Purpose: The heat shock protein 90 (Hsp90) chaperone plays an important role in transformation by regulating the conformational maturation and stability of oncogenic kinases and transcription factors. Ansamycins, such as 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), inhibit Hsp90 function; induce the degradation of Hsp90 client proteins such as HER2, and have shown activity in early clinical trials. However, the utility of these drugs has been limited by their hepatotoxicity, poor solubility, and poorly tolerated formulations. Experimental Design: We determined the pharmacodynamic and antitumor properties of a novel, synthetic Hsp90 inhibitor, SNX-2112, in cell culture and xenograft models of HER kinased ependent cancers. Results: We show in a panel of tumor cell lines that SNX-2112 and its prodrug SNX-5542 are Hsp90 inhibitors with properties and potency similar to that of 17-AAG, including: degradation of HER2, mutant epidermal growth factor receptor, and other client proteins, inhibition of extracellular signal-regulated kinase and Akt activation, and induction of a Rb-dependent G 1 arrest with subsequent apoptosis. SNX-5542 can be administered to mice orally on a daily schedule. Following oral administration, SNX-5542 is rapidly converted to SNX-2112, which accumulates in tumors relative to normal tissues. A single dose of SNX-5542 causes HER2 degradation and inhibits its downstream signaling for up to 24 h, and daily dosing results in regression of HER2-dependent xenografts. SNX-5542 also shows greater activity than 17-AAG in a nonŝ mall cell lung cancer xenograft model expressing mutant EGFR. Conclusions: These results suggest that Hsp90 inhibition with SNX-2112 (delivered as a prodrug) may represent a promising therapeutic strategy for tumors whose growth and survival is dependent on Hsp90 clients.Heat shock protein 90 (Hsp90) is a protein chaperone that functions to promote the maturation and conformational stabilization of a subset of cellular proteins important in transducing proliferative and survival signals. Hsp90 clients include protein kinases (e.g., HER2, Raf-1, Akt, and Cdk4), steroid receptors (e.g., androgen receptor and estrogen receptor), and transcription factors (e.g., Hif1a; refs.

Objective. To evaluate the ability of SNX-7081, a novel small molecule inhibitor of Hsp90, to block components of inflammation, including cytokine production, protein kinase activity, and angiogenic signaling. A close analog was evaluated in preclinical in vivo models of rheumatoid arthritis (RA).Methods. SNX-7081 binding to Hsp90 was characterized in Jurkat cells and RA synovial fibroblasts (RASFs). Inhibition of NF-B nuclear translocation was evaluated in cellular systems, using lipopolysaccharide (LPS), tumor necrosis factor ␣, or interleukin-1␤ stimulation. Suppression of cytokine production in THP-1 cells, human umbilical vein endothelial cells, and RASFs was studied. Disruption of MAPK signaling cascades by SNX-7081 following growth factor stimulation was assessed. SNX-7081 was tested in 2 relevant angiogenesis assays: platelet-derived growth factor activation of fibroblasts and LPS-induced nitric oxide (NO) release in J774 macrophages. A close analog, SNX-4414, was evaluated in rat collagen-induced arthritis and adjuvant-induced arthritis, following oral treatment.Results. SNX-7081 showed strong binding affinity to Hsp90 and expected induction of Hsp70. NF-B nuclear translocation was blocked by SNX-7081 at nanomolar concentrations, and cytokine production was potently inhibited. Growth factor activation of ERK and JNK signaling was significantly reduced by SNX-7081. NO production was also sharply inhibited. In animal models, SNX-4414 fully inhibited paw swelling and improved body weight. Scores for inflammation, pannus formation, cartilage damage, and bone resorption returned to normal.Conclusion. The present results demonstrate that a small molecule Hsp90 inhibitor can impact inflammatory disease processes. The strong in vivo efficacy observed with SNX-4414 provides preclinical validation for consideration of Hsp90 inhibitors in the treatment of RA.

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed from an unbiased screen to identify protein targets for a diverse compound library. These indol-4-one and indazol-4-one derived 2-aminobenzamides showed strong binding affinity to Hsp90, and optimized analogues exhibited nanomolar antiproliferative activity across multiple cancer cell lines. Heat shock protein 70 (Hsp70) induction and specific client protein degradation in cells on treatment with the inhibitors supported Hsp90 inhibition as the mechanism of action. Computational chemistry and X-ray crystallographic analysis of selected member compounds clearly defined the protein-inhibitor interaction and assisted the design of analogues. 4-[6,6-Dimethyl-4-oxo-3-(trifluoromethyl)-4,5,6,7-tetrahydro-1H-indazol-1-yl]-2-[(trans-4-hydroxycyclohexyl)amino]benzamide (SNX-2112, 9) was identified as highly selective and potent (IC(50) Her2 = 11 nM, HT-29 = 3 nM); its prodrug amino-acetic acid 4-[2-carbamoyl-5-(6,6-dimethyl-4-oxo-3-trifluoromethyl-4,5,6,7-tetrahydro-indazol-1-yl)-phenylamino]-cyclohexyl ester methanesulfonate (SNX-5422, 10) was orally bioavailable and efficacious in a broad range of xenograft tumor models (e.g. 67% growth delay in a HT-29 model) and is now in multiple phase I clinical trials.

