Discovery of Pemigatinib: A Potent and Selective Fibroblast Growth Factor Receptor (FGFR) Inhibitor.

Aberrant activation of FGFR has been linked to the pathogenesis of many tumor types. Selective inhibition of FGFR has emerged as a promising approach for cancer treatment. Herein, we describe the discovery of compound 38 (INCB054828, pemigatinib), a highly potent and selective inhibitor of FGFR1, FGFR2, and FGFR3 with excellent physiochemical properties and pharmacokinetic profiles. Pemigatinib has received accelerated approval from the U.S. Food and Drug Administration for the treatment of adults with previously treated, unresectable locally advanced or metastatic cholangiocarcinoma with a FGFR2 fusion or other rearrangement. Additional clinical trials are ongoing to evaluate pemigatinib in patients with FGFR alterations.

INCB054828 (pemigatinib), a potent and selective inhibitor of fibroblast growth factor receptors 1, 2, and 3, displays activity against genetically defined tumor models.

Alterations in fibroblast growth factor receptor (FGFR) genes have been identified as potential driver oncogenes. Pharmacological targeting of FGFRs may therefore provide therapeutic benefit to selected cancer patients, and proof-of-concept has been established in early clinical trials of FGFR inhibitors. Here, we present the molecular structure and preclinical characterization of INCB054828 (pemigatinib), a novel, selective inhibitor of FGFR 1, 2, and 3, currently in phase 2 clinical trials. INCB054828 pharmacokinetics and pharmacodynamics were investigated using cell lines and tumor models, and the antitumor effect of oral INCB054828 was investigated using xenograft tumor models with genetic alterations in FGFR1, 2, or 3. Enzymatic assays with recombinant human FGFR kinases showed potent inhibition of FGFR1, 2, and 3 by INCB054828 (half maximal inhibitory concentration [IC50] 0.4, 0.5, and 1.0 nM, respectively) with weaker activity against FGFR4 (IC50 30 nM). INCB054828 selectively inhibited growth of tumor cell lines with activation of FGFR signaling compared with cell lines lacking FGFR aberrations. The preclinical pharmacokinetic profile suggests target inhibition is achievable by INCB054828 in vivo with low oral doses. INCB054828 suppressed the growth of xenografted tumor models with FGFR1, 2, or 3 alterations as monotherapy, and the combination of INCB054828 with cisplatin provided significant benefit over either single agent, with an acceptable tolerability. The preclinical data presented for INCB054828, together with preliminary clinical observations, support continued investigation in patients with FGFR alterations, such as fusions and activating mutations.

Phosphorylation State-Dependent High Throughput Screening of the c-Met Kinase.

High-throughput screening (HTS) of ~50,000 chemical compounds against phosphorylated and unphosphorylated c-Met, a tyrosine kinase receptor for hepatocyte growth factor (HGF), was carried out in order to compare hit rates, hit potencies and also to explore scaffolds that might serve as potential leads targeting only the unphosphorylated form of the enzyme. The hit rate and potency for the confirmed hit molecules were higher for the unphosphoryalted form of c-Met. While the target of small molecule inhibitor discovery efforts has traditionally been the phosphorylated form, there are now examples of small molecules that target unphosphorylated kinases. Screening for inhibitors of unphosphorylated kinases may represent a complementary approach for prioritizing chemical scaffolds for hit-to-lead follow ups.

Isothiazolidinone inhibitors of PTP1B containing imidazoles and imidazolines.

The structure-based design and synthesis of isothiazolidinone (IZD) inhibitors of PTP1B containing imidazoles and imidazolines and their modification to interact with the B site of PTP1B are described here. The X-ray crystal structures of 3I and 4I complexed with PTP1B were solved and revealed the inhibitors are interacting extensively with the B site of the enzyme.

High-throughput determination of mode of inhibition in lead identification and optimization.

After finishing the primary high-throughput screening, the screening team is often faced with thousands of hits to be evaluated further. Effective filtering of these hits is crucial in identifying leads. Mode of inhibition (MOI) study is extremely useful in validating whether the observed compound activity is specific to the biological target. In this article, the authors describe a high-throughput MOI determination method for evaluating thousands of compounds using an existing screening infrastructure. Based on enzyme or receptor kinetics theory, the authors developed the method by measuring the ratio of IC(50) or percent inhibition at 2 carefully chosen substrate or ligand concentrations to define an inhibitor as competitive, uncompetitive, or noncompetitive. This not only facilitates binning of HTS hits according to their MOI but also greatly expands HTS utility in support of the medicinal chemistry team's lead optimization practice. Three case studies are presented to demonstrate how the method was applied successfully in 3 discovery programs targeting either an enzyme or a G-protein-coupled receptor.

Structural basis for inhibition of protein-tyrosine phosphatase 1B by isothiazolidinone heterocyclic phosphonate mimetics.

