Laura Lingardo

Characterization of PF-4708671, a novel and highly specific inhibitor of p70 ribosomal S6 kinase (S6K1).

S6K1 (p70 ribosomal S6 kinase 1) is activated by insulin and growth factors via the PI3K (phosphoinositide 3-kinase) and mTOR (mammalian target of rapamycin) signalling pathways. S6K1 regulates numerous processes, such as protein synthesis, growth, proliferation and longevity, and its inhibition has been proposed as a strategy for the treatment of cancer and insulin resistance. In the present paper we describe a novel cell-permeable inhibitor of S6K1, PF-4708671, which specifically inhibits the S6K1 isoform with a Ki of 20 nM and IC50 of 160 nM. PF-4708671 prevents the S6K1-mediated phosphorylation of S6 protein in response to IGF-1 (insulin-like growth factor 1), while having no effect upon the PMA-induced phosphorylation of substrates of the highly related RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated kinase) kinases. PF-4708671 was also found to induce phosphorylation of the T-loop and hydrophobic motif of S6K1, an effect that is dependent upon mTORC1 (mTOR complex 1). PF-4708671 is the first S6K1-specific inhibitor to be reported and will be a useful tool for delineating S6K1-specific roles downstream of mTOR.

Discovery of (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile (PF-06463922), a macrocyclic inhibitor of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) with preclinical brain exposure and broad-spectrum potency against ALK-resistant mutations.

Although crizotinib demonstrates robust efficacy in anaplastic lymphoma kinase (ALK)-positive non-small-cell lung carcinoma patients, progression during treatment eventually develops. Resistant patient samples revealed a variety of point mutations in the kinase domain of ALK, including the L1196M gatekeeper mutation. In addition, some patients progress due to cancer metastasis in the brain. Using structure-based drug design, lipophilic efficiency, and physical-property-based optimization, highly potent macrocyclic ALK inhibitors were prepared with good absorption, distribution, metabolism, and excretion (ADME), low propensity for p-glycoprotein 1-mediated efflux, and good passive permeability. These structurally unusual macrocyclic inhibitors were potent against wild-type ALK and clinically reported ALK kinase domain mutations. Significant synthetic challenges were overcome, utilizing novel transformations to enable the use of these macrocycles in drug discovery paradigms. This work led to the discovery of 8k (PF-06463922), combining broad-spectrum potency, central nervous system ADME, and a high degree of kinase selectivity.

Design of Potent and Selective Inhibitors to Overcome Clinical Anaplastic Lymphoma Kinase Mutations Resistant to Crizotinib.

Crizotinib (1), an anaplastic lymphoma kinase (ALK) receptor tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration in 2011, is efficacious in ALK and ROS positive patients. Under pressure of crizotinib treatment, point mutations arise in the kinase domain of ALK, resulting in resistance and progressive disease. The successful application of both structure-based and lipophilic-efficiency-focused drug design resulted in aminopyridine 8e, which was potent across a broad panel of engineered ALK mutant cell lines and showed suitable preclinical pharmacokinetics and robust tumor growth inhibition in a crizotinib-resistant cell line (H3122-L1196M).

Discovery of Novel, Potent, and Selective Inhibitors of 3-Phosphoinositide-Dependent Kinase (PDK1)

Analogues substituted with various amines at the 6-position of the pyrazine ring on (4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)pyrazin-2-ylmethanone were discovered as potent and selective inhibitors of PDK1 with potential as anticancer agents. An early lead with 2-pyridine-3-ylethylamine as the pyrazine substituent showed moderate potency and selectivity. Structure-based drug design led to improved potency and selectivity against PI3Kα through a combination of cyclizing the ethylene spacer into a saturated, five-membered ring and substituting on the 4-position of the aryl ring with a fluorine. ADME properties were improved by lowering the lipophilicity with heteroatom replacements in the saturated, five-membered ring. The optimized analogues have a PDK1 Ki of 1 nM and >100-fold selectivity against PI3K/AKT-pathway kinases. The cellular potency of these analogues was assessed by the inhibition of AKT phosphorylation (T308) and by their antiproliferation activity against a number of tumor cell lines.

Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays.

The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.

Development of hepatitis C virus chimeric replicons for identifying broad spectrum NS3 protease inhibitors

Several potent inhibitors of hepatitis C virus (HCV) NS3/4A protease have been identified that show great clinical potential against genotype 1. Due to the tremendous genetic diversity that exists among HCV isolates, development of broad spectrum inhibitors is challenging. With a limited number of lab strains available for preclinical testing, new tools are required for assessing protease inhibitor activity. We developed a chimeric replicon system for evaluating NS3 protease inhibitor activity against naturally occurring isolates. NS3/4A genes were cloned from the plasma of HCV-infected individuals and inserted into lab strain replicons, replacing the native sequences. The chimeric reporter replicons were transfected into Huh 7.5 cells, their replication monitored by luciferase assays, and their susceptibilities to inhibitors determined. Viable chimeras expressing heterologous genotypes 1, 2, 3, and 4 protease domains were identified that exhibited varying susceptibilities to inhibitors. Protease inhibitor spectrums observed against the chimeric replicon panel strongly correlated with published enzymatic and clinical results. This cell-based chimeric replicon system can be used to characterize the activities of protease inhibitors against diverse natural isolates and may improve the ability to predict dose and clinical efficacy.

