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Targeted inhibition of the molecular chaperone Hsp90 results in the simultaneous blockade of multiple oncogenic signaling pathways and has, thus, emerged as an attractive strategy for the development of novel cancer therapeutics. Ganetespib (formerly known as STA-9090) is a unique resorcinolic triazolone inhibitor of Hsp90 that is currently in clinical trials for a number of human cancers. In the present study, we showed that ganetespib exhibits potent in vitro cytotoxicity in a range of solid and hematologic tumor cell lines, including those that express mutated kinases that confer resistance to small-molecule tyrosine kinase inhibitors. Ganetespib treatment rapidly induced the degradation of known Hsp90 client proteins, displayed superior potency to the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), and exhibited sustained activity even with short exposure times. In vivo, ganetespib showed potent antitumor efficacy in solid and hematologic xenograft models of oncogene addiction, as evidenced by significant growth inhibition and/or regressions. Notably, evaluation of the microregional activity of ganetespib in tumor xenografts showed that ganetespib was efficiently distributed throughout tumor tissue, including hypoxic regions >150 μm from the microvasculature, to inhibit proliferation and induce apoptosis. Importantly, ganetespib showed no evidence of cardiac or liver toxicity. Taken together, this preclinical activity profile indicates that ganetespib may have broad application for a variety of human malignancies, and with select mechanistic and safety advantages over other first- and second-generation Hsp90 inhibitors. Mol Cancer Ther; 11(2); 475–84. ©2011 AACR.

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KRAS is the predominantly mutated RAS isoform, accounting for 85% of RAS mutations in cancers. KRAS G12C mutation occurs in approximately 14% of lung adenocarcinomas and 3% of colorectal adenocarcinomas. Several drugs have been developed that inhibit the function of KRAS proteins with a “G12C” mutation. The response rate to these drugs in patients with KRAS G12C-mutated NSCLC is approximately 40%. Primary resistance to G12C inhibitors in these patients may be explained in part by a lack of dependency on KRAS signaling. Despite the clinical benefit observed in some patients treated with G12C inhibitors, acquired resistance to single-agent inhibitor therapy eventually develops in most patients. KRAS G12C-mutated NSCLC cell lines treated with G12C- specific inhibitor demonstrated reactivation of the down-stream MAPK pathway as a mechanism of acquired resistance. As a terminal kinase in this pathway, inhibition of ERK1/2 activity could potentially be instrumental in overcoming G12C inhibitor resistance. Moreover, aurora kinase A (AURKA) has been identified as necessary for cancer cell escape from G12C inhibitor-induced quiescence, which may be due to a stabilizing reaction between AURKA, KRAS G12C and the downstream effector CRAF. JSI-1187 is an oral reversible ERK1/2 inhibitor, and VIC-1911 is an oral selective AURKA inhibitor. To evaluate the effects of the concomitant inhibition of JSI-1187 or VIC-1911 with sotorasib, we assessed the response in 4 G12C-mutated cell lines, including two NSCLC and two CRC. Loewe synergy scores calculated showed values between 0.5 to 14.3 in these cell lines, which signified a synergistic effect of JSI-1187 or VIC-1911 combined with sotorasib. JSI-1187 or VIC-1911 combined with sotorasib or adagrasib demonstrated marked enhancement of the antitumor effect compared with single-agent treatment in a H2122 mouse xenograft model. Additionally, the antitumor growth effect of JSI-1187 and sotorasib was significantly better than the combination of the MEK inhibitor, trametinib, and sotorasib. Greater inhibition of tumor growth was also observed for the combination vs. monotherapy in H358 and SW837 CRC- bearing mouse models. Furthermore, KRAS G12C-mutated patient-derived xenografts were used to investigate a synergistic effect. Combined treatment with VIC-1911 and sotorasib demonstrated greater tumor growth inhibition than either monotherapy alone in a NSCLC PDX model. In addition, the combination of JSI-1187 and sotorasib demonstrated a significant increase in tumor growth inhibition in a CRC PDX model. Taken together, our preclinical studies suggest that the combination of an ERK inhibitor, JSI-1187, or an AURKA inhibitor, VIC-1911, may potentially overcome the primary resistance and prevent or delay the acquired resistance to G12C inhibitors. Citation Format: Lei Zhang, Ao Li, Shanzhong Jian, Chengyi Du, Wan Zhu, Yue Tao, Meiwei Wang, Wen Xu, Wenshan Hao, Linda Paradiso, Thomas Myers, Keizo Koya, Jintao Zhang. Efficacy of JSI-1187 or VIC-1911 in combination with KRAS G12C inhibitors for the treatment of KRAS G12C-mutated non-small cell lung cancer and colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 429.

For secreted proteins, proper protein folding is essential not only for biological function but also for secretion itself. Proteins with folding problems are trapped in the endoplasmic reticulum (ER) and are eventually degraded in the cytoplasm. In this study, we exploited co-expression of an artificial fusion protein, based on the sequence of a DnaJ protein, which could interact as co-chaperones in the Hsp70-based protein-folding system, with target recombinant secreted proteins to enhance their production and secretion. The J-domain sequence or a fragment thereof was conjugated to a target protein–binding domain that was capable of binding to a portion of the target-protein sequence. Production of many of the target proteins was significantly upregulated when co-expressed with the J-domain fusion protein. Surprisingly, the enhancement of secretion was observed even when the J-domain had a mutation in the HPD motif, which is necessary for J-protein–Hsp70 interactions, suggesting the phenomenon observed is independent on functional J-protein–Hsp70 interactions. This technology has great potential for not only enhancing the production of recombinant proteins, but also to treat conformational diseases such as cystic fibrosis, and Alpha-1 antitrypsin deficiency.

