer cells was not accompanied by inhibition of telomerase activity or telomere shortening. Furthermore, cells with an alternative mechanism for lengthening of telomeres were also sensitive to MKT-077 and showed enhanced formation of alternative mechanism for lengthening of telomeres-associated PML bodies in their nuclei. The data suggested that inhibition of telomerase activity is unlikely to be a prime cause of MKT-077-induced toxicity in cancer cells.

Selective toxicity of MKT-077 to cancer cells is mediated by its binding to the hsp70 family protein mot-2 and reactivation of p53 function.[Pubmed:11156371]

Cancer Res. 2000 Dec 15;60(24):6818-21.

MKT-077, a cationic rhodacyanine dye analogue has been under preclinical cancer therapeutical trials because of its selective toxicity to cancer cells. Its cellular targets and mechanism of action remain poorly understood. Here we report that MKT-077 binds to an hsp70 family member, mortalin (mot-2), and abrogates its interactions with the tumor suppressor protein, p53. In cancer cells, but not in normal cells, MKT-077 induced release of wild-type p53 from cytoplasmically sequestered p53-mot-2 complexes and rescued its transcriptional activation function. Thus, MKT-077 may be particularly useful for therapy of cancers with wild-type p53.

Treatment of Ras-induced cancers by the F-actin cappers tensin and chaetoglobosin K, in combination with the caspase-1 inhibitor N1445.[Pubmed:10526670]

Cancer J Sci Am. 1999 Sep-Oct;5(5):293-300.

UNLABELLED: For transforming normal fibroblasts to malignant cells, oncogenic Ras mutants such as v-Ha-ras require Rho family GTPases (Rho, Rac, and CDC42) that are responsible for controlling actin-cytoskeleton organization. Ras activates Rac through a PI-3 kinase-mediated pathway. Rac causes uncapping of actin filaments (F-actin) at the plus-ends, through phosphatidylinositol 4,5 bisphosphate (PIP2), and eventually induces membrane ruffling. Several distinct F-actin/PIP2-binding proteins, such as gelsolin, which severs and caps the plus-ends of actin filaments, or HS1, which cross-links actin filaments, have been shown to suppress v-Ha-Ras-induced malignant transformation when they are overexpressed. Interestingly, an F-actin cross-linking drug (photosensitizer) called MKT-077 suppresses Ras transformation. Thus, an F-actin capping/severing drug might also have an anticancer potential. PURPOSE: This study was conducted to determine first whether Ras-induced malignant phenotype (anchorage-independent growth) is suppressed by overexpression of the gene encoding a large plus-end F-actin capping protein called tensin and second to test the anti-Ras potential of a unique fungal antibiotic (small compound) called chaetoglobosin K (CK) that also caps the plus-ends of actin filaments. METHODS AND RESULTS: DNA transfection with a retroviral vector carrying the tensin cDNA was used to overexpress tensin in v-Ha-Ras-transformed NIH 3T3 cells. All stable tensin transfectants rarely formed colonies in soft agar, indicating that tensin suppresses the anchorage-independent growth. The anti-Ras action of CK was determined by incubating the Ras-transformants in the presence of CK in soft agar. Two microM CK almost completely inhibited their colony formation, indicating that CK also suppresses the malignant phenotype. However, unlike tensin, CK causes an apoptosis of Ras-transformed NIH 3T3 cells and, less effectively, of normal NIH 3T3 cells, indicating that CK has an F-actin capping-independent side effect(s). CK-induced apoptosis is at least in part caused by CK-induced inhibition of the kinase PKB/AKT. However, a specific ICE/caspase-1 inhibitor called N1445 completely abolished the CK-induced apoptosis by reactivating PKB, but without affecting the CK-induced suppression of Ras transformation. CONCLUSIONS: Like the F-actin cross-linking drug MKT-077, the F-actin capping drug CK may be useful for the treatment of Ras-associated cancers if it is combined with the ICE inhibitor N1445, which abolishes the side effect of CK. Our observations that two distinct F-actin capping molecules (i.e., tensin and CK) suppress Ras-induced malignant phenotype strongly suggest, if not prove, that capping of actin filaments at the plus-ends alone is sufficient to block one of the Ras signaling pathways essential for its oncogenicity. This notion is compatible with the fact that Ras induces the uncapping of actin filaments at the plus-ends through the Rac/PIP2 pathway.