Rational antibody design for undruggable targets using kinetically controlled biomolecular probes.

Several important drug targets, e.g., ion channels and G protein-coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics-sensitive druggability probes in native-state and disease-relevant proteins. By using low-Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.

A novel multi-CDK inhibitor P1446A-05 restricts melanoma growth and produces synergistic effects in combination with MAPK pathway inhibitors.

Nearly 100% of melanomas have a defect in the p16(INK4A):cyclin D-CDK4/6:RB pathway, leading to abnormal cell cycle control and unregulated cellular proliferation. Here, we report that P1446A-05, a novel multi-CDK inhibitor has significant inhibitory activity against cutaneous and uveal melanoma. Mechanistic studies revealed that P1446A-05 inhibits phosphorylation targets of CDK members, and induces cell cycle arrest and apoptosis irrespective of melanoma genotype or phenotype. Additionally, we show preclinical evidence that P1446A-05 can synergize with other small molecule inhibitors previously studied in melanoma. Collectively, these data demonstrate that targeting cell cycle and transcriptional CDKs with a small molecule multi-CDK inhibitor is a viable approach for developing novel anti-melanoma therapeutics.

Cyclin-Dependent Kinase Inhibitor P1446A Induces Apoptosis in a JNK/p38 MAPK-Dependent Manner in Chronic Lymphocytic Leukemia B-Cells.

CDK (cyclin-dependent kinase) inhibitors have shown remarkable activity in CLL, where its efficacy has been linked to inhibition of the transcriptional CDKs (7 and 9) and deregulation of RNA polymerase and short-lived pro-survival proteins such as MCL1. Furthermore, ER (endoplasmic reticulum) stress has been implicated in CDK inhibition in CLL. Here we conducted a pre-clinical study of a novel orally active kinase inhibitor P1446A in CLL B-cells. P1446A inhibited CDKs at nanomolar concentrations and induced rapid apoptosis of CLL cells in vitro, irrespective of chromosomal abnormalities or IGHV mutational status. Apoptosis preceded inactivation of RNA polymerase, and was accompanied by phosphorylation of stress kinases JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase). Pharmacologic inhibitors of JNK/p38 MAPK conferred protection from P1446A-mediated apoptosis. Treatment with P1446A led to a dramatic induction of NOXA in a JNK-dependent manner, and sensitized CLL cells to ABT-737, a BH3-mimetic. We observed concurrent activation of apoptosis stress-inducing kinase 1 (ASK1) and its interaction with inositol-requiring enzyme 1 (IRE1) and tumor necrosis factor receptor-associated factor 2 (TRAF2) in CLL cells treated with P1446A, providing insights into upstream regulation of JNK in this setting. Consistent with previous reports on limited functionality of ER stress mechanism in CLL cells, treatment with P1446A failed to induce an extensive unfolded protein response. This study provides rationale for additional investigations of P1446A in CLL.

A novel SND1-BRAF fusion confers resistance to c-Met inhibitor PF-04217903 in GTL16 cells through [corrected] MAPK activation.

Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling.