Discovery of a Potent (4 R,5 S)-4-Fluoro-5-methylproline Sulfonamide Transient Receptor Potential Ankyrin 1 Antagonist and Its Methylene Phosphate Prodrug Guided by Molecular Modeling.

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed in sensory neurons where it functions as an irritant sensor for a plethora of electrophilic compounds and is implicated in pain, itch, and respiratory disease. To study its function in various disease contexts, we sought to identify novel, potent, and selective small-molecule TRPA1 antagonists. Herein we describe the evolution of an N-isopropylglycine sulfonamide lead (1) to a novel and potent (4 R,5 S)-4-fluoro-5-methylproline sulfonamide series of inhibitors. Molecular modeling was utilized to derive low-energy three-dimensional conformations to guide ligand design. This effort led to compound 20, which possessed a balanced combination of potency and metabolic stability but poor solubility that ultimately limited in vivo exposure. To improve solubility and in vivo exposure, we developed methylene phosphate prodrug 22, which demonstrated superior oral exposure and robust in vivo target engagement in a rat model of AITC-induced pain.

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.

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.

Rate-Determining and Rate-Limiting Steps in the Clearance and Excretion of a Potent and Selective p21-Activated Kinase Inhibitor: A Case Study of Rapid Hepatic Uptake and Slow Elimination in Rat.

BACKGROUND: Significant under-prediction of in vivo clearance in rat was observed for a potent p21-activated kinase (PAK1) inhibitor, GNE1. OBJECTIVE: Rate-determining (rapid uptake) and rate-limiting (slow excretion) steps in systemic clearance and elimination of GNE1, respectively, were evaluated to better understand the cause of the in vitro-in vivo (IVIV) disconnect. METHODS: A series of in vivo, ex vivo, and in vitro experiments were carried out: 1) the role of organic cation transporters (Oct or Slc22a) was investigated in transporter knock-out and wild-type animals with or without 1-aminobenzotriazole (ABT) pretreatment; 2) the concentration-dependent hepatic extraction ratio was determined in isolated perfused rat liver; and 3) excreta were collected from both bile duct cannulated and non-cannulated rats after intravenous injection. RESULTS: After intravenous dosing, the rate-determining step in clearance was found to be mediated by the active uptake transporter, Oct1. In cannulated rats, biliary and renal clearance of GNE1 accounted for only approximately 14 and 16% of the total clearance, respectively. N-acetylation, an important metabolic pathway, accounted for only about 10% of the total dose. In non-cannulated rats, the majority of the dose was recovered in feces as unchanged parent (up to 91%) overnight following intravenous administration. CONCLUSION: Because the clearance of GNE1 is mediated through uptake transporters rather than metabolism, the extrahepatic expression of Oct1 in kidney and intestine in rat likely plays an important role in the IVIV disconnect in hepatic clearance prediction. The slow process of intestinal secretion is the rate-limiting step for in vivo clearance of GNE1.

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.

Structure-Based Design of GNE-495, a Potent and Selective MAP4K4 Inhibitor with Efficacy in Retinal Angiogenesis.

Diverse biological roles for mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) have necessitated the identification of potent inhibitors in order to study its function in various disease contexts. In particular, compounds that can be used to carry out such studies in vivo would be critical for elucidating the potential for therapeutic intervention. A structure-based design effort coupled with property-guided optimization directed at minimizing the ability of the inhibitors to cross into the CNS led to an advanced compound 13 (GNE-495) that showed excellent potency and good PK and was used to demonstrate in vivo efficacy in a retinal angiogenesis model recapitulating effects that were observed in the inducible Map4k4 knockout mice.

Leveraging the Pre-DFG Residue Thr-406 To Obtain High Kinase Selectivity in an Aminopyrazole-Type PAK1 Inhibitor Series.