A facile and convenient synthetic method for the anticancer agent elesclomol and its analogues, N′1,N′3-dialkyl-N′1,N′3-bis(arylcarbonothioyl)malonohydrazides, by the direct coupling of N-alkyl-N-(substituted)benzothiohydrazides and substituted malonic acids using propylphosphonic anhydride (T3P®) under very mild conditions has been developed.

Elesclomol is a novel anticancer agent that has been evaluated in a number of late stage clinical trials. A new and convenient synthesis of elesclomol and its copper complex is described. X-ray crystallographic characterization and the electrochemical properties of the elesclomol copper(II) complex are discussed. The copper(II) cation is coordinated in a highly distorted square-planar geometry to each of the sulphur and amide nitrogen atoms of elesclomol. Electrochemical measurements demonstrate that the complex undergoes a reversible one-electron reduction at biologically accessible potentials. In contrast the free elesclomol is found electrochemically inactive. This evidence is in strong support of the mechanism of action we proposed for the anticancer activity of elesclomol.

In human trials certain heat shock protein 90 (Hsp90) inhibitors, including 17-DMAG and NVP-AUY922, have caused visual disorders indicative of retinal dysfunction; others such as 17-AAG and ganetespib have not. To understand these safety profile differences we evaluated histopathological changes and exposure profiles of four Hsp90 inhibitors, with or without clinical reports of adverse ocular effects, using a rat retinal model. Retinal morphology, Hsp70 expression (a surrogate marker of Hsp90 inhibition), apoptotic induction and pharmacokinetic drug exposure analysis were examined in rats treated with the ansamycins 17-DMAG and 17-AAG, or with the second-generation compounds NVP-AUY922 and ganetespib. Both 17-DMAG and NVP-AUY922 induced strong yet restricted retinal Hsp70 up-regulation and promoted marked photoreceptor cell death 24h after the final dose. In contrast, neither 17-AAG nor ganetespib elicited photoreceptor injury. When the relationship between drug distribution and photoreceptor degeneration was examined, 17-DMAG and NVP-AUY922 showed substantial retinal accumulation, with high retina/plasma (R/P) ratios and slow elimination rates, such that 51% of 17-DMAG and 65% of NVP-AUY922 present at 30min post-injection were retained in the retina 6h post-dose. For 17-AAG and ganetespib, retinal elimination was rapid (90% and 70% of drugs eliminated from the retina at 6h, respectively) which correlated with lower R/P ratios. These findings indicate that prolonged inhibition of Hsp90 activity in the eye results in photoreceptor cell death. Moreover, the results suggest that the retina/plasma exposure ratio and retinal elimination rate profiles of Hsp90 inhibitors, irrespective of their chemical class, may predict for ocular toxicity potential.

A series of N ′ 1 , N ′ 3 -dialkyl- N ′ 1 , N ′ 3 -di(alkylcarbonothioyl) malonohydrazides have been designed and synthesized as anticancer agents by targeting oxidative stress and Hsp70 induction. Structure–activity relationship (SAR) studies lead to the discovery of STA-4783 (elesclomol), a novel small molecule that has been evaluated in a number of clinical trials as an anticancer agent in combination with Taxol.

Among heat shock proteins, mortalin has been linked to the pathogenesis of Parkinson's disease. In the present work a rat model of Parkinson's disease was used to analyze the expression of striatal proteins and, more specifically, mortalin expression. The possible involvement of mortalin in Parkinson's disease pathogenesis was further investigated by utilizing an electrophysiological approach and pharmacological inhibition of mortalin in both the physiological and the parkinsonian states. Proteomic analysis was used to investigate changes in striatal protein expression in the 6-hydroxydopamine rat model of Parkinson's disease. The electrophysiological effects of MKT-077, a rhodamine-123 analogue acting as an inhibitor of mortalin, were measured by field potential recordings from corticostriatal brain slices obtained from control, sham-operated, and 6-hydroxydopamine–denervated animals. Slices in the presence of rotenone, an inhibitor of mitochondrial complex I, were also analyzed. Proteomic analysis revealed downregulation of mortalin in the striata of 6-hydroxydopamine–treated rats in comparison with sham-operated animals. MKT-077 reduced corticostriatal field potential amplitude in physiological conditions, inducing membrane depolarization and inward current in striatal medium spiny neurons. In addition, we observed that concentrations of MKT-077 not inducing any electrophysiological effect in physiological conditions caused significant changes in striatal slices from parkinsonian animals as well as in slices treated with a submaximal concentration of rotenone. These findings suggest a critical link between mortalin function and mitochondrial activity in both physiological and pathological conditions mimicking Parkinson's disease. © 2011 Movement Disorder Society

A series of novel indolizine 2-oxoacetamides were designed and synthesized as PDE4 inhibitors. Preliminary SAR of this new class of compounds revealed key structural features required for high potency. Compounds 1ab and 2a are among the most potent inhibitors of PDE4 with low single nM IC50. Cellular activity was demonstrated by the inhibition of TNFα production from human PBMC with IC50 ranging from 14 to 72 nM. Docking analyses suggest the OH group in 1ab enhance the binding via an H-bond interaction with the PDE4 enzyme.

A number of 3-acylated indolizines were synthesized in good to excellent yields by a newly established reaction between picolinium salts and the methylsulfate salt of A (R = Me), the adduct formed from DMF–Me2SO4 as the key reagent. The low cost, short reaction time, mild reaction condition, and easy purification of the products make this an attractive new method for the synthesis of indolizine compounds. A variety of functional groups (nitro, cyano, ester, methoxy, and halogens) were well tolerated under the reaction conditions.

