Design and pharmacology of a highly specific dual FMS and KIT kinase inhibitor

Abstract

Inflammation and cancer, two therapeutic areas historically addressed by separate drug discovery efforts, are now coupled in treatment approaches by a growing understanding of the dynamic molecular dialogues between immune and cancer cells. Agents that target specific compartments of the immune system, therefore, not only bring new disease modifying modalities to inflammatory diseases, but also offer a new avenue to cancer therapy by disrupting immune components of the microenvironment that foster tumor growth, progression, immune evasion, and treatment resistance. McDonough feline sarcoma viral (v-fms) oncogene homolog (FMS) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) are two hematopoietic cell surface receptors that regulate the development and function of macrophages and mast cells, respectively. We disclose a highly specific dual FMS and KIT kinase inhibitor developed from a multifaceted chemical scaffold. As expected, this inhibitor blocks the activation of macrophages, osteoclasts, and mast cells controlled by these two receptors. More importantly, the dual FMS and KIT inhibition profile has translated into a combination of benefits in preclinical disease models of inflammation and cancer.

Fig. 1.

Scaffold-based discovery of PLX647 and structural basis of its dual-binding specificity. Two chemical series were derived from the 7-azaindole scaffold. They represent different strategies for realizing a hydrogen bond interaction with the backbone NH group of the DFG motif.

Fig. 2.

PLX647 reduces macrophage accumulation in UUO kidney and blood monocytes. (A) Immunostaining identified large numbers of interstitial F4/80+ macrophages (brown) in the UUO kidney of a vehicle-treated mouse, and a significant proportion of these macrophages expressed the Ki67 antigen (blue) in their nuclei, indicating that they were proliferating. (B) Macrophage accumulation and proliferation was substantially reduced in the UUO kidneys of mice treated with PLX647. (C) Immunostaining was used to assess the accumulation of F4/80+ macrophages in mouse kidneys. Compared with nonligated control kidneys, UUO kidneys showed a 6- to 10-fold increase in the area of renal cortex containing macrophages at day 7. Treatment with PLX647 reduced the macrophage levels in UUO kidneys to a level similar to that found in control kidneys. (D) Peripheral blood monocytes from UUO mice were purified by density centrifugation and assessed for CD68 expression by flow cytometry. Treatment with PLX647 for 7 d reduced circulating blood monocytes by 65% (**P < 0.01, ***P < 0.001).

Fig. 3.

The effect of PLX647 on mouse CIA. (A) Clinical arthritic scores over the course of disease induction and treatment. After a second collagen/adjuvant emulsion injection on day 21, the mice were scored daily for signs of arthritis. One of the five possible scores was assigned for each limb: 0 = no visible effects of arthritis, 1 = edema and/or erythema of one digit, 2 = edema and/or erythema of two joints, 3 = edema and/or erythema of more than two joints, and 4 = severe arthritis of the entire paw and digits. The final score for each animal was the sum of the scores for all four limbs (ranging from 0 to 16). (B) Effect of disease and treatment on joint histology. The histology of the joint was graded using the following scores: 0, normal; 1, minimal; 2, mild; 3, moderate; and 4, severe. Treatment groups that showed significant changes (P < 0.05) relative to the vehicle-treated disease control are indicated by an asterisk.

Fig. 4.

PLX647 inhibits cancer bone pain and osteolysis. (A) Significant allodynia developed in the MRMT-1 inoculated animals at day-14 testing. PLX647 showed a significant reversal of allodynia compared with the vehicle group (P < 0.01). (B) Bone histopathology results of TRAP5b staining and osteolysis measures. Histopathology scoring criteria: 0, normal; 1, mild; 2, moderate; and 3, severe. (C and D) Representative micro-computerized tomography scans of the proximal ends of the tibiae of rats receiving MRMT-1/vehicle-po/saline-sc and MRMT-1/PLX647-po/saline-sc. PLX647 30 mg/kg BID protects rat from cancer-induced bone erosion.