Discovery of KRB-456, a KRAS G12D Switch-I/II Allosteric Pocket Binder That Inhibits the Growth of Pancreatic Cancer Patient-derived Tumors.

Currently, there are no clinically approved drugs that directly thwart mutant KRAS G12D, a major driver of human cancer. Here, we report on the discovery of a small molecule, KRB-456, that binds KRAS G12D and inhibits the growth of pancreatic cancer patient-derived tumors. Protein nuclear magnetic resonance studies revealed that KRB-456 binds the GDP-bound and GCP-bound conformation of KRAS G12D by forming interactions with a dynamic allosteric binding pocket within the switch-I/II region. Isothermal titration calorimetry demonstrated that KRB-456 binds potently to KRAS G12D with 1.5-, 2-, and 6-fold higher affinity than to KRAS G12V, KRAS wild-type, and KRAS G12C, respectively. KRB-456 potently inhibits the binding of KRAS G12D to the RAS-binding domain (RBD) of RAF1 as demonstrated by GST-RBD pulldown and AlphaScreen assays. Treatment of KRAS G12D-harboring human pancreatic cancer cells with KRB-456 suppresses the cellular levels of KRAS bound to GTP and inhibits the binding of KRAS to RAF1. Importantly, KRB-456 inhibits P-MEK, P-AKT, and P-S6 levels in vivo and inhibits the growth of subcutaneous and orthotopic xenografts derived from patients with pancreatic cancer whose tumors harbor KRAS G12D and KRAS G12V and who relapsed after chemotherapy and radiotherapy. These results warrant further development of KRB-456 for pancreatic cancer. SIGNIFICANCE: There are no clinically approved drugs directly abrogating mutant KRAS G12D. Here, we discovered a small molecule, KRB-456, that binds a dynamic allosteric binding pocket within the switch-I/II region of KRAS G12D. KRB-456 inhibits P-MEK, P-AKT, and P-S6 levels in vivo and inhibits the growth of subcutaneous and orthotopic xenografts derived from patients with pancreatic cancer. This discovery warrants further advanced preclinical and clinical studies in pancreatic cancer.

Dual Farnesyl and Geranylgeranyl Transferase Inhibitor Thwarts Mutant KRAS-Driven Patient-Derived Pancreatic Tumors.

PURPOSE: Mutant KRAS is a major driver of pancreatic oncogenesis and therapy resistance, yet KRAS inhibitors are lacking in the clinic. KRAS requires farnesylation for membrane localization and cancer-causing activity prompting the development of farnesyltransferase inhibitors (FTIs) as anticancer agents. However, KRAS becomes geranylgeranylated and active when cancer cells are treated with FTIs. To overcome this geranylgeranylation-dependent resistance to FTIs, we designed FGTI-2734, a RAS C-terminal mimetic dual FT and geranylgeranyltransferase-1 inhibitor (GGTI). EXPERIMENTAL DESIGN: Immunofluorescence, cellular fractionation, and gel shift assays were used to assess RAS membrane association, Western blotting to evaluate FGTI-2734 effects on signaling, and mouse models to demonstrate its antitumor activity. RESULTS: FGTI-2734, but not the selective FTI-2148 and GGTI-2418, inhibited membrane localization of KRAS in pancreatic, lung, and colon human cancer cells. FGTI-2734 induced apoptosis and inhibited the growth in mice of mutant KRAS-dependent but not mutant KRAS-independent human tumors. Importantly, FGTI-2734 inhibited the growth of xenografts derived from four patients with pancreatic cancer with mutant KRAS (2 G12D and 2 G12V) tumors. FGTI-2734 was also highly effective at inhibiting, in three-dimensional cocultures with resistance promoting pancreatic stellate cells, the viability of primary and metastatic mutant KRAS tumor cells derived from eight patients with pancreatic cancer. Finally, FGTI-2734 suppressed oncogenic pathways mediated by AKT, mTOR, and cMYC while upregulating p53 and inducing apoptosis in patient-derived xenografts in vivo. CONCLUSIONS: The development of this novel dual FGTI overcomes a major hurdle in KRAS resistance, thwarting growth of patient-derived mutant KRAS-driven xenografts from patients with pancreatic cancer, and as such it warrants further preclinical and clinical studies.

GSK3 suppression upregulates ß-catenin and c-Myc to abrogate KRas-dependent tumors.