Abstract 2180: Drug-like inhibitors of SGK1: Discovery and optimization of low molecular weight fragment leads

Triple negative (ER, PR and HER2/neu negative) breast cancers (TNBC) constitute 15-24% of breast cancers, but account for a disproportionate share of mortality. TNBC tumors are more aggressive, lack a targeted therapy, and are prone to relapse and metastasis after cytotoxic drug treatments, the only drug therapy currently available. Serum and glucocorticoid-regulated kinase 1 (SGK1) promotes the growth, invasiveness and chemo-resistance of cancer cells and is overexpressed in 48% of breast cancers, yet is not detected in normal breast tissue. In particular, SGK1 is overexpressed in TNBC cells and knockdown of SGK1 blocks the proliferation and invasiveness of TNBC cells. Thus, SGK1 is an attractive targeted therapy for TNBC. In the SGK1 inhibitors that have been reported previously, poor physicochemical properties have prevented these inhibitors from being active in vivo and advancing to the clinic. To discover drug-like inhibitors of SGK1, we used our technology platform (Leap-To-Lead™) and a fragment-based screening approach to find SGK1 inhibitor scaffolds. Low molecular weight inhibitors like ours are more readily optimized for potency while retaining drug-like properties. From this screen, we identified 11 scaffolds as novel inhibitors of SGK1. Four scaffold series had clear structure activity relationships (SAR). To illustrate the potential of these scaffolds, we carried out a limited synthetic expansion around one scaffold and improved potency more than 600-fold (SGK1 IC50 = 1 μM; LE = 0.4). Several compounds dose-dependently inhibited the proliferation of a TNBC cell line (MDA-MB-231) and inhibited the phosphorylation of the biomarker protein N-Myc downstream regulated 1 (NDRG-1). Unlike previously reported SGK1 inhibitors, our fragment lead compounds show excellent cellular penetration in Caco-2 assays. In summary, a fragment-based screening approach was used to identify four novel scaffolds with clear SAR trends. A single scaffold series was further optimized and shows anti-proliferative activity and biomarker modulation in a TNBC cell line. These SGK1 inhibitor series represent excellent starting points for further SAR studies and development of a novel preclinical candidate. Citation Format: James Zapf, Todd Meyer, Warren Wade, Laura Lingardo, Ayse Batova, Gordon Alton, Peter Pallai. Drug-like inhibitors of SGK1: Discovery and optimization of low molecular weight fragment leads. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2180.

Abstract LB-16: Discovery of novel SGK1 inhibitors by a fragment-based approach: Generating patentable, chemically tractable and ligand efficient leads

Triple negative (ER, PR and HER2/neu negative) breast cancer (TNBC) can have an unfavorable prognosis compared to other breast cancers. TNBC is more difficult to treat since most targeted breast cancer therapies are directed at one of the three receptors. Aggressive combinatorial therapies are usually required and the cytotoxic agents used in these regimens frequently loose effectiveness with time. Serum and glucocorticoid-regulated kinase 1 (SGK1) (a serine/threonine protein kinase), promotes the growth and invasiveness of breast cancer cells. SGK1 is a key protein regulated by the glucocorticoid receptor (GR). In TNBC tumors, high expression/activity in the GR/SGK axis correlates with shorter survival times and resistance to chemotherapy. The small numbers of SGK1 inhibitors reported in the literature have resulted from screening against other kinases (such as CHK1/azaindoles) and thus are not selective and lack patentable chemical diversity. We describe the identification of novel SGK1 inhibitors using a fragment-based library screening approach. The BioBlocks Leap-To-Lead platform contains a library of novel fragment-like compounds that span a range of polarities and sizes without compromising on flexibility. For example, this 275 compound library has an average molecule weight of 160 Da; AlogP, 1.1; hydrogen bond donors, 1.1; hydrogen bond acceptors, 2.2, and rotatable bonds, 0.7 yet still contains 114 unique rings. The library was screened against fully activated human SGK1 in an ADP-Glo assay using physiologically relevant peptide substrate and an optimal ATP concentration to also identify non-classical kinase inhibitor mode binders. The fragment library was screened at 2 mM and hits were profiled in IC50 dose response. From these, several novel chemical matter hit compounds were selected that had ligand efficiency values greater than 0.3 and spanned IC50 values from 75 µM to 2000 µM. In summary, a fragment-based screening approach was used to identify several novel scaffolds that represent both classical and non-classical kinase inhibitor binding modes as hit-to-lead starting points for the optimization of novel, potent and selective SGK1 inhibitors. Citation Format: Gordon Alton, Warren Wade, Laura Lingardo, Ayse Batova, Tom Smart, Chris Buhr, James W. Zapf, Peter Pallai. Discovery of novel SGK1 inhibitors by a fragment-based approach: Generating patentable, chemically tractable and ligand efficient leads. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-16. doi:10.1158/1538-7445.AM2014-LB-16