To increase kinase selectivity in an aminopyrazole-based PAK1 inhibitor series, analogues were designed to interact with the PAK1 deep-front pocket pre-DFG residue Thr-406, a residue that is hydrophobic in most kinases. This goal was achieved by installing lactam head groups to the aminopyrazole hinge binding moiety. The corresponding analogues represent the most kinase selective ATP-competitive Group I PAK inhibitors described to date. Hydrogen bonding with the Thr-406 side chain was demonstrated by X-ray crystallography, and inhibitory activities, particularly against kinases with hydrophobic pre-DFG residues, were mitigated. Leveraging hydrogen bonding side chain interactions with polar pre-DFG residues is unprecedented, and similar strategies should be applicable to other appropriate kinases.

Fragment-based identification and optimization of a class of potent pyrrolo[2,1-f][1,2,4]triazine MAP4K4 inhibitors.

MAP4K4 has been shown to regulate key cellular processes that are tied to disease pathogenesis. In an effort to generate small molecule MAP4K4 inhibitors, a fragment-based screen was carried out and a pyrrolotriazine fragment with excellent ligand efficiency was identified. Further modification of this fragment guided by X-ray crystal structures and molecular modeling led to the discovery of a series of promising compounds with good structural diversity and physicochemical properties. These compounds exhibited single digit nanomolar potency and compounds 35 and 44 achieved good in vivo exposure.

Discovery of selective 4-Amino-pyridopyrimidine inhibitors of MAP4K4 using fragment-based lead identification and optimization.

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is a serine/threonine kinase implicated in the regulation of many biological processes. A fragment-based lead discovery approach was used to generate potent and selective MAP4K4 inhibitors. The fragment hit pursued in this article had excellent ligand efficiency (LE), an important attribute for subsequent successful optimization into drug-like lead compounds. The optimization efforts eventually led us to focus on the pyridopyrimidine series, from which 6-(2-fluoropyridin-4-yl)pyrido[3,2-d]pyrimidin-4-amine (29) was identified. This compound had low nanomolar potency, excellent kinase selectivity, and good in vivo exposure, and demonstrated in vivo pharmacodynamic effects in a human tumor xenograft model.

Rare neurological diseases: a practical approach to management.

Although neurologists are frequently faced with the management of rare diseases, there is little generic guidance for the approach to management. There are complexities with respect to diagnosis, counselling, treatment and monitoring which are idiosyncratic to rare diseases. Here we use a case report as the basis for discussion of the management of rare neurological diseases. We discuss current issues, guidance from regulatory bodies, and offer practical tips for diagnosis, treatment and monitoring, including the use of decision tree analysis. We offer a generic algorithm to aid neurologists when facing rare conditions.

Discovery of dihydrothieno- and dihydrofuropyrimidines as potent pan Akt inhibitors.

Herein we report the discovery and synthesis of a novel series of dihydrothieno- and dihydrofuropyrimidines (2 and 3) as potent pan Akt inhibitors. Utilizing previous SAR and analysis of the amino acid sequences in the binding site we have designed inhibitors displaying increased PKA and general kinase selectivity with improved tolerability compared to the progenitor pyrrolopyrimidine (1). A representative dihydrothieno compound (34) was advanced into a PC3-NCI prostate mouse tumor model in which it demonstrated a dose-dependent reduction in tumor growth and stasis when dosed orally daily at 200 mg/kg.

GDC-0449-a potent inhibitor of the hedgehog pathway.

SAR for a wide variety of heterocyclic replacements for a benzimidazole led to the discovery of functionalized 2-pyridyl amides as novel inhibitors of the hedgehog pathway. The 2-pyridyl amides were optimized for potency, PK, and drug-like properties by modifications to the amide portion of the molecule resulting in 31 (GDC-0449). Amide 31 produced complete tumor regression at doses as low as 12.5mg/kg BID in a medulloblastoma allograft mouse model that is wholly dependent on the Hh pathway for growth and is currently in human clinical trials, where it is initially being evaluated for the treatment of BCC.

