William T. Windsor

Discovery of a Novel ERK Inhibitor with Activity in Models of Acquired Resistance to BRAF and MEK Inhibitors

The high frequency of activating RAS or BRAF mutations in cancer provides strong rationale for targeting the mitogen-activated protein kinase (MAPK) pathway. Selective BRAF and MAP-ERK kinase (MEK) inhibitors have shown clinical efficacy in patients with melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the extracellular signal-regulated kinase (ERK) signaling pathway. Here, we describe the identification and characterization of SCH772984, a novel and selective inhibitor of ERK1/2 that displays behaviors of both type I and type II kinase inhibitors. SCH772984 has nanomolar cellular potency in tumor cells with mutations in BRAF, NRAS, or KRAS and induces tumor regressions in xenograft models at tolerated doses. Importantly, SCH772984 effectively inhibited MAPK signaling and cell proliferation in BRAF or MEK inhibitor-resistant models as well as in tumor cells resistant to concurrent treatment with BRAF and MEK inhibitors. These data support the clinical development of ERK inhibitors for tumors refractory to MAPK inhibitors.

Dinaciclib (SCH 727965), a Novel and Potent Cyclin-Dependent Kinase Inhibitor

Cyclin-dependent kinases (CDK) are key positive regulators of cell cycle progression and attractive targets in oncology. SCH 727965 inhibits CDK2, CDK5, CDK1, and CDK9 activity in vitro with IC50 values of 1, 1, 3, and 4 nmol/L, respectively. SCH 727965 was selected as a clinical candidate using a functional screen in vivo that integrated both efficacy and safety parameters. Compared with flavopiridol, SCH 727965 exhibits superior activity with an improved therapeutic index. In cell-based assays, SCH 727965 completely suppressed retinoblastoma phosphorylation, which correlated with apoptosis onset and total inhibition of bromodeoxyuridine incorporation in >100 tumor cell lines of diverse origin and background. Moreover, short exposures to SCH 727965 were sufficient for long-lasting cellular effects. SCH 727965 induced regression of established solid tumors in a range of mouse models following intermittent scheduling of doses below the maximally tolerated level. This was associated with modulation of pharmacodynamic biomarkers in skin punch biopsies and rapidly reversible, mechanism-based effects on hematologic parameters. These results suggest that SCH 727965 is a potent and selective CDK inhibitor and a novel cytotoxic agent. Mol Cancer Ther; 9(8); 2344–53. ©2010 AACR.

Azapeptides as inhibitors of the hepatitis C virus NS3 serine protease.

Truncation and substitution SAR studies of azapeptide-based inhibitors of the Hepatitis C virus (HCV) NS3 serine protease have been performed. These azapeptides were designed from the HCV polyproteins NS5A-NS5B trans cleavage junction and contained an azaamino acid residue at the P1 position. These azapeptides exhibited predominantly non-acylating, competitive inhibition, contrary to classical azapeptides.

3-Phosphoinositide-Dependent Protein Kinase-1 Regulates Proliferation and Survival of Cancer Cells with an Activated Mitogen-Activated Protein Kinase Pathway

Engagement of cell surface receptor tyrosine kinases by insulin and growth factors activates phosphatidylinositol 3-kinase (PI3K) and generates the second messenger, phosphatidylinositol 3,4,5-trisphosphate. This second messenger leads to the recruitment of 3-phosphoinositide–dependent protein kinase-1 (PDK1) to the proximal side of the plasma membrane, which results in the activation of AKT kinase. In addition, PDK1 can phosphorylate numerous other kinases, including p90RSK, a kinase downstream of mitogen-activated protein kinase (MAPK) that is important for cell proliferation and survival. Previous studies have shown that the loss of PDK1 sensitizes tumor cells to chemotherapeutic agents and radiation but have not focused on delineating the contribution of PDK1 to pathway-specific mutations associated with various cancers other than the PI3K/AKT pathway. In this study, we show that the reduction of PDK1 by RNAi in melanoma and colon cancer cell lines activated in the MAPK pathway results in significant cell growth inhibition and apoptosis. Furthermore, PDK1 reduction in tumor cells resulted in impaired PAK kinase signaling, altered actin polymerization, and reduced cell migration. These studies show that PDK1 plays a pivotal role in MAPK and PI3K signaling in tumor cells. Mol Cancer Res; 8(3); 421–32

Discovery of Novel, Dual Mechanism ERK Inhibitors by Affinity Selection Screening of an Inactive Kinase