Mutant KRas is a significant driver of human oncogenesis and confers resistance to therapy, underscoring the need to develop approaches that disable mutant KRas-driven tumors. Because targeting KRas directly has proven difficult, identifying vulnerabilities specific for mutant KRas tumors is an important alternative approach. Here we show that glycogen synthase kinase 3 (GSK3) is required for the in vitro and in vivo growth and survival of human mutant KRas-dependent tumors but is dispensable for mutant KRas-independent tumors. Further, inhibiting phosphorylation of GSK3 substrates c-Myc on T58 and ß-catenin on S33/S37/T41 and their subsequent upregulation contribute to the antitumor activity of GSK3 inhibition. Importantly, GSK3 blockade inhibits the in vivo growth of G12D, G12V, and G12C mutant KRas primary and metastatic patient-derived xenografts from pancreatic cancer patients who progressed on chemo- and radiation therapies. This discovery opens new avenues to target mutant KRas-dependent cancers.

Distinct generation, pharmacology, and distribution of sphingosine 1-phosphate and dihydrosphingosine 1-phosphate in human neural progenitor cells.

In vivo and in vitro studies suggest a crucial role for Sphingosine 1-phosphate (S1P) and its receptors in the development of the nervous system. Dihydrosphingosine 1-phosphate (dhS1P), a reduced form of S1P, is an agonist at S1P receptors, but the pharmacology and physiology of dhS1P has not been widely studied. The mycotoxin fumonisin B1 (FB(1)) is a potent inhibitor of ceramide synthases and causes selective accumulation of dihydrosphingosine and dhS1P. Recent studies suggest that maternal exposure to FB(1) correlates with the development of neural tube defects (NTDs) in which the neural epithelial progenitor cell layers of the developing brain fail to fuse. We hypothesize that the altered balance of S1P and dhS1P in neural epithelial cells contributes to the developmental effects of FB(1). The goal of this work was first to define the effect of FB(1) exposure on levels of sphingosine and dh-sphingosine and their receptor-active 1-phosphate metabolites in human embryonic stem cell-derived neural epithelial progenitor (hES-NEP) cells; and second, to define the relative activity of dhS1P and S1P in hES-NEP cells. We found that dhS1P is a more potent stimulator of inhibition of cAMP and Smad phosphorylation than is S1P in neural progenitors, and this difference in apparent potency may be due, in part, to more persistent presence of extracellular dhS1P applied to human neural progenitors rather than a higher activity at S1P receptors. This study establishes hES-NEP cells as a useful human in vitro model system to study the mechanism of FB(1) toxicity and the molecular pharmacology of sphingolipid signaling. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Development of a phosphatase-resistant, L-tyrosine derived LPA1/LPA3 dual antagonist.

Lysophosphatidic acid (LPA) is a bioactive compound that has gained attention due to its role in neoplastic diseases. Our group has developed a potent dual LPA1/LPA3 receptor antagonist, VPC51098 (LPA1 IC(50) = 84 nM, LPA1 IC(50) = 48 nM) that contained a labile phosphate head group. This lability has impaired our evaluation of our scaffold of LPA receptor antagonists in vivo. We wished to replace the phosphate with a potentially more stable head group while retaining potency at both LPA1 and LPA3 to facilitate future in vivo studies. We tested in vitro potency of all head groups including α-methylene, α-fluoromethylene, α-hydroxymethylene; vinyl phosphonates; α-fluoro vinyl phosphonates. The most potent compound was found to be a low micromolar inhibitor VPC51299 that contained a vinyl phosphonate and possessed a half-life of approximately 90 min in rats when dosed intravenously. Herein, we describe the synthesis and initial biological evaluation of these compounds.

Sphingosine kinase type 1 inhibition reveals rapid turnover of circulating sphingosine 1-phosphate.

S1P (sphingosine 1-phosphate) is a signalling molecule involved in a host of cellular and physiological functions, most notably cell survival and migration. S1P, which signals via a set of five G-protein-coupled receptors (S1P1-S1P5), is formed by the action of two SphKs (sphingosine kinases) from Sph (sphingosine). Interfering RNA strategies and SphK1 (sphingosine kinase type 1)-null (Sphk1-/-) mouse studies implicate SphK1 in multiple signalling cascades, yet there is a paucity of potent and selective SphK1 inhibitors necessary to evaluate the effects of rapid onset inhibition of this enzyme. We have identified a set of submicromolar amidine-based SphK1 inhibitors and report using a pair of these compounds to probe the cellular and physiological functions of SphK1. In so doing, we demonstrate that our inhibitors effectively lower S1P levels in cell-based assays, but we have been unable to correlate SphK1 inhibition with changes in cell survival. However, SphK1 inhibition did diminish EGF (epidermal growth factor)-driven increases in S1P levels and Akt (also known as protein kinase B)/ERK (extracellular-signal-regulated kinase) phosphorylation. Finally, administration of the SphK1 inhibitor to wild-type, but not Sphk1-/-, mice resulted in a rapid decrease in blood S1P levels indicating that circulating S1P is rapidly turned over.

