SBIR-STTR Award

Focal Adhesion Kinase (FAK) is a major cancer drug target that is overexpressed in multiple tumor types. FAK is a critical regulator of tumor survival, invasion, proliferation, metastasis, and immune evasion. Current FAK inhibitors that target the ATP-binding pocket of the kinase domain do no effectively inhibit FAK in cancer because FAK also functions as a scaffolding protein. The Focal Adhesion Targeting (FAT) domain of FAK is an interesting alternative drug target due to its requirement for FAK localization, activity, and downstream effects. Disruption and mutation of the FAT domain causes significant effects on tumor cell apoptosis, proliferation, invasion, and metastasis. Specifically, the FAT domain interacts with the alpha helical LD2 and LD4 motifs of Paxillin to promote its biological effects. The structure of the FAT-Paxillin complex has been solved by x-ray crystallography however has been challenging to target with small molecules. In this project, we will use hydrocarbon stapled alpha helical peptides that have the advantage of enhanced proteolytic stability, cell permeability, and potent inhibition of the entire protein interaction interface. We have preliminary data of Stapled Peptide 3 showing low micromolar inhibition of FAK-Paxillin binding and NMR/SPR data validating the binding site of the peptide. In the first specific aim, we will perform structure-activity relationships (SAR) on stapled alpha helical peptides for enhanced binding and competitive inhibition. We will perform SAR on stapled peptides by changing hydrocarbon stapling strategy, modifying N- and C-terminal amino acids, and adding alternative amino acids. In addition, we will utilize molecular modeling to optimize peptide-protein contacts, synthesize stapled peptides of homologous peptide sequences, and characterize biophysical/biochemical properties of stapled peptides. In the second specific aim, we will characterize and optimize lead peptides for cellular effects. We will perform robust assays to measure cell permeability of stapled peptides, characterize peptides for protease resistance and effects on membrane lysis, and test peptides in cellular efficacy assays to assess the effects of stapled peptides on cancer cells. In the third specific aim, we will test lead peptides with in vitro DMPK assays and preliminary in vivo efficacy models. We will characterize peptides using plasma binding, metabolic stability, and CYP inhibition/assays. We will also test lead peptides in mouse xenograft models alone and in combination with chemotherapy. In all, these specific aims will be used to discover peptide inhibitors of FAK non-catalytic function that can be the basis for future clinical development.

Public Health Relevance Statement:
PROJECT NARRATIVE This research will identify innovative peptide-based drugs that target alternative regions of the cancer protein focal adhesion kinase (FAK). It will enhance technologies to target protein-protein interactions in cancer cells and improve upon current therapies that target FAK. Furthermore, this application will help establish peptide chemistry that can be used to create cell permeable peptide drugs.

Project Terms:
Amino Acids; aminoacid; inhibitor/antagonist; inhibitor; Antineoplastic Agents; anticancer drug; anticancer agent; anti-cancer drug; Tumor-Specific Treatment Agents; Neoplastic Disease Chemotherapeutic Agents; Cancer Drug; Antineoplastics; Antineoplastic Drugs; Anti-Cancer Agents; Binding Sites; Reactive Site; Combining Site; Biological Assay; Biologic Assays; Bioassay; Assay; Biophysics; biophysical sciences; biophysical principles; biophysical foundation; bone; Malignant Neoplasms; neoplasm/cancer; malignancy; Malignant Tumor; Cancers; Motility; Cellular Motility; Cellular Migration; Cell Movement; Cell Migration; Cell Locomotion; cell motility; Cell Body; Cells; Chemistry; Clinical Trials; Crystallization; Hydroxyldaunorubicin; Hydroxyl Daunorubicin; Doxorubicina; Adriamycine; 14-Hydroxydaunomycin; Doxorubicin; drug/agent; Pharmaceutic Preparations; Medication; Drugs; Pharmaceutical Preparations; Enzyme Gene; Enzymes; flow cytophotometry; Flow Microfluorometry; Flow Microfluorimetry; Flow Cytofluorometry; Flow Cytofluorometries; Flow Cytometry; Future; Hydrocarbons; In Vitro; heavy metal lead; heavy metal Pb; Pb element; Lead; Murine; Mice Mammals; Mice; Mus; genome mutation; Genetic defect; Genetic Change; Genetic Alteration; Mutation; tumor cell metastasis; cancer metastasis; Secondary Tumor; Secondary Neoplasm; Metastatic Tumor; Metastatic Neoplasm; Metastatic Mass; Metastatic Lesion; Metastasize; Metastasis; Neoplasm Metastasis; Proteolytic Enzymes; Proteinases; Proteases; Protease Gene; Peptidases; Esteroproteases; Peptide Hydrolases; Peptides; Permeability; Phenotype; Transphosphorylases; Phosphotransferase Gene; Kinases; Phosphotransferases; Reticuloendothelial System, Serum, Plasma; Plasma Serum; Blood Plasma; Plasma; tyrosyl protein kinase; hydroxyaryl protein kinase; Tyrosylprotein Kinase; Tyrosine-Specific Protein Kinase; Tyrosine-Protein Kinase Receptor EEK; Tyrosine Kinase; Protein Tyrosine Kinase EEK; Ephrin Type-A Receptor 8 Precursor; Ephrin Type-A Receptor 8; EPH-and ELK-Related Kinase; EPH- and ELK-Related Tyrosine Kinase; Protein Tyrosine Kinase; Proteins; Publishing; Research; social role; Role; structure function relationship; chemical structure function; Structure-Activity Relationship; Testing; focal adhesion-associated protein tyrosine kinase pp125FAK; focal adhesion protein tyrosine kinase; focal adhesion kinase; endogenous substrate pp120; PTK2 Protein Tyrosine Kinase 2; Focal Adhesion Kinase 1; Immunofluorescence; Immunofluorescence Immunologic; Measures; paxillin; Apoptosis; Programmed Cell Death; Apoptosis Pathway; base; improved; Clinical; Phase; Biological; Biochemical; Series; Receptor Protein-Tyrosine Kinases; Tyrosine Kinase Receptors; Tyrosine Kinase Linked Receptors; Transmembrane Receptor Protein Tyrosine Kinase; Receptor Tyrosine Kinase Gene; PTK Receptors; X-Ray Crystallography; Xray Crystallography; X-Ray/Neutron Crystallography; X-Ray Diffraction Crystallography; X Ray Crystallographies; Single Crystal Diffraction; Chemicals; Solid Neoplasm; Solid Tumor; Therapeutic; angiogenesis; Metabolic; cancer cell; Malignant Cell; scaffold; scaffolding; Complex; Side; Membrane; membrane structure; neoplastic cell; Tumor Cell; yeast two hybrid system; Yeast One/Two-Hybrid System; Yeast One Hybrid System; Two Hybrid; molecular modeling; Molecular Models; Molecular Modeling Protein/Amino Acid Biochemistry; Molecular Modeling Nucleic Acid Biochemistry; leupaxin; Structure; novel; Modeling; Property; protein protein interaction; drug development; Focal Adhesions; Focal Contacts; Cell-Matrix Adherens Junctions; Adhesion Plaques; protein aminoacid sequence; peptide sequence; peptide aminoacid sequence; Enhancement Technology; Molecular Interaction; Binding; kinase inhibitor; Lysis; Cytolysis; small molecule; Data; Immune Cell Suppression; Preclinical Testing; pre-clinical testing; in vivo; Scaffolding Protein; Xenograft Model; Modification; resistance mechanism; resistant mechanism; Development; developmental; BT 474; BT474; migration; N-terminal; NH2-terminal; innovation; innovative; innovate; Resistance; resistant; C-terminal; chemotherapy; tumor; MDA MB 231; MDA-MB231; MDA-231; overexpression; overexpress; FDA approved; in vitro testing; comparative efficacy; compare efficacy; Drug Targeting; targeted treatment; targeted therapy; targeted therapeutic agents; targeted therapeutic; targeted drug treatments; targeted drug therapy; peptide drug; therapeutic peptide; clinical development; Immune Evasion; lead optimization