Abstract 753: Novel, potent and selective small molecule inhibitors of 3-phosphoinositide-dependent kinase (PDK1)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a crucial role in cell growth, proliferation and survival. Genomic aberrations in the PI3K pathway, such as mutational activation of PI3Kα or loss-of-function of the tumor suppressor PTEN, have been closely linked to the development and progression of a wide range of cancers. Inhibition of the key targets in the pathway, PI3K, AKT, mTOR & PDK1, may provide an effective treatment of cancer. In an effort to discover compounds that inhibit PDK1, we have developed a series of 3-Carbonyl-4-Amino-Pyrrolopyrimidine (CAP) compounds that are selective and potent PDK1 inhibitors. Early screening led to a viable starting point, PF-03772304, (4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-(6-methylamino-pyrazin-2-yl)-methanone, which has an IC50 of 94 nM for PDK1 and a ligand efficiency of 0.42. While potent, this lead was not selective against PI3K. Using structure-based drug design, this lead was modified to expand into the selectivity pocket of PDK1 (under the G-Loop), leading to the identification of a potent and pathway-selective compound, PF-05017255 ((4-Amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-{6-[(3S,4R)-4-(4-fluoro-phenyl)-tetrahydro-furan-3-ylamino]-pyrazin-2-yl}-methanone). PF-05017255 has a Ki of 0.6 nM for PDK1 and is more than 400-fold selective against other PI3K pathway kinases: PI3Kα, AKT, S6K and mTOR. For even greater kinase selectivity, we sought to lower the clogP of our lead (clogP for PF-05017255 is 3.0) to reduce the contribution from the hydrophobic effect. These efforts led to PF-05168899 (1-{(2R,3R)-3-[6-(4-Amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonyl)-pyrazin-2-ylamino]-2-phenyl-pyrrolidin-1-yl}-ethanone) with a Ki of 0.4 nM for PDK1, a clogP of 2.1, and greater than 1000-fold selectivity against PI3Kα, AKT, S6K, mTOR, CDK2, CHK1 and PAK4. PF-05168899 also showed little inhibitory effect (<50% at 1 uM) against 33 of 35 kinases in a broader panel, demonstrating significant inhibition only against CHK2 (94%) and AuroraB (54%). In addition, the most potent analogs (e.g. PF-05168889) inhibited the phosphorylation of AKT at the residue threonine 308 (IC50 40-200 nM) in a variety of cancer cell lines (e.g. H460, A549). The design, synthesis and SAR of this chemical series will be described. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 753.

Abstract 4482: Novel and selective small molecule inhibitors of 3-phosphoinositide-dependent kinase-1 inhibit the PDK-1/AKT signaling pathway and cell proliferation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Receptor tyrosine kinases (RTKs), PTEN (phosphatase and tensin homolog deleted on chromosome 10), and PIK3CA (encodes the p110α subunit of phosphatidylinositol 3-kinase [PI3K]) frequently contribute to tumor progression through their ability to regulate the intracellular level of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-phosphoinositide-dependent kinase-1 (PDK-1), a serine/threonine kinase, activates the catalytic domain of numerous kinases by phosphorylating their T-loop sites. PDK-1 activity is required for activation of AKT, p70S6K, and RSK which lead to cell proliferation and transformation. The interaction of the pleckstrin homology (PH) domain of AKT with the membrane bound PIP3 confers a conformational change in AKT, allowing PDK-1 to phosphorylate AKT at the residue threonine-308 (T308). This T-loop activation at T308, along with the phosphorylation of the serine-473 residue by mTOR, fully activates the AKT pathway. Although the roles of many PDK-1 substrates have yet to be characterized, the oncogenic activity of aberrant PI3K pathway signaling through PDK-1 has been extensively validated. We have developed a series of 3-Carbonyl-4-Amino-Pyrrolopyrimidne (CAP) compounds that are potent inhibitors of human PDK-1 (full length and kinase domain) which demonstrate more than 100-fold selectivity against P70S6K, PI3K, AKT, and mTOR. In this study, representative compounds from the CAP series were used to perform a variety of anti-tumor assays. We demonstrate PDK-1 compounds inhibit the phosphorylation of T308 on AKT as well as downstream molecules of the PI3K pathway, such as S6 ribosomal protein (S6RP) in breast, lung and colon cancer cell lines harboring a PI3KCA mutation. Additionally, blockade of AKT and molecules in the PI3K pathway leads to the inhibition of cell proliferation and cell transformation in cancer cells. Our data suggest that the inhibition of PDK-1 activity is sufficient to induce anti-tumor activity in cancer cells through the PI3K-PDK1-AKT axis, and that a potent and specific PDK-1 inhibitor could potentially be developed as a therapeutic agent against several cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4482.