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The monogenic defects in specific lysosomal enzymes in mucopolysaccharidosis (MPS) III lead to lysosomal storage of glycosaminoglycans and complex CNS and somatic pathology, for which the detailed mechanisms remain unclear. In this study, serum samples from patients with MPS IIIA (age 2-9 yr) and MPS IIIB (2-13 yr) and healthy controls (age 2-9 yr) were assayed by global metabolomics profiling of 658 metabolites using mass spectrometry. Significant alterations were detected in 423 metabolites in all MPS III patients, of which 366 (86.5%) decreased and 57 (13.5%) increased. Similar profiles were observed when analyzing data from MPS IIIA and MPS IIIB samples separately, with only limited age variations in 36 metabolites. The observed metabolic disturbances in MPS III patients involve virtually all major pathways of amino acid (101/150), peptide (17/21), carbohydrate (19/23), lipid (221/325), nucleotide (15/25), energy (8/9), vitamins and co-factors (8/21), and xenobiotics (34/84) metabolism. Notably, detected serum metabolite decreases involved all key amino acids, all major neurotransmitter pathways, and broad neuroprotective compounds. The elevated metabolites are predominantly lipid derivatives, and also include cysteine metabolites and a fibrinogen peptide fragment, consistent with the status of oxidative stress and inflammation in MPS III. This study demonstrates that the lysosomal glycosaminoglycans storage triggers profound metabolic disturbances in patients with MPS III disorders, leading to severe functional depression of virtually all metabolic pathways, which emerge early during the disease progression. Serum global metabolomics profiling may provide an important and minimally invasive tool for better understanding the disease mechanisms and identification of potential biomarkers for MPS III.

While genomic sequencing routinely identifies oncogenic alterations for the majority of cancers, many tumors harbor no discernable driver lesion. Here, we describe the exceptional molecular phenotype of a genomically quiet kidney tumor, clear cell papillary renal cell carcinoma (CCPAP). In spite of a largely wild-type nuclear genome, CCPAP tumors exhibit severe depletion of mitochondrial DNA (mtDNA) and RNA and high levels of oxidative stress, reflecting a shift away from respiratory metabolism. Moreover, CCPAP tumors exhibit a distinct metabolic phenotype uniquely characterized by accumulation of the sugar alcohol sorbitol. Immunohistochemical staining of primary CCPAP tumor specimens recapitulates both the depletion of mtDNA-encoded proteins and a lipid-depleted metabolic phenotype, suggesting that the cytoplasmic clarity in CCPAP is primarily related to the presence of glycogen. These results argue for non-genetic profiling as a tool for the study of cancers of unknown driver.

Dysregulated metabolism is a hallmark of cancer, manifested through alterations in metabolites. We performed metabolomic profiling on 138 matched clear cell renal cell carcinoma (ccRCC)/normal tissue pairs and found that ccRCC is characterized by broad shifts in central carbon metabolism, one-carbon metabolism, and antioxidant response. Tumor progression and metastasis were associated with metabolite increases in glutathione and cysteine/methionine metabolism pathways. We develop an analytic pipeline and visualization tool (metabolograms) to bridge the gap between TCGA transcriptomic profiling and our metabolomic data, which enables us to assemble an integrated pathway-level metabolic atlas and to demonstrate discordance between transcriptome and metabolome. Lastly, expression profiling was performed on a high-glutathione cluster, which corresponds to a poor-survival subgroup in the ccRCC TCGA cohort.

A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K(m) for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability. An X-ray crystal structure for the complex of our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site approximately 13A from the ATP binding site. Preliminary data is presented for several of these compounds.

From HTS lead 1, a novel benzoisoquinolinone class of ATP-competitive Chk1 inhibitors was devised and synthesized via a photochemical route. Using X-ray crystallography as a guide, potency was rapidly enhanced through the installation of a tethered basic amine designed to interact with an acidic residue (Glu91) in the enzyme pocket. Further SAR was explored at the solvent front and near to the H1 pocket and resulted in the discovery of low MW, sub-nanomolar inhibitors of Chk1.

