Potent and selective inhibitors of receptor-interacting protein kinase 1 that lack an aromatic back pocket group.

Receptor-interacting protein kinase 1 (RIPK1), a key component of the cellular necroptosis pathway, has gained recognition as an important therapeutic target. Pharmacologic inhibition or genetic inactivation of RIPK1 has shown promise in animal models of disease ranging from acute ischemic conditions, chronic inflammation, and neurodegeneration. We present here a class of RIPK1 inhibitors that is distinguished by a lack of a lipophilic aromatic group present in most literature inhibitors that typically occupies a hydrophobic back pocket of the protein active site. Despite not having this ubiquitous feature of many known RIPK1 inhibitors, we were able to obtain compounds with good potency, kinase selectivity, and pharmacokinetic properties in rats. The use of the lipophilic yet metabolically stable pentafluoroethyl group was critical to balancing the potency and properties of optimized analogs.

Synthesis and evaluation of a series of 4-azaindole-containing p21-activated kinase-1 inhibitors.

A series of 4-azaindole-containing p21-activated kinase-1 (PAK1) inhibitors was prepared with the goal of improving physicochemical properties relative to an indole starting point. Indole 1 represented an attractive, non-basic scaffold with good PAK1 affinity and cellular potency but was compromised by high lipophilicity (clogD=4.4). Azaindole 5 was designed as an indole surrogate with the goal of lowering logD and resulted in equipotent PAK1 inhibition with a 2-fold improvement in cellular potency over 1. Structure-activity relationship studies around 5 identified additional 4-azaindole analogs with superior PAK1 biochemical activity (Ki <10nM) and up to 24-fold selectivity for group I over group II PAKs. Compounds from this series showed enhanced permeability, improved aqueous solubility, and lower plasma protein binding over indole 1. The improvement in physicochemical properties translated to a 20-fold decrease in unbound clearance in mouse PK studies for azaindole 5 relative to indole 1.

Design and Development of a Series of Potent and Selective Type II Inhibitors of CDK8.

Using Sorafenib as a starting point, a series of potent and selective inhibitors of CDK8 was developed. When cocrystallized with CDK8 and cyclin C, these compounds exhibit a Type-II (DMG-out) binding mode.

Chemically Diverse Group I p21-Activated Kinase (PAK) Inhibitors Impart Acute Cardiovascular Toxicity with a Narrow Therapeutic Window.

p21-activated kinase 1 (PAK1) has an important role in transducing signals in several oncogenic pathways. The concept of inhibiting this kinase has garnered significant interest over the past decade, particularly for targeting cancers associated with PAK1 amplification. Animal studies with the selective group I PAK (pan-PAK1, 2, 3) inhibitor G-5555 from the pyrido[2,3-d]pyrimidin-7-one class uncovered acute toxicity with a narrow therapeutic window. To attempt mitigating the toxicity, we introduced significant structural changes, culminating in the discovery of the potent pyridone side chain analogue G-9791. Mouse tolerability studies with this compound, other members of this series, and compounds from two structurally distinct classes revealed persistent toxicity and a correlation of minimum toxic concentrations and PAK1/2 mediated cellular potencies. Broad screening of selected PAK inhibitors revealed PAK1, 2, and 3 as the only overlapping targets. Our data suggest acute cardiovascular toxicity resulting from the inhibition of PAK2, which may be enhanced by PAK1 inhibition, and cautions against continued pursuit of pan-group I PAK inhibitors in drug discovery.

Structure-Guided Design of Group I Selective p21-Activated Kinase Inhibitors.

The p21-activated kinases (PAKs) play important roles in cytoskeletal organization, cellular morphogenesis, and survival and have generated significant attention as potential therapeutic targets for cancer. Following a high-throughput screen, we identified an aminopyrazole scaffold-based series that was optimized to yield group I selective PAK inhibitors. A structure-based design effort aimed at targeting the ribose pocket for both potency and selectivity led to much-improved group I vs II selectivity. Early lead compounds contained a basic primary amine, which was found to be a major metabolic soft spot with in vivo clearance proceeding predominantly via N-acetylation. We succeeded in identifying replacements with improved metabolic stability, leading to compounds with lower in vivo rodent clearance and excellent group I PAK selectivity.