An efficient synthesis of the novel microtubule inhibitor STA-5312 (3-[(4-cyanophenyl)methyl]-N-(3-methyl-5-isothiazolyl)-α-oxo-1-indolizineacetamide) was developed. A novel DMF/Me2SO4 directed regioselective synthesis of the 3-(4-cyanobenzoyl)-indolizine (4) was a critical transformation within the four-step process. Alternatively, a CuCl mediated synthesis of 3-(4-cyanobenzyl)indolizine (5) was also developed. All intermediates were obtained in high quality and were used directly for the next step without extensive purification. The drug substance itself was purified by recrystallization from a mixture of THF and water, resulting in a high purity product (HPLC >98%). The process was applied successfully in the manufacturing of kilograms of GMP API.

A facile and convenient synthetic method for the anticancer agent elesclomol and its analogues, N′1,N′3-dialkyl-N′1,N′3-bis(arylcarbonothioyl)malonohydrazides, by the direct coupling of N-alkyl-N-(substituted)benzothiohydrazides and substituted malonic acids using propylphosphonic anhydride (T3P®) under very mild conditions has been developed.

Background. MKT-077, a delocalized lipophilic cation, accumulates in mitochondria according to the negative potential inside the mitochondria and exerts cytotoxicity. We investigated the antitumor activity and spectrum of MKT-077 in fresh surgical specimens. Methods. Single cell suspensions of fresh surgical specimens obtained from patients with gastric, colon, and hepatocellular carcinoma were treated with MKT-077 at a cutoff concentration of 30 μg/ml. Cell viability was detected by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. Results. When the cutoff concentration was 30 μg/ml, the efficacy rates (sensitive cases/evaluated cases in percent) of MKT-077 were 52.6% for gastric cancer, 77.8% for colon cancer, and 35.7% for hepatocellular carcinoma, resulting in an overall efficacy rate of 52.4% (22/42). This was higher than the rates for other agents, including mitomycin C, doxorubicin, 5-fluorouracil, and cisplatin. The antitumor spectrum of MKT-077 was correlated with that of doxorubicin, while the antitumor spectra of mitomycin C, 5-fluorouracil, and cisplatin were completely independent of that of MKT-077. Conclusion. Since MKT-077 seemed to have a different antitumor spectrum from that of the conventionally available agents, further investigation should be conducted, including studies of the combined effect of MKT-077 and other antitumor agents.

MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4- oxothiazolidin-2-ylidenemethyl] pyridinium chloride), a novel rhodacyanine dye in phase I/II clinical trials, may provide a new approach to cancer therapy based on the accumulation in the mitochondria of the cells of certain carcinomas, for example, those of the colon, breast and pancreas. To support the development of MKT-077 for clinical application as an intravenous (i.v.) therapy, we investigated the metabolic fate of [14C]MKT-077 in BDF1 mice as well as the distribution of MKT-077 in experimental LS174T tumor-bearing mice using a high-performance liquid chromatography (HPLC) method. The plasma levels of 14C after i.v. administration of [14C]MKT-077 declined in a triphasic manner. In the first distribution phase, the levels of 14C decreased with a T1/2 of approximately 5 min. In the second and terminal phase, the T1/2 of 14C was 2.8-4.6 h and 16.2 h, respectively. Cmax (1 min after injection) increased from 0.3 to 1.5 microg/ml linearly, but less than proportionately between the doses. The AUC(0-infinity) at 0.3, 1 and 3 mg/kg were 0.030 +/- 0.002, 0.60 +/- 0.12 and 1.73 +/- 0.25 microg x h/ml, respectively. Plasma clearance was approximately 1.8 l/h per kg (at doses of 1 and 3 mg/kg). The steady state volume of distribution (6.8 and 25.1 l/kg) indicated that MKT-077 distributed as a lipid-soluble molecule. The mean residence time (MRT) was 4.1 (at a dose of 1 mg/kg) and 14.1 h (at a dose of 3 mg/kg). In the first rapid phase (5 min after dosing), 14C radioactivity was detected in most of the tissues and organs, most strongly in the kidney cortex, and not in the central nervous system and testes. In the terminal phase (24 h after dosing), 14C contents increased in the intestinal tract, and in the kidney and liver were nearly to the background level. After i.v. bolus administration at a dose of 3 mg/kg of [14C]MKT-077, the predominant route of elimination of the radioactivity was via the feces, and recoveries of total radioactivity in urine and feces corresponded to 33.5% and 61.1%, respectively. More than 60% was recovered within 24 h and 95% within 1 week. MKT-077 was primarily excreted in unmetabolized form with five unidentified metabolites found in the urine and plasma. Intact MKT-077 was retained in the tumor tissue longer than in plasma and kidney in LS174T tumor-bearing mice receiving MKT-077 at an i.v. therapeutic dose (10 mg/kg). This accumulation decreased very slowly, suggesting that the high membrane potentials of tumor cell mitochondria may help retain the drug in tumors.