Through a comparison of X-ray co-crystallographic data for 1 and 2 in the Chek1 active site, it was hypothesized that the affinity of the indolylquinolinone series (2) for Chek1 kinase would be improved via C6 substitution into the hydrophobic region I (HI) pocket. An efficient route to 6-bromo-3-indolyl-quinolinone (9) was developed, and this series was rapidly optimized for potency by modification at C6. A general trend was observed among these low nanomolar Chek1 inhibitors that compounds with multiple basic amines, or elevated polar surface area (PSA) exhibited poor cell potency. Minimization of these parameters (basic amines, PSA) resulted in Chek1 inhibitors with improved cell potency, and preliminary pharmacokinetic data are presented for several of these compounds.

The MNU-induced model of ER+ mammary cancers in Sprague-Dawley rats has routinely been employed in screening for chemopreventive agents. We recently reported that metformin was ineffective in preventing mammary cancers in rats on a standard diet (Thompson, et al, Cancer Prev. Res, 8, 231-9, 2015). In this study, we evaluated the carcinogenic response in Vehicle-treated rats and rats receiving Metformin (150 mg/Kg BW/day) on standard Teklad or isocaloric High-Fructose diets. In the first study, rats were placed on Teklad or High-Fructose diet at 43 days of age. At 100 days of age, animals were administered a single dose of MNU. Animals were administered Metformin or tamoxifen beginning 5 days later. Rats were palpated weekly for mammary tumors and sacrificed at 300 days of age. Body weights between groups were not different. Tumor multiplicity and tumor weights were: Vehicle-Teklad,1.15 and 1.44g; Vehicle- High-Fructose, 2.10 and 2.94g; Metformin-Teklad, 1.25 and 1.91g; Metformin -High-Fructose, 1.00 and1.99g. Tamoxifen resulted in the prevention of all tumors. High-Fructose alone resulted in a 2X increase in tumor multiplicity (PP0.05). In the second study, which parallels the standard MNU model, rats were also placed on Teklad or High-Fructose diet at 43 days of age. At 50 days of age, rats were administered a single dose of MNU. Rats on High-Fructose diet were administered either Vehicle or Metformin, while rats on Teklad diet were given only Vehicle; beginning 5 days later. Rats were palpated weekly and sacrificed at 170 days of age. Body weights between groups were not different. Tumor multiplicity and tumor weights were: Vehicle-High-Fructose, 6.00 and 7.74g; Metformin High-Fructose, 5.00 and 6.17g; and Vehicle-Teklad, 2.70 and 5.75g. As indicated, Vehicle-treated rats on High-Fructose diet had twice the number of cancers as Vehicle-Teklad diet (P Citation Format: Mark S. Miller, Clinton J. Grubbs, Steve Stirdivant, Ronald A. Lubet. Comparison of high-fructose and standard diets in methylnitrosourea (MNU)-induced ER+ mammary cancers: Altered carcinogenesis, differential effects of metformin, and metabolomic differences [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5255. doi:10.1158/1538-7445.AM2017-5255

In order to rapidly screen and select lead candidates for in vivo evaluation of lipid nanoparticles (LNPs) for systemic small interfering RNA (siRNA) delivery, an in vitro assay amenable to high-throughput screening (HTS) is developed. The strategy is to mimic the in vivo experience of LNPs after systemic administration, such as interactions with serum components, exposure to endosomal pH environments, and interactions with endosomal membrane lipids. It is postulated that the amount of siRNA released from LNPs after going through these treatments can be used as a screening tool to rank order the effectiveness of siRNA delivery by lipid nanoparticles in vivo. LNPs were incubated with 50% serum from different species (i.e. mouse, rat, or rhesus) at 37°C. The resulting samples were then reacted with anionic, endosomal-mimicking lipids at different pHs. The amount of siRNA released from LNPs was determined using spectrophotometry employing the fluorescent indicator SYBR Gold. Our results indicated that the amount of siRNA liberated was highly dependent upon the species of serum used and the pH to which it was exposed. LNPs treated with mouse serum showed higher levels of siRNA release, as did those subjected to endosomal pH (6.0), compared to physiological pH. Most interestingly, a good correlation between the amount of siRNA released and the in vivo efficacy was observed. In conclusion, an in vitro siRNA release assay was developed to screen and rank order LNPs for in vivo evaluation.