A chemoproteomics-based drug discovery strategy is presented that utilizes a highly parallel screening platform, encompassing more than 1000 targets, with a focused chemical library prior to target selection. This chemoproteomics-based process enables a data-driven selection of both the biological target and chemical hit after the screen is complete. The methodology has been exemplified for the purine binding proteome (proteins utilizing ATP, NAD, FAD). Screening of an 8000 member library yielded over 1500 unique protein-ligand interactions, which included novel hits for the oncology target Hsp90. The approach, which also provides broad target selectivity information, was used to drive the identification of a potent and orally active Hsp90 inhibitor, SNX-5422, which is currently in phase 1 clinical studies.

Background Although both men and women use e-cigarettes, most preclinical nicotine research has focused on its effects in male rodents following injection. The goals of the present study were to develop an effective e-cigarette nicotine delivery system, to compare results to those obtained after subcutaneous (s.c.) injection, and to examine sex differences in the model. Methods Hypothermia and locomotor suppression were assessed following aerosol exposure or s.c. injection with nicotine in female and male mice. Subsequently, plasma and brain concentrations of nicotine and cotinine were measured. Results Passive exposure to nicotine aerosol produced concentration-dependent and mecamylamine reversible hypothermic and locomotor suppressant effects in female and male mice, as did s.c. nicotine injection. In plasma and brain, nicotine and cotinine concentrations showed dose/concentration-dependent increases in both sexes following each route of administration. Sex differences in nicotine-induced hypothermia were dependent upon route of administration, with females showing greater hypothermia following aerosol exposure and males showing greater hypothermia following injection. In contrast, when they occurred, sex differences in nicotine and cotinine levels in brain and plasma consistently showed greater concentrations in females than males, regardless of route of administration. Discussion In summary, the e-cigarette exposure device described herein was used successfully to deliver pharmacologically active doses of nicotine to female and male mice. Further, plasma nicotine concentrations following exposure were similar to those after s.c. injection with nicotine and within the range observed in human smokers. Future research on vaped products can be strengthened by inclusion of translationally relevant routes of administration.

The pharmacological effects of tobacco products are primarily mediated by nicotine; however, research suggests that several non-nicotine tobacco constituents may alter the reinforcing effects of nicotine. This study evaluated the reinforcing effects of aqueous solutions of smoke/aerosol condensate from cigarettes, little cigars, electronic cigarettes (e-cigarettes), and waterpipe tobacco in a self-administration procedure to determine if abuse liability of these tobacco products differed. Adult male Sprague-Dawley rats (n = 64 total) were trained to self-administer intravenous nicotine (30 μg/kg/infusion) on a fixed ratio 5 schedule of reinforcement. Following nicotine dose-effect assessment (1, 7.5, 15, and 30 μg/kg/infusion), rats were given access to smoke/aerosol condensate derived from their assigned tobacco product. Rats responded for smoke/aerosol condensate containing 1, 7.5, 15, and 30 μg/kg/infusion nicotine, with the ratio of nicotine:non-nicotine constituents held constant across doses for each tobacco product. Responding for nicotine or smoke/aerosol condensate was also assessed on a progressive ratio schedule of reinforcement. Cigarette, little cigar, and e-cigarette smoke/aerosol condensates shifted the nicotine dose-effect curve leftward, whereas waterpipe tobacco smoke condensate shifted the dose-effect curve rightward. Smoke/aerosol condensate from all tobacco products produced similar levels of responding compared to nicotine alone during the progressive ratio phase. Results suggest that non-nicotine constituents in cigarettes, little cigars, and e-cigarettes differentially enhance nicotine’s reinforcing potency. In contrast, waterpipe tobacco blunted nicotine’s reinforcing potency, suggesting that it may contain unique constituents that dampen nicotine’s reinforcing effects.

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Hydroxyurea (HU) is a valuable therapy for individuals with sickle cell anemia. With increased use of HU in children and throughout their lives, it is important to understand the potential effects of HU therapy on their development and fertility. Thus, studies were conducted to identify appropriate doses to examine long-term effects of prenatal and early postnatal HU exposure and to understand kinetics of HU at various life stages. Pregnant Sprague Dawley dams were administered HU (0-150 mg/kg/day) via oral gavage from gestation days 17 to 21 and during lactation. Pups were dosed with the same dose as their respective dam starting on postnatal day (PND) 10 and up to PND 34. There was minimal maternal toxicity, and no significant effects on littering at any dose of HU. Starting on PND 16, offspring displayed skin discoloration and alopecia at doses ≥75 mg/kg/day and lower body weight compared to controls at doses ≥100 mg/kg/day. Gestational transfer of HU was observed, but there was minimal evidence of lactational transfer. Our toxicokinetic studies suggest that the internal dose in offspring may be altered due to age, but not due to sex. The plasma area under the curve, a measure of systemic exposure, at doses tolerated by offspring was threefold to sevenfold lower than the internal therapeutic dose in humans. Therefore, strategies to establish clinically relevant exposures in animal studies are needed. Overall, these data are useful for the design of appropriate nonclinical studies in the future to evaluate the consequences of long-term HU treatment starting in childhood.