We have previously reported that rhodacyanine dyes, such as 1 and 2, exhibited a potent inhibitory effect on the growth of several tumor cells and that 4-oxothiazolidine (rhodanine) was an essential moiety for antitumor activity. On the basis of our foregoing work, two types of rhodacyanine dyes, which categorized into class I and II depending on the methine length, were synthesized and evaluated as a novel antitumor agent. Attention was particularly focused on the structure-activity study of two heteroaromatic rings. In class I, where the A rings were conjugated to rhodanine via two methine groups, compounds 1, 20, 23, and 24 were found to be efficacious in tumor-bearing nude mice model study, but they did not have the chemical properties (stability, solubility) suitable for clinical use. In contrast, in class II, where the A rings were directly conjugated to rhodanine, compounds 13 and 25, which possessed a benzothiazole moiety for the A ring, exhibited the favorable biological and chemical properties. Therefore, we decided to have a benzothiazole moiety as the A ring and introduce various heterocyclic groups for the B ring. As a result, the pyridinium ring was selected as the optimal moiety for the B ring (compound 13). Further, the variation of counteranion had a profound effect on solubility in water without influence on antitumor activity. Chloride anion was selected as the favorable anion with respect to synthetic method as well as solubility in water. Our study finally led us to the identification of compound 3 (MKT 077, 1-ethyl-2-[[3-ethyl-5-(methylbenzothiazolin-2-ylidene)-4-oxothi azolidin-2 -ylidene]methyl]pyridinium chloride) as the candidate for clinical trials and is currently subjected to further investigation as a potent antitumor agent in phase I clinical trial for the treatment of solid tumors.

Targeted inhibition of the molecular chaperone Hsp90 results in the simultaneous blockade of multiple oncogenic signaling pathways and has, thus, emerged as an attractive strategy for the development of novel cancer therapeutics. Ganetespib (formerly known as STA-9090) is a unique resorcinolic triazolone inhibitor of Hsp90 that is currently in clinical trials for a number of human cancers. In the present study, we showed that ganetespib exhibits potent in vitro cytotoxicity in a range of solid and hematologic tumor cell lines, including those that express mutated kinases that confer resistance to small-molecule tyrosine kinase inhibitors. Ganetespib treatment rapidly induced the degradation of known Hsp90 client proteins, displayed superior potency to the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), and exhibited sustained activity even with short exposure times. In vivo, ganetespib showed potent antitumor efficacy in solid and hematologic xenograft models of oncogene addiction, as evidenced by significant growth inhibition and/or regressions. Notably, evaluation of the microregional activity of ganetespib in tumor xenografts showed that ganetespib was efficiently distributed throughout tumor tissue, including hypoxic regions >150 μm from the microvasculature, to inhibit proliferation and induce apoptosis. Importantly, ganetespib showed no evidence of cardiac or liver toxicity. Taken together, this preclinical activity profile indicates that ganetespib may have broad application for a variety of human malignancies, and with select mechanistic and safety advantages over other first- and second-generation Hsp90 inhibitors. Mol Cancer Ther; 11(2); 475–84. ©2011 AACR.

Among heat shock proteins, mortalin has been linked to the pathogenesis of Parkinson's disease. In the present work a rat model of Parkinson's disease was used to analyze the expression of striatal proteins and, more specifically, mortalin expression. The possible involvement of mortalin in Parkinson's disease pathogenesis was further investigated by utilizing an electrophysiological approach and pharmacological inhibition of mortalin in both the physiological and the parkinsonian states. Proteomic analysis was used to investigate changes in striatal protein expression in the 6-hydroxydopamine rat model of Parkinson's disease. The electrophysiological effects of MKT-077, a rhodamine-123 analogue acting as an inhibitor of mortalin, were measured by field potential recordings from corticostriatal brain slices obtained from control, sham-operated, and 6-hydroxydopamine-denervated animals. Slices in the presence of rotenone, an inhibitor of mitochondrial complex I, were also analyzed. Proteomic analysis revealed downregulation of mortalin in the striata of 6-hydroxydopamine-treated rats in comparison with sham-operated animals. MKT-077 reduced corticostriatal field potential amplitude in physiological conditions, inducing membrane depolarization and inward current in striatal medium spiny neurons. In addition, we observed that concentrations of MKT-077 not inducing any electrophysiological effect in physiological conditions caused significant changes in striatal slices from parkinsonian animals as well as in slices treated with a submaximal concentration of rotenone. These findings suggest a critical link between mortalin function and mitochondrial activity in both physiological and pathological conditions mimicking Parkinson's disease.

A novel 1,8-bridged fluorenophane (4a) is found to assume an $lsquoinward-folded’ conformation with the bulky tert-butyl group located in the cavity, a situation which is different from the conformation of the corresponding dithiafluorenophane (3a).

Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement-dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC-induced cell death. Similar results were obtained with MKT-077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT-077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre-treatment of cells with MKT-077 also reduces the extent of MAC-mortalin vesiculation following a sublytic complement attack. In the presence of MKT-077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT-077 on complement-mediated lysis of HCT116 p53(+/+) and p53(-/-) cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement-mediated hemolysis of rabbit erythrocytes as well as zinc-induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement-dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy.

We report herein the SAR studies of a series of indole- and indolizine-glyoxylamides that demonstrate substantial in vitro anti-proliferative activities against cancer cell lines, including multidrug resistance (MDR) phenotypes. The in vitro cytotoxic effects have been demonstrated across a wide array of tumor types of various origins (e.g., breast, colon, uterine).

Among a number of DMF dialkyl acetals investigated for the regioselective synthesis of 3-acylindolizines, the di-t-butyl acetal, via its iminium intermediate readily formed in situ, provides the highest chemoselectivity for the intermolecular cyclization of picolinium salts. DMF di-t-butyl acetal was applied to the syntheses of a variety of 3-acylated indolizines including alkyl, aryl, and heteroaryl substituents.

We report herein the SAR studies of a series of indole-imidazole compounds. that demonstrate substantial in vitro anti-proliferative activities against cancer cell lines, including multidrug resistance (MDR) phenotypes. The in vitro cytotoxic effects have been demonstrated across a wide array of tumor types, including hematologic and solid tumor cell lines of various origins (e.g., leukemia, breast, colon, and uterine).