An affinity-based mass spectrometry screening technology was used to identify novel binders to both nonphosphorylated and phosphorylated ERK2. Screening of inactive ERK2 identified a pyrrolidine analogue 1 that bound to both nonphosphorylated and phosphorylated ERK2 and inhibited ERK2 kinase activity. Chemical optimization identified compound 4 as a novel, potent, and highly selective ERK1,2 inhibitor which not only demonstrated inhibition of phosphorylation of ERK substrate p90RSK but also demonstrated inhibition of ERK1,2 phosphorylation on the activation loop. X-ray cocrystallography revealed that upon binding of compound 4 to ERK2, Tyr34 undergoes a rotation (flip) along with a shift in the poly-Gly rich loop to create a new binding pocket into which 4 can bind. This new binding mode represents a novel mechanism by which high affinity ATP-competitive compounds may achieve excellent kinase selectivity.

Development of a fluorescence polarization bead-based coupled assay to target different activity/conformation states of a protein kinase.

Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP™)-based couple™d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z′ of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC™ and validated with the protein kinase C inhibitor staurosporine. The IC50 value generated was comparable to the value obtained by the radioactive 33P-γ-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.

Purification and crystallization of a complex between human interferon γ receptor (extracellular domain) and human interferon γ

X‐ray diffraction quality crystals have been obtained from a complex between interferon γ and the extracellular domain of its high‐affinity cell surface receptor. The crystals were obtained from interferon γ/interferon γ receptor complexes purified by size exclusion chromatography. Diffraction quality crystals required analyzing these complex samples by isoelectric focusing gels to select purified complex fractions devoid of unbound interferon γ. These studies used interferon γ receptor engineered with an eight amino acid N‐terminal deletion to eliminate heterogeneity generated due to proteolytic cleavage. In addition, the receptor was expressed in an E. coli secretion cell line which eliminated the need to refold the protein. Hexagonal crystals were grown from 1.6 M ammonium phosphate solutions and belong to a spacegroup of P6522 with unit cell dimensions a = 145.9 Å and c = 180.3 Å. These crystals diffract to at least 2.9 Å resolution when exposed to synchrotron radiation. SDS PAGE analysis of the crystals demonstrated that both interferon γ and the receptor were present. Analysis of the x‐ray diffraction data revealed that the crystals contain complexes with a stoichiometry of 2:1 receptor: ligand within the crystallographic asymmetric unit and consist of approximately 55% solvent.

Modulating the interaction between CDK2 and cyclin A with a quinoline-based inhibitor

A new class of quinoline-based kinase inhibitors has been discovered that both disrupt cyclin dependent 2 (CDK2) interaction with its cyclin A subunit and act as ATP competitive inhibitors. The key strategy for discovering this class of protein-protein disrupter compounds was to screen the monomer CDK2 in an affinity-selection/mass spectrometry-based technique and to perform secondary assays that identified compounds that bound only to the inactive CDK2 monomer and not the active CDK2/cyclin A heterodimer. Through a series of chemical modifications the affinity (Kd) of the original hit improved from 1 to 0.005μM.

In Vitro Kinetic Profiling of Hepatitis C Virus NS3 Protease Inhibitors by Progress Curve Analysis

Kinetic profiling of drug binding to its target reveals important mechanistic parameters including drug-target residence time. In this chapter, we focus on global progress curve analysis as a convenient method for kinetic profiling. Detailed guidelines with pros and cons for various experimental designs and data analysis are provided. Kinetic profiling of Boceprevir and Telaprevir is illustrated.

Double-stranded DNA-induced localized unfolding of HCV NS3 helicase subdomain 2

The NS3 helicase of the hepatitis C virus (HCV) unwinds double‐stranded (ds) nucleic acid (NA) in an NTP‐dependent fashion. Mechanistic details of this process are, however, largely unknown for the HCV helicase. We have studied the binding of dsDNA to an engineered version of subdomain 2 of the HCV helicase (d2ΔNS3h) by NMR and circular dichroism. Binding of dsDNA to d2ΔNS3h induces a local unfolding of helix (α3), which includes residues of conserved helicase motif VI (Q460RxxRxxR467), and strands (β1 and β8) from the central β‐sheet. This also occurs upon lowering the pH (4.4) and introducing an R461A point mutation, which disrupt salt bridges with Asp 412 and Asp 427 in the protein structure. NMR studies on d2ΔNS3h in the partially unfolded state at low pH map the dsDNA binding site to residues previously shown to be involved in single‐stranded DNA binding. Sequence alignment and structural comparison suggest that these Arg–Asp interactions are highly conserved in SF2 DEx(D/H) proteins. Thus, modulation of these interactions by dsNA may allow SF2 helicases to switch between conformations required for helicase function.