Disruption of angiogenesis and tumor growth with an orally active drug that stabilizes the inactive state of PDGFRβ/B-RAF

Abstract

Kinases are known to regulate fundamental processes in cancer including tumor proliferation, metastasis, neovascularization, and chemoresistance. Accordingly, kinase inhibitors have been a major focus of drug development, and several kinase inhibitors are now approved for various cancer indications. Typically, kinase inhibitors are selected via high-throughput screening using catalytic kinase domains at low ATP concentration, and this process often yields ATP mimetics that lack specificity and/or function poorly in cells where ATP levels are high. Molecules targeting the allosteric site in the inactive kinase conformation (type II inhibitors) provide an alternative for developing selective inhibitors that are physiologically active. By applying a rational design approach using a constrained aminotriazole scaffold predicted to stabilize kinases in the inactive state, we generated a series of selective type II inhibitors of PDGFRβ and B-RAF, important targets for pericyte recruitment and endothelial cell survival, respectively. These molecules were designed in silico and screened for antivascular activity in both cell-based models and a Tg (fli1-EGFP) zebrafish embryogenesis model. Dual inhibition of PDGFRβ and B-RAF cellular signaling demonstrated synergistic antiangiogenic activity in both zebrafish and murine models of angiogenesis, and a combination of previously characterized PDGFRβ and RAF inhibitors validated the synergy. Our lead compound was selected as an orally active molecule with favorable pharmacokinetic properties which demonstrated target inhibition in vivo leading to suppression of murine orthotopic tumors in both the kidney and pancreas.R AF is an important convergent point downstream of FGFR and VEGFR2 signaling in endothelial cells and has previously been shown to play a critical role in endothelial cell survival during angiogenesis (1-3). PDGFRβ is a receptor tyrosine kinase that is essential for promoting proper pericyte function, which stabilizes blood vessels and enables vessel maturation (4-6). We rationalized that inhibition of both RAF and PDGFRβ would produce a potent antiangiogenic effect by targeting the two primary cell types involved in angiogenesis and vascular remodeling, endothelial cells and pericytes, respectively. As such, we designed compounds predicted to inhibit both RAF and PDGFRβ in a selective manner.The recent approval of imatinib (7, 8) (1) and sorafenib (9) (2), inhibitors which target PDGFRβ (10) and/or B-RAF (11, 12), has created much enthusiasm for small molecules that stabilize the inactive kinase conformation (13-15). These two molecules were cocrystallized with their respective targets, B-RAF (16) and Abl (17) kinase domains, and shown to interact in part with the allosteric site in the "DFG-out" conformation, referred to as type II inhibition. Based on the binding mode of sorafenib and imatinib, we synthesized an amino-triazole scaffold designed to target the allosteric site of both PDGFRβ and B-RAF using a combination of in silico screening and in vitro bioass...

Targeting Olfactomedin-like 3 Inhibits Tumor Growth by Impairing Angiogenesis and Pericyte Coverage

“…Inhibition of PDGFb signaling can indirectly reduce tumor vascularization but does not necessarily retard tumor growth (38). Several studies have shown that the dual targeting of endothelial cells and pericytes is more efficient than targeting either cell type alone, even in established or drug-resistant tumors (39)(40)(41). Therefore, blocking Olfml3 holds promise for more effective strategies to control tumor growth by targeting a single molecule that affects 2 distinct cell types within the tumor microenvironment.…”

Sorafenib-Induced Apoptosis of Chronic Lymphocytic Leukemia Cells Is Associated with Downregulation of RAF and Myeloid Cell Leukemia Sequence 1 (Mcl-1)

“…KG5 is a kinase inhibitor of RAF, PDGFR α and β, FLT3 and KIT (29), whereas KG1 targets PDGFR α and β, FLT3 and KIT but not RAF (29). Vatalanib targets KIT, PDGFR and VEGFR (40).…”

“…To identify which other targets of sorafenib are critical for CLL cell viability, and therefore might contribute to sorafenib-mediated cytotoxicity, we compared a set of inhibitors sharing overlapping targets with sorafenib for their ability to abrogate CLL cell viability ( Figure 7A). We tested KG5, a kinase inhibitor of RAF signaling through B-RAF and C-RAF in addition to PDGFR α and β, FLT3 and KIT (29), and KG1, a kinase inhibitor that targets all of these kinases except B-and C-RAF (29) and compared their activity on CLL cell viability. In CLL cells cultured alone (Figure 7B) or in the presence of MSCs (Figures 7C, D), KG5 but not KG1 induced CLL cell apoptosis.…”

Pericyte-targeting drug delivery and tissue engineering

“…[41][42][43][44] Karen et al 41 investigated the effects of PC-targeting small molecules, more specifically, whether valproic acid, a histone deacetylase inhibitor, influenced PC proliferation. Other small molecules including trichostatic A and 2-Et-4-Me-Penta were also tested in comparison with valproic acid.…”

“…The efforts to synthesize novel small molecule drugs have increased since imatinib and sorafenib showed the potential to target PDGFR-β and B-RAF, stabilizing inactive kinase conformations, thus disrupting only PC activity. 42,44 As an example of PC-targeting synthetic small molecules, amino triazole scaffolds act at an allosteric conformational site on kinase due to hydrophobic and specific hydrogen bonding. Using an allosteric approach, synthesized novel small molecules with an amino triazole functional group selectively disrupted angiogenesis and tumor growth by stabilizing the inactive site of PRGFR-β and B-RAF in orthotropic pancreatic and renal carcinoma models.…”