BACKGROUND AND OBJECTIVES 1-Ethyl-2-?[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-+ ++oxothiazolidin-2-ylidenemethyl?pyridium chloride (MKT-077, formerly known as FJ776), a delocalized lipophilic cation, is known to accumulate in the mitochondria, according to the negative potential inside the mitochondria, and exert its cytotoxicity. METHODS The single-cell suspensions of human cancer cell lines, human spleen cells, and fresh cancer specimens obtained from patients with gastric carcinoma were used for the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. RESULTS The antitumor activity of MKT-077 was dose and concentration related, and 50% inhibitory concentrations (IC50) ranged from 1.7 to 14.3 microg/ml, with a mean +/- standard deviation (SD) of 8.4 +/- 4.6 microg/ml. The IC50 of fresh surgical spleen-cell specimens ranged from 0.34 microg/ml to >100 microg/ml in a 48 h incubation, with a mean +/- SD of 66.5 +/- 37.7 microg/ml. When the antitumor activity of MKT-077 was compared between gastric cancer cells and spleen cells obtained from the same patient, the concentration-dependent antitumor activity of this agent was obvious in the cancer cells, while no significant cytotoxicity was observed in the spleen cells. The fresh surgical specimens of gastric cancer showed higher sensitivity to MKT-077 than did spleen cells at a concentration of 30 microg/ml, with a statistically significant difference at P < 0.05. CONCLUSIONS The selective antitumor activity of MKT-077 was confirmed using fresh surgical specimens and warrants further investigation.

Rhodacyanine dyes and several analogous delocalized lipophilic cations (DLCs) were synthesized and evaluated as novel antitumor agents. Rhodacyanine dye consists of two heteroaromatic rings such as thiazoles at both termini of the conjugate systems and 4-oxothiazolidine (rhodanine) in the middle of it. Compounds with such a unique double-conjugate structure were found to inhibit the growth of several tumor cell lines, such as colon carcinoma CX-1, and to exhibit relatively low toxicity against normal kidney cell line CV-1 (e.g., IC50(CX-1) = 50 nM, IC50(CV-1) = 17.3 microM; selectivity index = 346 for compound 5). These compounds were also found to be efficacious in the tumor-bearing nude mice model (e.g., against human melanoma LOX; T/C (%) = 168 for compound 5). Structural modifications on rhodacyanine, including deletion of a heteroaromatic ring involved in the merocyanine conjugate system and replacement of rhodanine with a structurally related moiety such as 4-oxoimidazolidine or 4-oxo-1,3-dithiolane, resulted in a loss of the selectivity and/or the activity. Our current structure-activity studies imply that the double-conjugate system with a rhodanine moiety is essential for the selective activity of rhodacyanine dyes, and we find this class of compounds as unique antitumor agents candidates.

In addition to regulating a number of oncogenic client proteins, the Hsp90 molecular chaperone also controls the folding of key signaling molecules required to maintain normal cell function in many organs, including the retina. In human clinical trials Hsp90 inhibition has been associated with visual disorders including blurred vision, flashes, delayed light/dark accommodation, and photophobia. These adverse effects involving injury to the retina may be attributable to photoreceptor degeneration and cell death, as previously reported in dogs following repeated doses of PF-04929113. In contrast, ganetespib, a potent Hsp90 inhibitor currently in phase II/III trials, has demonstrated promising clinical activity without manifesting ocular toxicity. This difference in vision deficits between ganetespib and other Hsp90 inhibitors likely depends on a number of contributing factors. In this study, we examined the relationship between retinal drug distribution profiles and photoreceptor degeneration in male SD rats treated with17-DMAG, 17-AAG, and STA-9056 (an Hsp90 inhibitor with comparable in vitro activity to 17-DMAG). All compounds were tested in short-term studies at 1–3 dose levels administered i.v., At necropsy, eyes were dissected and processed for histopathological examination. In subsets of animals, the retinal tissues, along with plasma and cerebrospinal fluid (CSF) samples, were collected for analysis. Our results indicate that all compounds evaluated showed greater exposure in the retinal tissue compared to plasma and CSF. 17-DMAG, for which visual changes have been reported in clinical subjects, produced marked photoreceptor cell death and was associated with a slow elimination rate (at 6 hrs post-dose, 50% of the drug present at 30 min remained in the retina) and a high retina/plasma (R/P) ratio. In contrast, and consistent with the absence of clinically-reported visual changes, 17-AAG at the maximum tolerated dose did not produce detectable photoreceptor injury. At 6 hr post dose, 94% of 17-AAG had been eliminated from the retina resulting in a low R/P ratio. Finally, STA-9056 showed 79% drug elimination at 6 hrs and an R/P ratio that was moderately low. Photoreceptor degeneration was not observed at doses of STA-9056 that are active in animal tumor models, and only minimal degeneration was seen at a higher dose. Our findings suggest that the R/P exposure ratio and elimination rate profiles play crucial roles in ocular toxicity and can be used as indicators of potential Hsp90 inhibitor-induced damage in rats. In summary, Hsp90 plays an important role in the retina and prolonged Hsp90 inhibition can lead to vision disorders. However, ocular toxicity may be successfully minimized by administration of Hsp90 inhibitors with favorable drug properties that include, although not necessarily limited to, lower retina/plasma exposure ratios and faster retinal elimination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C212.