Crystal structures of protein-tyrosine phosphatase 1B in complex with compounds bearing a novel isothiazolidinone (IZD) heterocyclic phosphonate mimetic reveal that the heterocycle is highly complementary to the catalytic pocket of the protein. The heterocycle participates in an extensive network of hydrogen bonds with the backbone of the phosphate-binding loop, Phe(182) of the flap, and the side chain of Arg(221). When substituted with a phenol, the small inhibitor induces the closed conformation of the protein and displaces all waters in the catalytic pocket. Saturated IZD-containing peptides are more potent inhibitors than unsaturated analogs because the IZD heterocycle and phenyl ring directly attached to it bind in a nearly orthogonal orientation with respect to each other, a conformation that is close to the energy minimum of the saturated IZD-phenyl moiety. These results explain why the heterocycle is a potent phosphonate mimetic and an ideal starting point for designing small nonpeptidic inhibitors.

Structure-based design and discovery of protein tyrosine phosphatase inhibitors incorporating novel isothiazolidinone heterocyclic phosphotyrosine mimetics.

Structure-based design led to the discovery of novel (S)-isothiazolidinone ((S)-IZD) heterocyclic phosphotyrosine (pTyr) mimetics that when incorporated into dipeptides are exceptionally potent, competitive, and reversible inhibitors of protein tyrosine phosphatase 1B (PTP1B). The crystal structure of PTP1B in complex with our most potent inhibitor 12 revealed that the (S)-IZD heterocycle interacts extensively with the phosphate binding loop precisely as designed in silico. Our data provide strong evidence that the (S)-IZD is the most potent pTyr mimetic reported to date.

Cellular pharmacology of D-d4FC, a nucleoside analogue active against drug-resistant HIV.

The backbone of effective highly active antiretroviral therapy regimens for the treatment of HIV infections currently contains at least two nucleosides. Among the features that influence the potency of each component of a regimen and the overall efficacy of the combination are the cellular uptake and bioconversion of nucleoside analogues to their active triphosphate form, and the extent of possible interactions in these steps that might occur when more than one nucleoside is used in a regimen. D-d4FC (Reverset), a new cytidine analogue with the ability to inhibit many nucleoside-resistant viral variants, was examined for these parameters. In phytohemaglutinin-stimulated human peripheral blood mononuclear cells, D-d4FC was taken up in a rapid (8 h to 50% maximal value), saturable (plateau above 10 microM parent nucleoside concentration) process, resulting in levels of D-d4FC triphosphate that should provide potent antiviral activity against a variety of virus genotypes. Based on measurement of antiviral effects in cell culture, additive and in some cases, synergistic interactions were observed with protease inhibitors, non-nucleoside reverse transcriptase inhibitors or other nucleosides, including cytidine analogues.

Synthesis, antiviral activity and pharmacokinetics of P1/P1' substituted 3-aminoindazole cyclic urea HIV protease inhibitors.

A series of P1/P1' substituted cyclic urea analogues were prepared in an attempt to increase the intra-cellular antiviral potency of the nonsymmetrical 3-aminoindazoles DMP 850 and DMP 851. The effect of alkyl substitution of the P1/P1' residues on cellular antiviral potency, protein binding, resistance profile and pharmacokinetics are described.

Potency of nonnucleoside reverse transcriptase inhibitors (NNRTIs) used in combination with other human immunodeficiency virus NNRTIs, NRTIs, or protease inhibitors.

Efavirenz and a series of related quinazolinone nonnucleoside inhibitors of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) were evaluated in a series of two-drug combinations with several nucleoside RT inhibitors (NRTIs), nonnucleoside RT inhibitors (NNRTIs), and protease inhibitors (PIs). These combinations were tested in an established HIV-1 RT enzyme assay and a cell-based yield reduction assay with HIV-1 (replicative form [RF])-infected MT-2 cells. Synergy, additivity, and antagonism were determined in the two different assay systems by the method of Chou and Talalay (T.-C. Chou and P. Talalay, Adv. Enzyme Reg. 22:27-55, 1984). Efavirenz, DPC082, DPC083, DPC961, and DPC963 used in combination with the NRTIs zidovudine and lamivudine acted synergistically to inhibit RT activity in the HIV-1 RT enzyme assay and additively to slightly synergistically to inhibit HIV-1 (RF) replication in the yield reduction assay. The five NNRTIs in combination with the PI nelfinavir acted additively in the yield reduction assay to inhibit HIV-1 replication. Interestingly, efavirenz in combination with a second NNRTI acted additively to inhibit HIV-1 RT function in the enzyme assay, while it acted antagonistically to inhibit HIV-1 (RF) replication in the yield reduction assay. These data suggest that antiretroviral combination regimens containing multiple NNTRIs should be given thorough consideration before being used.