A Novel Pan-RAS Inhibitor with a Unique Mechanism of Action Blocks Tumor Growth in Mouse Models of GI Cancer.

Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RAS WT cancer cells with activated RAS from upstream mutations were equally sensitive. Conversely, cells from normal tissues or RAS WT cancer cells harboring downstream BRAF mutations were insensitive. Insensitivity to ADT-007 was attributed to low activated RAS levels and metabolic deactivation by UDP-glucuronosyltransferases expressed in normal cells but repressed in RAS mutant cancer cells. Cellular, biochemical, and biophysical experiments show ADT-007 binds nucleotide-free RAS to block GTP activation of RAS and MAPK/AKT signaling. Local administration of ADT-007 strongly inhibited tumor growth in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer while activating innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for treating RAS-driven cancers. SIGNIFICANCE: ADT-007 is a 1 st -in-class pan-RAS inhibitor with ultra-high potency and unique selectivity for cancer cells with mutant or activated RAS capable of circumventing resistance and activating antitumor immunity. Further development of ADT-007 analogs or prodrugs with oral bioavailability as a generalizable monotherapy or combined with immunotherapy is warranted.

Suppression of Colon Tumorigenesis in Mutant Apc Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic ß-Catenin.

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-COX inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic ß-catenin, inhibited Wnt-induced nuclear translocation of ß-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc+/min-FCCC mouse model of colorectal cancer without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing colorectal cancer. PREVENTION RELEVANCE: PDE10 is overexpressed in colon tumors whereby inhibition activates cGMP/PKG signaling and suppresses Wnt/ß-catenin transcription to selectively induce apoptosis of colon cancer cells. ADT 061 is a novel PDE10 inhibitor that shows promising cancer chemopreventive activity and tolerance in a mouse model of colon cancer.

Phosphodiesterase 10A is overexpressed in lung tumor cells and inhibitors selectively suppress growth by blocking ß-catenin and MAPK signaling.

Phosphodiesterase 10A (PDE10) is a cyclic nucleotide (e.g. cGMP) degrading enzyme highly expressed in the brain striatum where it plays an important role in dopaminergic neurotransmission, but has limited expression and no known physiological function outside the central nervous system. Here we report that PDE10 mRNA and protein levels are strongly elevated in human non-small cell lung cancer cells and lung tumors compared with normal human airway epithelial cells and lung tissue, respectively. Genetic silencing of PDE10 or inhibition by small molecules such as PQ10 was found to selectively inhibit the growth and colony formation of lung tumor cells. PQ10 treatment of lung tumor cells rapidly increased intracellular cGMP levels and activated cGMP-dependent protein kinase (PKG) at concentrations that inhibit lung tumor cell growth. PQ10 also increased the phosphorylation of ß-catenin and reduced its levels, which paralleled the suppression of cyclin D1 and survivin but preceded the activation of PARP and caspase cleavage. PQ10 also suppressed RAS-activated RAF/MAPK signaling within the same concentration range and treatment period as required for cGMP elevation and PKG activation. These results show that PDE10 is overexpressed during lung cancer development and essential for lung tumor cell growth in which inhibitors can selectively induce apoptosis by increasing intracellular cGMP levels and activating PKG to suppress oncogenic ß-catenin and MAPK signaling.

Unique functional and structural properties of the LRRK2 protein ATP-binding pocket.