Targeted inhibition of the molecular chaperone heat shock protein 90 (Hsp90) results in the simultaneous blockade of multiple oncogenic signaling pathways and has thus emerged as an attractive strategy for the development of novel cancer therapeutics. Ganetespib (STA-9090) is a unique resorcinolic inhibitor of Hsp90 currently in clinical trials for a number of human cancers. Here we describe the key interaction of ganetespib with a number of amino acid residues in the ATP binding pocket of Hsp90 which results in high affinity binding. Ganetespib exhibits potent in vitro cytotoxicity in a range of solid and hematological tumor cell lines. By using a novel isotope-labeling scheme and LC-MS/MS detection technique, we have determined that Hsp90 occupancy by ganetespib in cancer cells is relatively fast under saturating conditions, reaching equilibrium within 5 minutes of ganetespib exposure. In vivo, ganetespib demonstrated strong single-agent activity in solid and hematological xenograft models, as evidenced by significant tumor growth inhibition and/or regression. Of note, evaluation of the microregional activity of ganetespib in tumor xenografts showed that ganetespib efficiently distributed throughout tumor tissue, including hypoxic regions >150 m from the microvasculature, to inhibit proliferation and induce apoptosis. Most importantly, ganetespib showed no evidence of cardiac or liver toxicity and exhibited minimal potential risk for CNS or ocular toxicities. Taken together, this preclinical activity profile suggests that ganetespib may have broad application for a variety of human malignancies and mechanistic and safety advantages over other first- and second-generation Hsp90 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B105.

Elesclomol is a first-in-class investigational drug that exerts potent anticancer activity through the elevation of reactive oxygen species (ROS) levels and is currently under clinical evaluation as a novel anticancer therapeutic. We recently demonstrated that elesclomol preferentially binds extracellular copper (Cu) and selectively transports this metal ion to the mitochondria of tumor cells to promote mitochondrial ROS generation and subsequent apoptosis. Here we report that elesclomol-induced copper transport and apoptosis is tumor selective. Comparative analysis using human PBMCs and the promyelocytic tumor cell line HL-60 demonstrated increased Cu levels in the mitochondrial fraction of HL-60 cells following elesclomol-Cu treatment, but not in donor PBMCs. Further, elesclomol-Cu induces ROS in HL-60-derived mitochondria but not in those isolated from PBMCs. These results suggest that elesclomol-Cu selectively targets cancer cell mitochondria to ultimately produce critical elevations in oxidative stress. To evaluate increased exposure to elesclomol-Cu in vivo, elesclomol was administered continuously to tumor-bearing mice using an Alzet pump at a clinically relevant dose. Upon release, elesclomol immediately chelates copper from the blood to form an elesclomol-Cu complex, achieving 10-fold higher levels of elesclomol-Cu compared to those following bolus injection. Even with this increased formation of elesclomol-Cu in situ, no signs of toxicity have been observed. In five different tumor xenograft models tested, elesclomol consistently demonstrated marked single agent activity with significant tumor growth suppression, indicating that increased elesclomol-Cu levels result in selective and enhanced antitumor efficacy. These findings highlight a unique mechanism of action of elesclomol and support potential single agent activity of this compound in a variety of tumor types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C168.

There is accumulating evidence that dysregulated JAK signaling occurs in a wide variety of cancer types. In particular, mutations in JAK2 can result in the constitutive activation of STAT transcription factors and lead to oncogenic growth. JAK kinases are established Hsp90 client proteins and here we show that the novel small molecule Hsp90 inhibitor ganetespib (formerly STA-9090) exhibits potent in vitro and in vivo activity in a range of solid and hematological tumor cells that are dependent on JAK2 activity for growth and survival. Of note, ganetespib treatment results in sustained depletion of JAK2, including the constitutively active JAK2(V617F) mutant, with subsequent loss of STAT activity and reduced STAT-target gene expression. In contrast, treatment with the pan-JAK inhibitor P6 results in only transient effects on these processes. Further differentiating these modes of intervention, RNA and protein expression studies show that ganetespib additionally modulates cell cycle regulatory proteins, while P6 does not. The concomitant impact of ganetespib on both cell growth and cell division signaling translates to potent antitumor efficacy in mouse models of xenografts and disseminated JAK/STAT-driven leukemia. Overall, our findings support Hsp90 inhibition as a novel therapeutic approach for combating diseases dependent on JAK/STAT signaling, with the multimodal action of ganetespib demonstrating advantages over JAK-specific inhibitors.

