Water Networks and Correlated Motions in Mutant Isocitrate Dehydrogenase 1 (IDH1) Are Critical for Allosteric Inhibitor Binding and Activity.

Point mutations in human isocitrate dehydrogenase 1 (IDH1) can drive malignancies, including lower-grade gliomas and secondary glioblastomas, chondrosarcomas, and acute myeloid leukemias. These mutations, which usually affect residue R132, ablate the normal activity of catalyzing the NADP+-dependent oxidation of isocitrate to α-ketoglutarate (αKG) while also acquiring a neomorphic activity of reducing αKG to d-2-hydroxyglutarate (D2HG). Mutant IDH1 can be selectively therapeutically targeted due to structural differences that occur in the wild type (WT) versus mutant form of the enzyme, though the full mechanisms of this selectivity are still under investigation. Here we probe the mechanistic features of the neomorphic activity and selective small molecule inhibition through a new lens, employing WaterMap and molecular dynamics simulations. These tools identified a high-energy path of water molecules connecting the inhibitor binding site with the αKG and NADP+ binding sites in mutant IDH1. This water path aligns spatially with the α10 helix from WT IDH1 crystal structures. Mutating residues at the termini of this water path specifically disrupted inhibitor binding and/or D2HG production, revealing additional key residues to consider in optimizing druglike molecules against mutant IDH1. Taken together, our findings from molecular simulations and mutant enzyme kinetic assays provide insight into how disrupting water paths through enzyme active sites can impact not only inhibitor potency but also substrate recognition and activity.

K-RasG12D Has a Potential Allosteric Small Molecule Binding Site.

KRAS is the most commonly mutated oncogene in human cancer, with particularly high mutation frequencies in pancreatic cancers, colorectal cancers, and lung cancers [Ostrem, J. M., and Shokat, K. M. (2016) Nat. Rev. Drug Discovery 15, 771-785]. The high prevalence of KRAS mutations and its essential role in many cancers make it a potentially attractive drug target; however, it has been difficult to create small molecule inhibitors of mutant K-Ras proteins. Here, we identified a putative small molecule binding site on K-RasG12D using computational analyses of the protein structure and then used a combination of computational and biochemical approaches to discover small molecules that may bind to this pocket, which we have termed the P110 site, due to its adjacency to proline 110. We confirmed that one compound, named K-Ras allosteric ligand KAL-21404358, bound to K-RasG12D, as measured by microscale thermophoresis, a thermal shift assay, and nuclear magnetic resonance spectroscopy. KAL-21404358 did not bind to four mutants in the P110 site, supporting our hypothesis that KAL-21404358 binds to the P110 site of K-RasG12D. This compound impaired the interaction of K-RasG12D with B-Raf and disrupted the RAF-MEK-ERK and PI3K-AKT signaling pathways. We synthesized additional compounds, based on the KAL-21404358 scaffold with more potent binding and greater aqueous solubility. In summary, these findings suggest that the P110 site is a potential site for binding of small molecule allosteric inhibitors of K-RasG12D.

Multivalent Small-Molecule Pan-RAS Inhibitors.

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.

Simplified silvestrol analogues with potent cytotoxic activity.

The complex natural products silvestrol (1) and episilvestrol (2) are inhibitors of translation initiation through binding to the DEAD-box helicase eukaryotic initiation factor 4A (eIF4A). Both compounds are potently cytotoxic to cancer cells in vitro, and 1 has demonstrated efficacy in vivo in several xenograft cancer models. Here we show that 2 has limited plasma membrane permeability and is metabolized in liver microsomes in a manner consistent with that reported for 1. In addition, we have prepared a series of analogues of these compounds where the complex pseudo-sugar at C6 has been replaced with chemically simpler moieties to improve drug-likeness. Selected compounds from this work possess excellent activity in biochemical and cellular translation assays with potent activity against leukemia cell lines.

Induction of a stringent metabolic response in intracellular stages of Leishmania mexicana leads to increased dependence on mitochondrial metabolism.

Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using (13)C-stable isotope resolved metabolomics and (2)H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid ß-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.

Synthesis of biotinylated episilvestrol: highly selective targeting of the translation factors eIF4AI/II.

Silvestrol (1) and episilvestrol (2) are protein synthesis inhibitors, and the former has shown efficacy in multiple mouse models of cancer; however, the selectivity of these potent cytotoxic natural products has not been described. Herein, it is demonstrated that eukaryotic initiation factors eIF4AI/II were the only proteins detected to bind silvestrol (1) and biotinylated episilvestrol (9) by affinity purification. Our study demonstrates the remarkable selectivity of these promising chemotherapeutics.