Pathogenic mutations in the LRRK2 gene can cause late-onset Parkinson disease. The most common mutation, G2019S, resides in the kinase domain and enhances activity. LRRK2 possesses the unique property of cis-autophosphorylation of its own GTPase domain. Because high-resolution structures of the human LRRK2 kinase domain are not available, we used novel high-throughput assays that measured both cis-autophosphorylation and trans-peptide phosphorylation to probe the ATP-binding pocket. We disclose hundreds of commercially available activity-selective LRRK2 kinase inhibitors. Some compounds inhibit cis-autophosphorylation more strongly than trans-peptide phosphorylation, and other compounds inhibit G2019S-LRRK2 more strongly than WT-LRRK2. Through exploitation of structure-activity relationships revealed through high-throughput analyses, we identified a useful probe inhibitor, SRI-29132 (11). SRI-29132 is exquisitely selective for LRRK2 kinase activity and is effective in attenuating proinflammatory responses in macrophages and rescuing neurite retraction phenotypes in neurons. Furthermore, the compound demonstrates excellent potency, is highly blood-brain barrier-permeant, but suffers from rapid first-pass metabolism. Despite the observed selectivity of SRI-29132, docking models highlighted critical interactions with residues conserved in many protein kinases, implying a unique structural configuration for the LRRK2 ATP-binding pocket. Although the human LRRK2 kinase domain is unstable and insoluble, we demonstrate that the LRRK2 homolog from ameba can be mutated to approximate some aspects of the human LRRK2 ATP-binding pocket. Our results provide a rich resource for LRRK2 small molecule inhibitor development. More broadly, our results provide a precedent for the functional interrogation of ATP-binding pockets when traditional approaches to ascertain structure prove difficult.

Synthesis and SAR of pyridothiazole substituted pyrimidine derived HCV replication inhibitors.

Introduction of a nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzothiazole inhibitor 1, resulted in the discovery of the more potent pyridothiazole analogues 3. The potency and PK properties of the compounds were attenuated by the introductions of various functionalities at the R(1), R(2) or R(3) positions of the molecule (compound 3). Inhibitors 38 and 44 displayed excellent potency, selectivity (GAPDH/MTS CC(50)), PK parameters in all species studied, and cross genotype activity.

New insights into the design of inhibitors of human S-adenosylmethionine decarboxylase: studies of adenine C8 substitution in structural analogues of S-adenosylmethionine.

S-adenosylmethionine decarboxylase (AdoMetDC) is a critical enzyme in the polyamine biosynthetic pathway and depends on a pyruvoyl group for the decarboxylation process. The crystal structures of the enzyme with various inhibitors at the active site have shown that the adenine base of the ligands adopts an unusual syn conformation when bound to the enzyme. To determine whether compounds that favor the syn conformation in solution would be more potent AdoMetDC inhibitors, several series of AdoMet substrate analogues with a variety of substituents at the 8-position of adenine were synthesized and analyzed for their ability to inhibit hAdoMetDC. The biochemical analysis indicated that an 8-methyl substituent resulted in more potent inhibitors, yet most other 8-substitutions provided no benefit over the parent compound. To understand these results, we used computational modeling and X-ray crystallography to study C(8)-substituted adenine analogues bound in the active site.

The 3-hydroxy group and 4,5-trans double bond of sphingomyelin are essential for modulation of galactosylceramide transmembrane asymmetry.

The structural features of SPM that control the transbilayer distribution of beta-GalCer in POPC vesicles were investigated by (13)C- and (31)P-NMR spectroscopy using lipid analogs that share physical similarities with GalCer or SPM. The SPM analogs included N-palmitoyl-4,5-dihydro-SPM, 3-deoxy-SPM, 1-alkyl-2-amidophosphatidylcholine, and dipalmitoylphosphatidylcholine, a popular model "raft lipid". The transbilayer distributions of the SPM analogs and SPM in POPC vesicles were similar by (31)P-NMR. To observe the dramatic change in GalCer transbilayer distribution that occurs when SPM is included in POPC vesicles, the 3-OH group, 4,5-trans double bond, and amide linkage all were required in SPM. However, inclusion of 2 and 10 mol % dihydroSPM in SPM/POPC (1:1) vesicles mitigated and completely abrogated the effect of SPM on the transbilayer distribution of GalCer. Despite sharing some structural features with GalCer and localizing preferentially to the inner leaflet of POPC vesicles, dimyristoylphosphatidylethanolamine did not undergo a change in transbilayer distribution when SPM was incorporated into the vesicles. The results support the hypothesis that specific interactions may be favored among select sphingolipids in curvature-stressed membranes and emphasize the potential importance of the SPM-dihydroSPM ratio in membrane fission and fusion processes associated with vesicle biogenesis and trafficking.

