Small molecule branched-chain ketoacid dehydrogenase kinase (BDK) inhibitors with opposing effects on BDK protein levels.

Branched chain amino acid (BCAA) catabolic impairments have been implicated in several diseases. Branched chain ketoacid dehydrogenase (BCKDH) controls the rate limiting step in BCAA degradation, the activity of which is inhibited by BCKDH kinase (BDK)-mediated phosphorylation. Screening efforts to discover BDK inhibitors led to identification of thiophene PF-07208254, which improved cardiometabolic endpoints in mice. Structure-activity relationship studies led to identification of a thiazole series of BDK inhibitors; however, these inhibitors did not improve metabolism in mice upon chronic administration. While the thiophenes demonstrated sustained branched chain ketoacid (BCKA) lowering and reduced BDK protein levels, the thiazoles increased BCKAs and BDK protein levels. Thiazoles increased BDK proximity to BCKDH-E2, whereas thiophenes reduced BDK proximity to BCKDH-E2, which may promote BDK degradation. Thus, we describe two BDK inhibitor series that possess differing attributes regarding BDK degradation or stabilization and provide a mechanistic understanding of the desirable features of an effective BDK inhibitor.

AMPK activation is sufficient to increase skeletal muscle glucose uptake and glycogen synthesis but is not required for contraction-mediated increases in glucose metabolism.

The AMP-activated protein kinase (AMPK) is a cellular sensor of energetics and when activated in skeletal muscle during contraction can impart changes in skeletal muscle metabolism. Therapeutics that selectively activate AMPK have been developed to lower glucose levels through increased glucose disposal rates as an approach to abrogate the hyperglycemic state of diabetes; however, the metabolic fate of glucose following AMPK activation remains unclear. We have used a combination of in vivo evaluation of glucose homeostasis and ex vivo skeletal muscle incubation to systematically evaluate metabolism following pharmacological activation of AMPK with PF-739, comparing this with AMPK activation through sustained intermittent electrical stimulation of contraction. These methods to activate AMPK result in increased glucose uptake but divergent metabolism of glucose: pharmacological activation results in increased glycogen accumulation while contraction-induced glucose uptake results in increased lactate formation and glucose oxidation. These results provide additional evidence to support a role for AMPK in control of skeletal muscle metabolism and additional insight into the potential for AMPK stimulation with small molecule direct activators.

Acyl Glucuronide Metabolites of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1 H-indole-3-carboxylic Acid (PF-06409577) and Related Indole-3-carboxylic Acid Derivatives are Direct Activators of Adenosine Monophosphate-Activated Protein Kinase (AMPK).

Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) α1ß1γ1 isoform have culminated in the identification of PF-06409577 (1), PF-06885249 (2), and PF-06679142 (3) as potential clinical candidates. Compounds 1-3 are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of 1-3. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of ß1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Cocrystallization of the AMPK α1ß1γ1 isoform with 1-3 and M1-M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates.

Activation of Liver AMPK with PF-06409577 Corrects NAFLD and Lowers Cholesterol in Rodent and Primate Preclinical Models.

Dysregulation of hepatic lipid and cholesterol metabolism is a significant contributor to cardiometabolic health, resulting in excessive liver lipid accumulation and ultimately non-alcoholic steatohepatitis (NASH). Therapeutic activators of the AMP-Activated Protein Kinase (AMPK) have been proposed as a treatment for metabolic diseases; we show that the AMPK ß1-biased activator PF-06409577 is capable of lowering hepatic and systemic lipid and cholesterol levels in both rodent and monkey preclinical models. PF-06409577 is able to inhibit de novo lipid and cholesterol synthesis pathways, and causes a reduction in hepatic lipids and mRNA expression of markers of hepatic fibrosis. These effects require AMPK activity in the hepatocytes. Treatment of hyperlipidemic rats or cynomolgus monkeys with PF-06409577 for 6weeks resulted in a reduction in circulating cholesterol. Together these data suggest that activation of AMPK ß1 complexes with PF-06409577 is capable of impacting multiple facets of liver disease and represents a promising strategy for the treatment of NAFLD and NASH in humans.

Activation of Skeletal Muscle AMPK Promotes Glucose Disposal and Glucose Lowering in Non-human Primates and Mice.

The AMP-activated protein kinase (AMPK) is a potential therapeutic target for metabolic diseases based on its reported actions in the liver and skeletal muscle. We evaluated two distinct direct activators of AMPK: a non-selective activator of all AMPK complexes, PF-739, and an activator selective for AMPK ß1-containing complexes, PF-249. In cells and animals, both compounds were effective at activating AMPK in hepatocytes, but only PF-739 was capable of activating AMPK in skeletal muscle. In diabetic mice, PF-739, but not PF-249, caused a rapid lowering of plasma glucose levels that was diminished in the absence of skeletal muscle, but not liver, AMPK heterotrimers and was the result of an increase in systemic glucose disposal with no impact on hepatic glucose production. Studies of PF-739 in cynomolgus monkeys confirmed translation of the glucose lowering and established activation of AMPK in skeletal muscle as a potential therapeutic approach to treat diabetic patients.