Characterization of a sphingosine 1-phosphate receptor antagonist prodrug.

Sphingosine 1-phosphate (S1P) is a phospholipid that binds to a set of G protein-coupled receptors (S1P(1)-S1P(5)) to initiate an array of signaling cascades that affect cell survival, differentiation, proliferation, and migration. On a larger physiological scale, the effects of S1P on immune cell trafficking, vascular barrier integrity, angiogenesis, and heart rate have also been observed. An impetus for the characterization of S1P-initiated signaling effects came with the discovery that FTY720 [fingolimod; 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] modulates the immune system by acting as an agonist at S1P(1). In the course of structure-activity relationship studies to better understand the functional chemical space around FTY720, we discovered conformationally constrained FTY720 analogs that behave as S1P receptor type-selective antagonists. Here, we present a pharmacological profile of a lead S1P(1/3) antagonist prodrug, 1-(hydroxymethyl)-3-(3-octylphenyl)cyclobutane (VPC03090). VPC03090 is phosphorylated by sphingosine kinase 2 to form the competitive antagonist species 3-(3-octylphenyl)-1-(phosphonooxymethyl)cyclobutane (VPC03090-P) as observed in guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays, with effects on downstream S1P receptor signaling confirmed by Western blot and calcium mobilization assays. Oral dosing of VPC03090 results in an approximate 1:1 phosphorylated/alcohol species ratio with a half-life of 30 h in mice. Because aberrant S1P signaling has been implicated in carcinogenesis, we applied VPC03090 in an immunocompetent mouse mammary cancer model to assess its antineoplastic potential. Treatment with VPC03090 significantly inhibited the growth of 4T1 primary tumors in mice. This result calls to attention the value of S1P receptor antagonists as not only research tools but also potential therapeutic agents.

Discovery, biological evaluation, and structure-activity relationship of amidine based sphingosine kinase inhibitors.

Sphingosine 1-phosphate (S1P), a potent phospholipid growth and trophic factor, is synthesized in vivo by two sphingosine kinases. Thus these kinases have been proposed as important drug targets for treatment of hyperproliferative diseases and inflammation. We report here a new class of amidine-based sphingosine analogues that are competitive inhibitors of sphingosine kinases exhibiting varying degrees of enzyme selectivity. These inhibitors display K(I) values in the submicromolar range for both sphingosine kinases and, in cultured vascular smooth muscle cells, decrease S1P levels and initiate growth arrest.

Synthesis and biological evaluation of sphingosine kinase substrates as sphingosine-1-phosphate receptor prodrugs.

In the search for bioactive sphingosine 1-phosphate (S1P) receptor ligands, a series of 2-amino-2-heterocyclic-propanols were synthesized. These molecules were discovered to be substrates of human-sphingosine kinases 1 and 2 (SPHK1 and SPHK2). When phosphorylated, the resultant phosphates showed varied activities at the five sphingosine-1-phosphate (S1P) receptors (S1P(1-5)). Agonism at S1P(1) was displayed in vivo by induction of lymphopenia. A stereochemical preference of the quaternary carbon was crucial for phosphorylation by the kinases and alters binding affinities at the S1P receptors. Oxazole and oxadiazole compounds are superior kinase substrates to FTY720, the prototypical prodrug immunomodulator, fingolimod (FTY720). The oxazole-derived structure was the most active for human SPHK2. Imidazole analogues were less active substrates for SPHKs, but more potent and selective agonists of the S1P(1) receptor; additionally, the imidazole class of compounds rendered mice lymphopenic.

Asymmetric synthesis of conformationally constrained fingolimod analogues--discovery of an orally active sphingosine 1-phosphate receptor type-1 agonist and receptor type-3 antagonist.

Compound 1 (FTY720, Fingolimod) represents a new generation of immunosuppressant that modulates lymphocyte trafficking by interacting with the S1P(1) receptor. Compound 1 also provides a template molecule for studying the molecular biology of S1P receptors and related enzymes (kinases and phosphatases). In this study, two conformationally constrained analogues of 1 ( 3a and 3c) were asymmetrically synthesized in high optical purity. In vitro assessment documented that both analogues are Sphk2 substrates, their phosphorylated species are potent S1P(1) receptor agonists, and 3a-P is a potent S1P 3 antagonist. After oral administration in mice, both compounds evoked lymphopenia, but their duration of action differed markedly.