Selective caveolin-1-dependent endocytosis of glycosphingolipids.

We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs in various cell types using pathway-specific inhibitors and colocalization studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on inhibitor studies, all GSLs tested were internalized predominantly (>80%) by a clathrin-independent, caveolar-related mechanism, regardless of cell type. In addition, fluorescent lactosylceramide (LacCer) colocalized with DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The internalization mechanism for GSLs was unaffected by varying the carbohydrate headgroup or sphingosine backbone chain length; however, a fluorescent phosphatidylcholine analog was not internalized via caveolae, suggesting that the GSL ceramide core may be important for caveolar uptake. Internalization of fluorescent LacCer was reduced 80-90% in cell types with low cav-1, but was dramatically stimulated by cav-1 overexpression. However, even in cells with low levels of cav-1, residual LacCer internalization was clathrin independent. In contrast, cholera toxin B subunit (CtxB), which binds endogenous GM1, was internalized via clathrin-independent endocytosis in cells with high cav-1 expression, whereas significant clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent GM1, normally internalized by clathrin-independent endocytosis in HeLa cells with low cav-1, was induced to partially internalize via the clathrin pathway in the presence of CtxB. These results suggest that GSL analogs are selectively internalized via a caveolar-related mechanism in most cell types, whereas CtxB may undergo "pathway switching" when cav-1 levels are low.

Sphingomyelin modulates the transbilayer distribution of galactosylceramide in phospholipid membranes.

The interrelationships among sphingolipid structure, membrane curvature, and glycosphingolipid transmembrane distribution remain poorly defined despite the emerging importance of sphingolipids in curved regions and vesicle buds of biomembranes. Here, we describe a novel approach to investigate the transmembrane distribution of galactosylceramide in phospholipid small unilamellar vesicles by (13)C NMR spectroscopy. Quantitation of the transbilayer distribution of [6-(13)C]galactosylceramide (99.8% isotopic enrichment) was achieved by exposure of vesicles to the paramagnetic ion, Mn(2+). The data show that [6-(13)C]galactosylceramide prefers (70%) the inner leaflet of phosphatidylcholine vesicles. Increasing the sphingomyelin content of the 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles shifted galactosylceramide from the inner to the outer leaflet. The amount of galactosylceramide localized in the inner leaflet decreased from 70% in pure 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles to only 40% in 1-palmitoyl-2-oleoyl-phosphatidylcholine/sphingomyelin (1:2) vesicles. The present study demonstrates that sphingomyelin can dramatically alter the transbilayer distribution of a monohexosylceramide, such as galactosylceramide, in 1-palmitoyl-2-oleoyl-phosphatidylcholine/sphingomyelin vesicles. The results suggest that sphingolipid-sphingolipid interactions that occur even in the absence of cholesterol play a role in controlling the transmembrane distributions of cerebrosides.

Structural elucidation of the specificity of the antibacterial agent triclosan for malarial enoyl acyl carrier protein reductase.

The human malaria parasite Plasmodium falciparum synthesizes fatty acids using a type II pathway that is absent in humans. The final step in fatty acid elongation is catalyzed by enoyl acyl carrier protein reductase, a validated antimicrobial drug target. Here, we report the cloning and expression of the P. falciparum enoyl acyl carrier protein reductase gene, which encodes a 50-kDa protein (PfENR) predicted to target to the unique parasite apicoplast. Purified PfENR was crystallized, and its structure resolved as a binary complex with NADH, a ternary complex with triclosan and NAD(+), and as ternary complexes bound to the triclosan analogs 1 and 2 with NADH. Novel structural features were identified in the PfENR binding loop region that most closely resembled bacterial homologs; elsewhere the protein was similar to ENR from the plant Brassica napus (root mean square for Calphas, 0.30 A). Triclosan and its analogs 1 and 2 killed multidrug-resistant strains of intra-erythrocytic P. falciparum parasites at sub to low micromolar concentrations in vitro. These data define the structural basis of triclosan binding to PfENR and will facilitate structure-based optimization of PfENR inhibitors.