Suppression of HER2/HER3-mediated growth of breast cancer cells with combinations of GDC-0941 PI3K inhibitor, trastuzumab, and pertuzumab.

PURPOSE: Oncogenic activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is prevalent in breast cancer and has been associated with resistance to HER2 inhibitors in the clinic. We therefore investigated the combinatorial activity of GDC-0941, a novel class I PI3K inhibitor, with standard-of-care therapies for HER2-amplified breast cancer. EXPERIMENTAL DESIGN: Three-dimensional laminin-rich extracellular matrix cultures of human breast cancer cells were utilized to provide a physiologically relevant approach to analyze the efficacy and molecular mechanism of combination therapies ex vivo. Combination studies were done using GDC-0941 with trastuzumab (Herceptin), pertuzumab, lapatinib (Tykerb), and docetaxel, the principal therapeutic agents that are either approved or being evaluated for treatment of early HER2-positive breast cancer. RESULTS: Significant GDC-0941 activity (EC(50) <1 micromol/L) was observed for >70% of breast cancer cell lines that were examined in three-dimensional laminin-rich extracellular matrix culture. Differential responsiveness to GDC-0941 as a single agent was observed for luminal breast cancer cells upon stimulation with the HER3 ligand, heregulin. Combined treatment of GDC-0941, trastuzumab, and pertuzumab resulted in growth inhibition, altered acinar morphology, and suppression of AKT mitogen-activated protein kinase (MAPK) / extracellular signed-regulated kinase (ERK) kinase and MEK effector signaling pathways for HER2-amplified cells in both normal and heregulin-supplemented media. The GDC-0941 and lapatinib combination further showed that inhibition of HER2 activity was essential for maximum combinatorial efficacy. PI3K inhibition also rendered HER2-amplified BT-474M1 cells and tumor xenografts more sensitive to docetaxel. CONCLUSIONS: GDC-0941 is efficacious in preclinical models of breast cancer. The addition of GDC-0941 to HER2-directed treatment could augment clinical benefit in breast cancer patients.

Pharmacodynamics of 2-[4-[(1E)-1-(hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridine-4-yl)-1H-pyrazol-1-yl]ethan-1-ol (GDC-0879), a potent and selective B-Raf kinase inhibitor: understanding relationships between systemic concentrations, phosphorylated mitogen-activated protein kinase kinase 1 inhibition, and efficacy.

The Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase signaling pathway is involved in cellular responses relevant to tumorigenesis, including cell proliferation, invasion, survival, and angiogenesis. 2-[4-[(1E)-1-(Hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridine-4-yl)-1H-pyrazol-1-yl]ethan-1-ol (GDC-0879) is a novel, potent, and selective B-Raf inhibitor. The objective of this study was to characterize the relationship between GDC-0879 plasma concentrations and tumor growth inhibition in A375 melanoma and Colo205 colon cancer xenografts and to understand the pharmacodynamic (PD) marker response requirements [phosphorylated (p)MEK1 inhibition] associated with tumor growth inhibition in A375 xenografts. Estimates of GDC-0879 plasma concentrations required for tumor stasis obtained from fitting tumor data to indirect response models were comparable, at 4.48 and 3.27 microM for A375 and Colo205 xenografts, respectively. This was consistent with comparable in vitro potency of GDC-0879 in both cell lines. The relationship between GDC-0879 plasma concentrations and pMEK1 inhibition in the tumor was characterized in A375 xenografts after oral doses of 35, 50, and 100 mg/kg. Fitting pMEK1 inhibition to an indirect response model provided an IC(50) estimate of 3.06 microM. pMEK1 inhibition was further linked to A375 tumor volume data from nine different GDC-0879 dosing regimens using an integrated pharmacokinetic-PD model. A simulated PD marker response curve plot of K (rate constant describing tumor growth inhibition) versus pMEK1 inhibition generated using pharmacodynamic parameters estimated from this model, showed a steep pMEK1 inhibition-response curve consistent with an estimated Hill coefficient of approximately equal 8. A threshold of >40% pMEK1 inhibition is required for tumor growth inhibition, and a minimum of approximately 60% pMEK1 inhibition is required for stasis in A375 xenografts treated with GDC-0879.

Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents.

Although Akt is known as a survival kinase, inhibitors of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway do not always induce substantial apoptosis. We show that silencing Akt1 alone, or any combination of Akt isoforms, can suppress the growth of tumors established from phosphatase and tensin homologue-null human cancer cells. Although these findings indicate that Akt is essential for tumor maintenance, most tumors eventually rebound. Akt knockdown or inactivation with small molecule inhibitors did not induce significant apoptosis but rather markedly increased autophagy. Further treatment with the lysosomotropic agent chloroquine caused accumulation of abnormal autophagolysosomes and reactive oxygen species, leading to accelerated cell death in vitro and complete tumor remission in vivo. Cell death was also promoted when Akt inhibition was combined with the vacuolar H(+)-adenosine triphosphatase inhibitor bafilomycin A1 or with cathepsin inhibition. These results suggest that blocking lysosomal degradation can be detrimental to cancer cell survival when autophagy is activated, providing rationale for a new therapeutic approach to enhancing the anticancer efficacy of PI3K-Akt pathway inhibition.

pHUSH: a single vector system for conditional gene expression.

BACKGROUND: Conditional expression vectors have become a valuable research tool to avoid artefacts that may result from traditional gene expression studies. However, most systems require multiple plasmids that must be independently engineered into the target system, resulting in experimental delay and an increased potential for selection of a cell subpopulation that differs significantly from the parental line. We have therefore developed pHUSH, an inducible expression system that allows regulated expression of shRNA, miRNA or cDNA cassettes on a single viral vector. RESULTS: Both Pol II and Pol III promoters have been successfully combined with a second expression cassette containing a codon-optimized tetracycline repressor and selectable marker. We provide examples of how pHUSH has been successfully employed to study the function of target genes in a number of cell types within in vitro and in vivo assays, including conditional gene knockdown in a murine model of brain cancer. CONCLUSION: We have successfully developed and employed a single vector system that enables Doxycycline regulated RNAi or transgene expression in a variety of in vitro and in vivo model systems. These studies demonstrate the broad application potential of pHUSH for conditional genetic engineering in mammalian cells.

Regulation of ERK3/MAPK6 expression by BRAF.

Several forms of cancer are characterized by frequent activating mutations in the serine/threonine kinase, BRAF. Substitution of glutamic acid for valine at codon 600 (V600E) accounts for approximately 90% of all BRAF activating mutations and leads to stimulation of kinase activity, downstream signaling, and cell transformation. To better understand the molecular pathogenesis induced by oncogenic BRAF signaling, we used microarray gene expression profiling to comprehensively analyze the BRAF-directed transcriptional program of cells expressing a conditionally active form of BRAFV600E. Several novel genes that affect proliferation, cell survival, angiogenesis and immune surveillance were identified as possible mediators of BRAF-induced oncogenic signaling. Moreover, we show that a MAPK family member, extracellular signal-regulated kinase-3 (ERK3/MAPK6) is highly expressed in response to BRAF signaling in this system. Cellular ERK3 protein is highly unstable and pharmacological inhibition of BRAF activity resulted in rapid ERK3 degradation. In melanoma cells, RNAi-mediated knockdown of endogenous BRAF or treatment with MEK inhibitors that prevent ERK1/2 activation led to a reduction in ERK3 levels, indicating that elevated ERK3 expression is mediated through MEK1/2 signaling. These results provide strong evidence for another mode by which BRAF can regulate the ERK protein kinase family and suggest ERK3 to be a potential pharmacodynamic marker for targeting BRAF signaling in melanoma.