Abstract 2899 STA-9090 is a potent, second generation, small-molecule Hsp90 inhibitor, with a chemical structure unrelated to the first-generation, ansamycin family of Hsp90 inhibitors. In preclinical in vitro and in vivo studies, STA-9090 has shown potency up to 100 times greater than the first-generation Hsp90 inhibitors against a wide range of solid and hematological cancer types including those resistant to imatinib, sunitinib, erlotinib, and dasatinib. STA-9090 is currently being evaluated two Phase 1 and four Phase 2 trials (non-small cell lung, GIST, colon, and gastric) in solid tumor cancers; and two trials in hematologic cancers. Additional Phase 2 trials in several other indications are planned for 2H 2010. Inhibition of Hsp90 by STA-9090 results in the destabilization of a broad range of oncogenic kinases often overexpressed or mutated in hematological cancers. For example, the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressed in the anaplastic large cell lymphoma (ALCL) cell line Karpas 299, is degraded rapidly in the presence of STA-9090 in vitro, resulting in the loss of viability. Similar results were shown in other NPM-ALK driven ALCL cells including SU-DHL-1 and SR-786 with IC50 less than 20 nM. Stability of other kinases common to hematological malignancies, such as Bcr-Abl, FLT3 and c-Kit, were also shown to be highly sensitive to STA-9090, resulting in potent cell death of cell lines addicted to signaling by these kinases. In vivo, STA-9090 was highly effective in a subcutaneous xenograft model of diffuse large B-cell lymphoma SU-DHL-4 with resulting %T/C values of 26, 4, -90 and -93 when dosed at 25, 50, 75 and 100 mg twice per week, respectively. Importantly, 75 and 100 mg/kg STA-9090 dosed 2 times per week for a total of 3 weeks (150 and 200 mg/kg weekly) resulted in 25% and 50% of the animals in each group being free of tumors by the end of the study, respectively. MV4-11, an AML (FLT3ITD) cell line, turned out to be one of the most sensitive xenograft models to STA-9090 treatment. STA-9090 at 100 mg/kg or125mg/kg once weekly was highly efficacious with 37.5% of mice achieving tumor free with acceptable toxicity at the end of the 3-week treatment period. In conclusion, STA-9090 exhibits preferable biological profiles both in vitro and in vivo in treating hematological malignances. Clinical studies for using STA-9090 both once weekly and twice weekly are ongoing. Disclosures: Ying: Synta Pharmaceuticals: Employment. Proia:Synta Pharmaceuticals: Employment. He:Synta Pharmaceur: Employment. Sang:Synta Pharmaceuticals: Employment. Foley:Synta Pharmaceuticals: former employee. Du:Synta Pharmaceuticals: former employee. Blackman:Synta Pharmaceuticals: Employment. Wada:Synta Pharmaceuticals: Employment. Sun:Synta Pharmaceuticals: Employment. Koya:Synta Pharmaceuticals: Employment.

Heat Shock Protein 90 (Hsp90) is a molecular chaperone that mediates the post-translational stability of its protein substrates (“client proteins”). Cancer cells are particularly sensitive to Hsp90 inhibition because many client proteins play critical oncogenic roles. Accordingly, the potent synthetic small-molecule Hsp90 inhibitor STA-9090 shows in vitro activity in the low nM range against a broad range of tumor cell lines and it currently is in multiple Phase 1 and Phase 2 clinical trials for solid tumor and hematologic malignancies. The JAK kinases are established Hsp90 client proteins and there is accumulating evidence that constitutive activation of JAK signaling occurs in a wide variety of cancer types. In particular, mutations in JAK2 can result in the constitutive phosphorylation and activation of the transcription factors STAT3 and STAT5, thereby leading to oncogenic growth. Here, we show, both in vitro and in vivo, that STA-9090 potently inhibits the proliferation of numerous solid tumor and hematological cancer cell lines that are dependent upon persistent JAK/STAT signaling for growth and survival. Western blot analysis demonstrates that STA-9090 blocks endogenous and IL-6-induced JAK/STAT signaling in these cells. Importantly, STA-9090 treatment leads to sustained depletion of JAK2, producing a prolonged loss of STAT phosphorylation/activity and reduced expression of STAT target genes such as the oncogenes PIM1/2 and SOCS1/3. In contrast, treatment with the pan-JAK kinase inhibitor P6 leads only to a transient effect on these processes. RNA profiling studies on HEL92.1.7 cells, which express the constitutively activated JAK2V617F mutation, demonstrate that STA-9090 has a more profound activity on these cells than P6. In addition to the JAK/STAT targets that both drugs influence, only STA-9090 treatment leads to the loss of cyclins and cyclin-dependent kinases. Such changes reflect the G1 and G2/M arrest typically induced by STA-9090 in multiple cancer cell types. Additional client proteins are affected as well and STA-9090 treatment leads to apoptosis. In vivo, xenograft models of human MV4-11 AML cells (which carry a FLT3-ITD mutation that drives constitutive activation of STAT5) demonstrate that a single dose of STA-9090 represses activation of STAT5 and suppresses expression of the cyclin-dependent kinase CDK1 for more than three days, leading to tumor regression. STAT5 signaling, but not CDK1 expression, returns by the sixth day without a change in tumor volume. This suggests that the coordinate loss of cell growth and cell division signals orchestrated by STA-9090, with only intermittent dosing, is responsible for reducing and maintaining tumor regression. Thus, inhibition of Hsp90 activity presents a new method for combating diseases dependent on constitutive JAK/STAT signaling, with STA-9090 having advantages over JAK-specific inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2640.

Elesclomol is a first-in-class investigational drug that exerts anticancer activity through the elevation of reactive oxygen species (ROS). Our previous studies revealed that elesclomol selectively chelates copper and generates ROS via reduction of Cu(II) to Cu(I). In a Phase 3 trial in metastatic melanoma, the level of baseline lactate dehydrogenase (LDH) in patients emerged as an important prognostic factor for treatment outcomes with elesclomol. Additional investigations were undertaken to evaluate the relation between elesclomol activity and LDH levels. Because the copper redox reactions necessary for elesclomol activity can be influenced by changes in cellular metabolic properties, we investigated whether elesclomol activity varies between normoxic conditions (active mitochondrial respiration, low LDH) and hypoxic conditions (decreased mitochondrial respiration, high LDH). To assess the influence of hypoxic conditions, we evaluated elesclomol activity under several situations in which cells express high levels of Hypoxia Inducible Factor-1α (HIF1α), a transcription factor that induces a complete set of glycolytic enzymes, including LDH, and switches off glycolytic carbon flow to mitochondria. First, an increase in the level of HIF1α protein was observed in M14 melanoma cells at low oxygen level or high cell density. Elesclomol showed decreased cytotoxicity under both of these conditions. Second, we evaluated lines within the same cancer phenotype but showing distinct levels of HIF1α, and found that the high-HIF1α-expressing Caki-2 renal cancer line was resistant to elesclomol, while lower-HIF1α-expressing renal cancer cell lines were sensitive to elesclomol. Third, we assessed HIF1α levels and elesclomol activity in cells treated with CoCl2, a chemical mimetic of hypoxia. High levels of HIF1α induction were present in M14 cells treated with CoCl2, and elesclomol was significantly less active in these cells. The addition of oxamate, which selectively inhibits LDHA and activates pyruvate entry into mitochondria, restored the activity of elesclomol in the CoCl2-treated cells. These results support the hypothesis that elesclomol is more active under normoxic conditions (low LDH) and less active under hypoxic conditions (high LDH), consistent with the clinical findings. The results may be important for identifying patient populations for future clinical development of elesclomol. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4545.