The interaction of bortezomib with multidrug transporters: implications for therapeutic applications in advanced multiple myeloma and other neoplasias.

PURPOSE: Bortezomib is an important agent in multiple myeloma treatment, but resistance in cell lines and patients has been described. The main mechanisms of resistance described in cancer fall into one of two categories, pharmacokinetic resistance (PK), e.g. over expression of drug efflux pumps and pharmacodynamic resistance, e.g. apoptosis resistance or altered survival pathways, where the agent reaches an appropriate concentration, but this fails to propagate an appropriate cell death response. Of the known pump mechanisms, P-glycoprotein (P-gp) is the best studied and considered to be the most important in contributing to general PK drug resistance. Resistance to bortezomib is multifactorial and there are conflicting indications that cellular overexpression of P-gp may contribute to resistance agent. Hence, better characterization of the interactions of this drug with classical resistance mechanisms should identify improved treatment applications. METHODS: Cell lines with different P-gp expression levels were used to determine the relationship between bortezomib and P-gp. Coculture system with stromal cells was used to determine the effect of the local microenvironment on the bortezomib-elacridar combination. To further assess P-gp function, intracellular accumulation of P-gp probe rhodamine-123 was utilised. RESULTS: In the present study, we show that bortezomib is a substrate for P-gp, but not for the other drug efflux transporters. Bortezomib activity is affected by P-gp expression and conversely, the expression of P-gp affect bortezomib's ability to act as a P-gp substrate. The local microenvironment did not alter the cellular response to bortezomib. We also demonstrate that bortezomib directly affects the expression and function of P-gp. CONCLUSIONS: Our findings strongly support a role for P-gp in bortezomib resistance and, therefore, suggest that combination of a P-gp inhibitor and bortezomib in P-gp positive myeloma would be a reasonable treatment combination to extend efficacy of this important drug.

Methyljasmonate displays in vitro and in vivo activity against multiple myeloma cells.

Jasmonates, plant stress hormones, have been demonstrated to be effective in killing various types of cancer cells. We therefore tested if methyljasmonate (MJ) has activity against multiple myeloma (MM) in vitro and in vivo. MM cell lines and primary MM tumour cells responded to MJ in vitro at concentrations that did not significantly affect normal haematopoietic cells, without stroma-mediated resistance. Brief MJ exposures of MM cells caused release of Hexokinase 2 (HK2) from mitochondria, rapid ATP depletion, perturbation of major intracellular signalling pathways, and ensuing mainly apoptotic cell death. Sensitivity to MJ correlated with lower cellular glucose consumption and lactate production, as well as lower intracellular protein levels of HK2, phosphorylated Voltage-dependent anion channel 2/3 (pVDAC2/3) and Aldo-keto reductase family 1 member C1 (AKR1C1), which represent potential biomarkers of responsiveness to MJ treatment, especially as AKR1C1 transcript levels also correlate with clinical outcome in bortezomib- or dexamethasone-treated MM patients. Interestingly, MJ synergized with bortezomib in vitro and prolonged survival of immunocompromised mice harbouring diffuse lesions of MM.1S cells compared to vehicle-treated mice (P = 0·0046). These studies indicate that jasmonates represent a new, promising strategy to treat MM.

Inhibition of tumor cells interacting with stromal cells by xanthones isolated from a Costa Rican Penicillium sp.

CR1642D, an endophytic isolate of Penicillium sp. collected from a Costa Rican rainforest, was identified through a high-throughput approach to identify natural products with enhanced antitumor activity in the context of tumor-stromal interactions. Bioassay-guided separation led to the identification of five xanthones (1-5) from CR1642D. The structures of the xanthone dimer penexanthone A (1) and monomer penexanthone B (2) were elucidated on the basis of spectroscopic analyses, including 2D NMR experiments. All of the compounds were tested against a panel of tumor cell lines in the presence and absence of bone marrow stromal cells. Compound 3 was the most active, with IC(50) values of 1-17 µM, and its activity was enhanced 2-fold against tumor cell line RPMI8226 in the presence of stromal cells (IC(50) 1.2 µM, but 2.4 µM without stromal cells).

Compartment-Specific Bioluminescence Imaging platform for the high-throughput evaluation of antitumor immune function.

Conventional assays evaluating antitumor activity of immune effector cells have limitations that preclude their high-throughput application. We adapted the recently developed Compartment-Specific Bioluminescence Imaging (CS-BLI) technique to perform high-throughput quantification of innate antitumor activity and to show how pharmacologic agents (eg, lenalidomide, pomalidomide, bortezomib, and dexamethasone) and autologous BM stromal cells modulate that activity. CS-BLI-based screening allowed us to identify agents that enhance or inhibit innate antitumor cytotoxicity. Specifically, we identified compounds that stimulate immune effector cells against some tumor targets but suppressed their activity against other tumor cells. CS-BLI offers rapid, simplified, and specific evaluation of multiple conditions, including drug treatments and/or cocultures with stromal cells and highlights that immunomodulatory pharmacologic responses can be heterogeneous across different types of tumor cells. This study provides a framework to identify novel immunomodulatory agents and to prioritize compounds for clinical development on the basis of their effect on antitumor immunity.