Kristin K. Brown

Type IIA topoisomerase inhibition by a new class of antibacterial agents

Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 Å crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor ‘bridges’ the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.

A Tale of Two Subunits: How the Neomorphic R132H IDH1 Mutation Enhances Production of αHG

Heterozygously expressed single-point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2, respectively) render these dimeric enzymes capable of producing the novel metabolite α-hydroxyglutarate (αHG). Accumulation of αHG is used as a biomarker for a number of cancer types, helping to identify tumors with similar IDH mutations. With IDH1, it has been shown that one role of the mutation is to increase the rate of conversion from αKG to αHG. To improve our understanding of the function of this mutation, we have detailed the kinetics of the normal (isocitrate to αKG) and neomorphic (αKG to αHG) reactions, as well as the coupled conversion of isocitrate to αHG. We find that the mutant IDH1 is very efficient in this coupled reaction, with the ability to form αHG from isocitrate and NADP(+). The wild type/wild type IDH1 is also able to catalyze this conversion, though it is much more sensitive to concentrations of isocitrate. This difference in behavior can be attributed to the competitive binding between isocitrate and αKG, which is made more favorable for αKG by the neomorphic mutation at arginine 132. Thus, each partial reaction in the heterodimer is functionally isolated from the other. To test whether there is a cooperative effect resulting from the two subunits being in a dimer, we selectively inactivated each subunit with a secondary mutation in the NADP/H binding site. We observed that the remaining, active subunit was unaffected in its associated activity, reinforcing the notion of each subunit being functionally independent. This was further demonstrated using a monomeric form of IDH from Azotobacter vinelandii, which can be shown to gain the same neomorphic reaction when a homologous mutation is introduced into that protein.

A human fatty acid synthase inhibitor binds β-ketoacyl reductase in the keto-substrate site

Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the β-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.

2,3,5-Trisubstituted pyridines as selective AKT inhibitors. Part II: Improved drug-like properties and kinase selectivity from azaindazoles

A novel series of AKT inhibitors containing 2,3,5-trisubstituted pyridines with novel azaindazoles as hinge binding elements are described. Among these, the 4,7-diazaindazole compound 2c has improved drug-like properties and kinase selectivity than those of indazole 1, and displays greater than 80% inhibition of GSK3beta phosphorylation in a BT474 tumor xenograft model in mice.

Efficient Biocatalytic Reductive Aminations by Extending the Imine Reductase Toolbox

Chiral secondary and tertiary amines are ubiquitous in pharmaceutical, fine, and specialty chemicals, but their synthesis typically suffers from significant sustainability and selectivity challenges. Biocatalytic alternatives, such as enzyme‐catalyzed reductive amination, offer several advantages over traditional chemistry, but industrial applicability has not yet been demonstrated. Herein, we report the use of cell lysates expressing imine reductases operating at 1:1 stoichiometry for a variety of amines and carbonyls. A collection of biocatalysts with diversity in coverage of small molecules and direct industrial applicability is presented.

Tetrasubstituted pyridines as potent and selective AKT inhibitors: Reduced CYP450 and hERG inhibition of aminopyridines.

The synthesis and evaluation of tetrasubstituted aminopyridines, bearing novel azaindazole hinge binders, as potent AKT inhibitors are described. Compound 14c was identified as a potent AKT inhibitor that demonstrated reduced CYP450 inhibition and an improved developability profile compared to those of previously described trisubstituted pyridines. It also displayed dose-dependent inhibition of both phosphorylation of GSK3beta and tumor growth in a BT474 tumor xenograft model in mice.

Abstract 5418: Rapid LDH5 inhibition reverses malignant metabolic phenotype and impairs survival of hepatocellular carcinoma cells .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Many cancer cells generate energy by rapidly converting glucose to lactate in the cytosol, a process termed aerobic glycolysis. This metabolic phenotype is recognized as one of the hallmarks of cancer and is enabled by lactate dehydrogenase (LDH), which catalyzes pyruvate to lactate inter-conversion. We find that hepatocellular carcinoma cells express micromolar quantities of LDH5 and that LDH5 protein down-regulation takes about 5 days allowing time for the cells to adapt their metabolism. Since metabolic processes happen in minutes, addressing consequences of LDH5 inhibition by protein down-regulation is inadequate. We screened the GSK compound library and identified a series of quinoline acids as NADH-competitive LDH5 inhibitors. Subsequent lead optimization yielded molecules with LDH5 inhibitory potencies as low as 2-3 nM and selectivity over LDH1 on the order of 10-100-fold. These molecules were cell-permeable and did not have any appreciable activity against a panel of approximately fifty common enzymes, receptors and ion channels, making them the most potent and selective LDH5 inhibitors identified to date. Using these tool inhibitors, we find that rapid chemical inhibition of LDH5 in Snu398 hepatocellular carcinoma cells results in profound inhibition of lactate production and increase in pyruvate as measured by mass spectrometric analysis. Real-time analysis by NMR spectroscopy of live Snu398 cells fed with 13C-labeled glucose demonstrated that chemical LDH5 inhibition led to a rapid decrease in glucose uptake and concomitant slow-down of lactate production. Comprehensive analysis of more than 500 metabolites upon LDH5 inhibition in Snu398 cells revealed that the cytosolic glycolysis pathway was significantly impeded with some up-stream intermediates increasing as much as 40-fold. As the cell lost its ability for cytosolic glucose processing, the TCA cycle activity increased indicating that pyruvate entered the mitochondria and restored their activity resulting in increased oxygen consumption upon LDH5 inhibition. Several pathways that rely on glycolytic and TCA intermediates were also upregulated, including fatty acid metabolism and pentose phosphate pathway. LDH5 inhibition also strongly potentiated PKM2 activity. These profound metabolic alterations greatly impaired cell survival and induced cell death in Snu398 cells. In summary, we have shown that rapid chemical inhibition of LDH5 leads to profound metabolic alterations and impairs cell survival in hepatocellular carcinoma cells making it a compelling strategy for treating solid tumors relying on aerobic glycolysis. Citation Format: Julia Billiard, Roland Annan, Jennifer Ariazi, Jacques Briand, Kristin Brown, Nino Campobasso, Subhas Chakravorty, Deping Chai, Mariela Colon, Elizabeth Davenport, Christopher Dodson, Nathan Gaul, Seth Gilbert, Anthony Jurewicz, Hong Lu, Dean McNulty, Jeanelle McSurdy-Freed, Lisa Miller, Kelvin Nurse, Paru Rao Nuthulaganti, Chad Quinn, Jessica Schneck, Gilbert Scott, Tony Shaw, Christian Sherk, Angela Smallwood, Sharon Sweitzer, James Villa, Gregory Waitt, Richard Wooster, Kevin Duffy. Rapid LDH5 inhibition reverses malignant metabolic phenotype and impairs survival of hepatocellular carcinoma cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5418. doi:10.1158/1538-7445.AM2013-5418