BACKGROUND: Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the folding, stability and activity of many kinases and signal transduction proteins that are critical for the proliferation and survival of cancer cells. These Hsp90 client proteins include wild type and drug-resistant forms of such clinically important drug targets as: BCR-ABL, BRAF, EGFR, HER2, KIT, MET and VEGFR1. STA-9090 is a highly potent and less toxic, next-generation Hsp90 inhibitor that is structurally unrelated to first-generation agents such as 17-AAG and IPI-504. STA-9090 induces proteasome-mediated degradation of Hsp90 client proteins preferentially in cancer versus normal cells, resulting in cell cycle arrest and apoptosis. STA-9090 is currently being evaluated in multiple Phase 1/2 clinical trials in sold tumor and hematologic malignancies, including an open-label, multi-center Phase 2 trial in patients with advanced NSCLC harboring wild type or activated forms of EGFR or KRAS. In anticipation of conducting clinical trials combining STA-9090 with therapeutic agents commonly employed to treat NSCLC, we have examined the ability of STA-9090 to enhance the in vitro and in vivo activity of paclitaxel, docetaxel, erlotinib and bevacizumab in preclinical models of lung cancer. RESULTS: In vitro cytotoxicity studies were conducted with the erlotinib-sensitive HCC827 (expressing EGFRdel746–750) and erlotinib-resistant NCI-H1975 (expressing EGFRL858R/T790M) NSCLC cell lines. Using the median-effect method of Chau and Talalay, STA-9090 was found to significantly enhance the in vitro cytotoxicity of the taxanes, paclitaxel and docetaxel, with combination index (CI) values ranging from 0.3-0.7, which is indicative of synergy rather than additive or antagonistic effects. Similarly, STA-9090 also displayed synergy (0.4-0.8 CI) with the EGFR inhibitor erlotinib against erlotinib-sensitive cells, but as expected, not against erlotinib-resistant cells. Consistent with these results, STA-9090 enhanced the in vivo efficacy of concomitantly dosed paclitaxel and docetaxel in the HCC827 and NCI-H1975 xenograft models. Further, STA-9090 also enhanced the in vivo efficacy of the anti-VEGF monoclonal antibody bevacizumab in these models. These combinations did not result in significant additional toxicity relative to the single agents alone, and pharmacokinetic analyses demonstrated that these findings were not due to drug-drug interactions. CONCLUSIONS: Our results demonstrate that STA-9090 potently synergizes with paclitaxel, docetaxel, erlotinib and bevacizumab, which are widely employed to treat NSCLC in the clinic. Hsp90 inhibition using STA-9090 represents a novel and promising approach to combination therapy for NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2637.

A novel 1,8-bridged fluorenophane (4a) is found to assume an $lsquoinward-folded’ conformation with the bulky tert-butyl group located in the cavity, a situation which is different from the conformation of the corresponding dithiafluorenophane (3a).

On prepare le compose du titre a partir du bis-chloromethyl-2,6 t-butyl-4 anisole et du bis-methylthio-2,5 dimethoxy-1,4benzene. La reduction par Zn en poudre conduit au derive tetrahydroxy correspondant qui se transforme a l'air en complexe de type quinhydrone

Summary Systemic chemotherapy using two-drug platinum-based regimens for the treatment of advanced stage non-small cell lung cancer (NSCLC) has largely reached a plateau of effectiveness. Accordingly, efforts to improve survival and quality of life outcomes have more recently focused on the use of molecularly targeted agents, either alone or in combination with standard of care therapies such as taxanes. The molecular chaperone heat shock protein 90 (Hsp90) represents an attractive candidate for therapeutic intervention, as its inhibition results in the simultaneous blockade of multiple oncogenic signaling cascades. Ganetespib is a non-ansamycin inhibitor of Hsp90 currently under clinical evaluation in a number of human malignancies, including NSCLC. Here we show that ganetespib potentiates the cytotoxic activity of the taxanes paclitaxel and docetaxel in NSCLC models. The combination of ganetespib with paclitaxel, docetaxel or another microtubule-targeted agent vincristine resulted in synergistic antiproliferative effects in the H1975 cell line in vitro. These benefits translated to improved efficacy in H1975 xenografts in vivo, with significantly enhanced tumor growth inhibition observed in combination with paclitaxel and tumor regressions seen with docetaxel. Notably, concurrent exposure to ganetespib and docetaxel improved antitumor activity in 5 of 6 NSCLC xenograft models examined. Our data suggest that the improved therapeutic indices are likely to be mechanistically multifactorial, including loss of pro-survival signaling and direct cell cycle effects resulting from Hsp90 modulation by ganetespib. Taken together, these findings provide preclinical evidence for the use of this combination to treat patients with advanced NSCLC. Electronic supplementary material The online version of this article (doi:10.1007/s10637-011-9790-6) contains supplementary material, which is available to authorized users.