Melanoma is the deadliest form of skin cancer, affecting an estimated 1.2 million Americans. The disease has a high propensity for dissemination, and metastatic melanoma has a dismal prognosis, with a median survival of only 5-8 months. Healthcare costs for melanoma in 2021 are projected to reach nearly $4 billion, with a rising incidence rate. Despite considerable efforts in recent years to develop more effective targeted and immunotherapies against melanoma, the 5-year survival rate for stage IV melanoma patients remains around 20%. Thus, there is a significant unmet clinical need for novel treatment strategies to improve outcomes for patients with melanoma. Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase and scaffolding protein considered to be a highly promising cancer drug target due to its involvement in multiple hallmarks of cancer, including migration, invasion, metastasis, apoptosis, proliferation, angiogenesis, and immune-cell suppression. FAK is overexpressed in 60-80% of multiple solid tumors, including breast, colon, ovarian, and pancreatic cancer, and is massively upregulated in human melanoma samples. Despite its potential as a therapeutic target, the FAK inhibitors developed to date only target the ATP-binding pocket of the kinase domain and have shown little clinical success. However, recent proof-of-principle studies have shown that the focal adhesion targeting (FAT) scaffolding domain of FAK is an essential regulator of melanoma survival, growth, and metastasis. Additionally, FAKnostics has preliminary data showing that FAT domain inhibition selectively decreases viability of NRAS-mutant melanoma cells, a subset of melanoma that represents 20-30% of all cases and has no effective targeted therapies. On the basis of these findings, FAKnostics has identified a first-in-class series of peptidic FAK inhibitors that directly target the FAT domain of FAK and have significant anti-cancer effects in NRAS- mutant melanoma. In Phase I, FAKnostics identified a stapled peptide candidate with ~5,000-fold higher binding affinity to the FAK FAT domain versus the native paxillin LD2 motif. Furthermore, we have demonstrated proof- of-concept of this approach by confirming in vivo efficacy in a syngeneic mouse model. Through this Phase II project, FAKnostics seeks to continue the development of this treatment approach through the following specific aims: 1) Optimize lead peptides through iterations of medicinal chemistry to improve cellular potency and drug- like properties; 2) Evaluate top optimized peptides in pharmacokinetic and in vivo efficacy studies to select preclinical candidate peptide; 3) Complete formulation studies of final clinical candidate peptide and evaluate safety/toxicology in rats; and 4) Determine patient population suitable for clinical trials using melanoma patient- derived xenograft and syngeneic efficacy modelsand determine optimal combinational treatment regimen. Upon successful completion of these aims, FAKnostics intends to initiate GMP manufacturing and GLP safety/toxicology studies in preparation for an FDA IND application and a clinical trial.

Public Health Relevance Statement:
Narrative FAKnostics is developing a first-in-class peptidic inhibitor of focal adhesion kinase (FAK) as a clinical candidate for the treatment of melanoma and other solid tumors. Melanoma is the most lethal form of skin cancer and is estimated to affect 1.2 million Americans. This project will further advance the development of a novel treatment strategy that aims to improve outcomes for patients with melanoma.