Design of Selective PAK1 Inhibitor G-5555: Improving Properties by Employing an Unorthodox Low-pK a Polar Moiety.

Signaling pathways intersecting with the p21-activated kinases (PAKs) play important roles in tumorigenesis and cancer progression. By recognizing that the limitations of FRAX1036 (1) were chiefly associated with the highly basic amine it contained, we devised a mitigation strategy to address several issues such as hERG activity. The 5-amino-1,3-dioxanyl moiety was identified as an effective means of reducing pK a and logP simultaneously. When positioned properly within the scaffold, this group conferred several benefits including potency, pharmacokinetics, and selectivity. Mouse xenograft PK/PD studies were carried out using an advanced compound, G-5555 (12), derived from this approach. These studies concluded that dose-dependent pathway modulation was achievable and paves the way for further in vivo investigations of PAK1 function in cancer and other diseases.

Lysophosphatidic acid induces glycodelin gene expression in cancer cells.

Glycodelin is a glycoprotein that has been suggested to be important in normal pregnancy and in malignancy. The regulation of its synthesis has not been studied. In this study, we report the induction of glycodelin gene expression by lysophosphatidic acid (LPA). We studied the effect of LPA (5, 10 and 25 microM) on glycodelin production in breast (MDA-MB-231), cervical (Hela), endometrial (RL-95), ovarian cancer (OVCAR-3) and erythroleukemia (K562) cells. There was a dose-dependent (5-25 microM) induction of glycodelin gene and protein expression in these cell types. LPA is a mimic of phorbol myristate acetate (PMA) action and is found to be elevated in high concentrations in the serum of cancer subjects. As glycodelin is an angiogenic protein with a potential immunosuppressive role, control of LPA synthesis might offer a potential target for intervention.

A whole cell assay to measure caspase-6 activity by detecting cleavage of lamin A/C.

Caspase-6 is a cysteinyl protease implicated in neurodegenerative conditions including Alzheimer's and Huntington's disease making it an attractive target for therapeutic intervention. A greater understanding of the role of caspase-6 in disease has been hampered by a lack of suitable cellular assays capable of specifically detecting caspase-6 activity in an intact cell environment. This is mainly due to the use of commercially available peptide substrates and inhibitors which lack the required specificity to facilitate development of this type of assay. We report here a 384-well whole-cell chemiluminescent ELISA assay that monitors the proteolytic degradation of endogenously expressed lamin A/C during the early stages of caspase-dependent apoptosis. The specificity of lamin A/C proteolysis by caspase-6 was demonstrated against recombinant caspase family members and further confirmed in genetic deletion studies. In the assay, plasma membrane integrity remained intact as assessed by release of lactate dehydrogenase from the intracellular environment and the exclusion of cell impermeable peptide inhibitors, despite the induction of an apoptotic state. The method described here is a robust tool to support drug discovery efforts targeting caspase-6 and is the first reported to specifically monitor endogenous caspase-6 activity in a cellular context.

A plate-based assay to measure cellular ERK substrate phosphorylation: utility for drug discovery of the MAPK-signaling cascade.

The Ras, Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling cascade is critically involved in cellular signaling with activating mutations in Ras and Raf present in many human tumors. Each constituent of this pathway is considered an important target for pharmaceutical intervention. The terminal kinase ERK is known to phosphorylate p90RSK among myriad substrates, yet robust plate-based high-throughput cellular assays monitoring such activity are not commercially available. In this study, we have utilized the Meso Scale Discovery platform to develop a plate-based assay to monitor the level of phosphorylation of p90RSK. This method is highly robust and can be used to evaluate a large number of inhibitors of ERK, MEK, or Raf in a variety of cellular backgrounds. Furthermore, this assay can be used to quantify the level of phospho-p90RSK in tumor lysates to function as a valuable pharmacodynamic readout.