Project Terms:
Affect; inhibitor/antagonist; inhibitor; Antineoplastic Agents; Anti-Cancer Agents; Antineoplastic Drugs; Antineoplastics; Cancer Drug; Neoplastic Disease Chemotherapeutic Agents; Tumor-Specific Treatment Agents; anti-cancer drug; anticancer agent; anticancer drug; malignant breast neoplasm; Breast Cancer; malignant breast tumor; Malignant Neoplasms; Cancers; Malignant Tumor; malignancy; neoplasm/cancer; Skin Cancer; Malignant Skin Neoplasm; malignant skin tumor; Cell membrane; Cytoplasmic Membrane; Plasma Membrane; plasmalemma; Cells; Cell Body; Pharmaceutical Chemistry; Medicinal Chemistry; Pharmaceutic Chemistry; Clinical Research; Clinical Study; Clinical Trials; Disease; Disorder; Drug Combinations; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Growth; Generalized Growth; Tissue Growth; ontogeny; Human; Modern Man; Immunotherapy; Immune mediated therapy; Immunologically Directed Therapy; immune therapeutic approach; immune therapeutic interventions; immune therapeutic regimens; immune therapeutic strategy; immune therapy; immune-based therapies; immune-based treatments; immuno therapy; Incidence; intravenous injection; Lead; Pb element; heavy metal Pb; heavy metal lead; Liposomes; Liposomal; melanoma; Malignant Melanoma; Neoplasm Metastasis; Metastasis; Metastasize; Metastatic Lesion; Metastatic Mass; Metastatic Neoplasm; Metastatic Tumor; Secondary Neoplasm; Secondary Tumor; cancer metastasis; tumor cell metastasis; Patients; Peptides; Permeability; Drug Kinetics; Pharmacokinetics; Pharmacology; Phosphotransferases; Kinases; Phosphotransferase Gene; Transphosphorylases; Rattus; Common Rat Strains; Rat; Rats Mammals; Role; social role; Safety; Solubility; Survival Rate; Toxicology; Treatment Protocols; Treatment Regimen; Treatment Schedule; Work; Focal Adhesion Kinase 1; PTK2 Protein Tyrosine Kinase 2; endogenous substrate pp120; focal adhesion kinase; focal adhesion protein tyrosine kinase; focal adhesion-associated protein tyrosine kinase pp125FAK; Drug Delivery Systems; Drug Delivery; Health Care Costs; Health Costs; Healthcare Costs; paxillin; Apoptosis; Apoptosis Pathway; Programmed Cell Death; base; improved; Acute; Clinical; Phase; Biochemical; Series; Active Sites; Evaluation; Funding; Metastatic Melanoma; Solid Neoplasm; Solid Tumor; angiogenesis; Metabolic; scaffolding; scaffold; Malignant Pancreatic Neoplasm; Pancreas Cancer; Pancreatic Cancer; pancreatic malignancy; Malignant neoplasm of pancreas; Hour; Complex; meetings; American; mutant; success; aqueous; Toxicities; Toxic effect; novel; Colon Cancer; Colonic Carcinoma; cancer in the colon; Colon Carcinoma; Regulation; Modeling; Sampling; Property; Adhesion Plaques; Cell-Matrix Adherens Junctions; Focal Contacts; Focal Adhesions; Malignant Ovarian Neoplasm; Malignant Ovarian Tumor; Malignant Tumor of the Ovary; Ovary Cancer; ovarian cancer; Malignant neoplasm of ovary; Molecular Interaction; Binding; kinase inhibitor; Tumor Load; Tumor Burden; Affinity; Data; Immune Cell Suppression; Melanoma Cell; in vitro Assay; in vivo; Cancer Etiology; Cancer Cause; Patient-Focused Outcomes; Patient outcome; Patient-Centered Outcomes; Scaffolding Protein; Small Business Technology Transfer Research; STTR; Preparation; Development; developmental; pre-clinical; preclinical; Advanced Development; efficacy evaluation; efficacy analysis; efficacy assessment; efficacy examination; evaluate efficacy; examine efficacy; nanoparticle; nano particle; nano-sized particle; nanosized particle; migration; Resistance; resistant; Oncogenic; mouse model; murine model; therapeutic target; therapy development; develop therapy; intervention development; treatment development; high risk; overexpression; overexpress; patient population; treatment strategy; animal efficacy; screening; Drug Targeting; targeted treatment; targeted drug therapy; targeted drug treatments; targeted therapeutic; targeted therapeutic agents; targeted therapy; Formulation; improved outcome; clinical candidate; efficacy study; anti-PD-1; aPD-1; aPD1; anti programmed cell death 1; anti-PD1; anti-programmed cell death protein 1; antiPD-1; antiPD1; aPD-1; aPD1; lead optimization; anti-cancer; anticancer; anti-CTLA4; aCTLA-4; aCTLA4; anti-CTLA-4; a-CTLA-4; a-CTLA4; aCTLA-4; aCTLA4; patient derived xenograft model; PDX model; Patient derived xenograft; Prognosis