Oncogenic BRAF is required for tumor growth and maintenance in melanoma models.

The usual paradigm for developing kinase inhibitors in oncology is to use a high-affinity proof-of-concept inhibitor with acceptable metabolic properties for key target validation experiments. This approach requires substantial medicinal chemistry and can be confounded by drug toxicity and off-target activities of the test molecule. As a better alternative, we have developed inducible short-hairpin RNA xenograft models to examine the in vivo efficacy of inhibiting oncogenic BRAF. Our results show that tumor regression resulting from BRAF suppression is inducible, reversible, and tightly regulated in these models. Analysis of regressing tumors showed the primary mechanism of action for BRAF to be increased tumor cell proliferation and survival. In a metastatic melanoma model, conditional BRAF suppression slowed systemic tumor growth as determined by in vivo bioluminescence imaging. Taken together, gain-of-function BRAF signaling is strongly associated with in vivo tumorigenicity, confirming BRAF as an important target for small-molecule and RNA interference-based therapeutics.

Preclinical evaluation of the tyrosine kinase inhibitor SU11248 as a single agent and in combination with "standard of care" therapeutic agents for the treatment of breast cancer.

SU11248 is an oral multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activities through targeting platelet-derived growth factor receptor, vascular endothelial growth factor receptor, KIT, and FLT3, the first three of which are expressed in human breast cancer and/or its supporting tissues. The purpose of the present studies was to demonstrate the potent anticancer activity of SU11248 alone or in combination with conventional cytotoxic agents against several distinct preclinical models of breast cancer. SU11248 was administered as a monotherapy to (1) mouse mammary tumor virus-v-Ha-ras mice and 7,12-dimethylbenz(a)anthracene-treated rats bearing mammary tumors and (2) mice bearing human breast cancer xenografts of s.c. MX-1 tumors and osseous metastasis of a MDA-MB-435-derived cell line (435/HAL-Luc). SU11248 was also administered in combination with docetaxel both in xenograft models and in combination with 5-fluorouracil and doxorubicin in the MX-1 model. SU11248 treatment potently regressed growth of mammary cancers in mouse mammary tumor virus-v-Ha-ras transgenic mice (82% regression) and 7,12-dimethylbenz(a)anthracene-induced mammary tumors in rats (99% regression at the highest dose; P < 0.05 for both). This agent also inhibited MX-1 tumor growth by 52%, with markedly enhanced anticancer effects when administered in combination with docetaxel, 5-fluorouracil, or doxorubicin compared with either agent alone (P < 0.05). SU11248 treatment in combination with docetaxel effectively prolonged survival of mice, with 435/HAL-Luc cancer xenografts established in bone compared with either agent alone (P < 0.05). These results demonstrate that SU11248 is effective in preclinical breast cancer models and suggest that it may be useful in the treatment of breast cancer in the clinic.

SU11248 inhibits KIT and platelet-derived growth factor receptor beta in preclinical models of human small cell lung cancer.

The purpose of this study was to evaluate the activity of the indolinone kinase inhibitor SU11248 against the receptor tyrosine kinase KIT in vitro and in vivo, examine the role of KIT in small cell lung cancer (SCLC), and anticipate clinical utility of SU11248 in SCLC. SU11248 is an oral, multitargeted tyrosine kinase inhibitor with direct antitumor and antiangiogenic activity through targeting platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor, KIT, and FLT3 receptors. Treatment of the KIT-expressing SCLC-derived NCI-H526 cell line in vitro with SU11248 resulted in dose-dependent inhibition of stem cell factor-stimulated KIT phosphotyrosine levels and proliferation. The biological significance of KIT inhibition was evaluated in vivo by treating mice bearing s.c. NCI-H526 tumors with SU11248 or another structurally unrelated KIT inhibitor, STI571 (Gleevec), which is also known to inhibit Bcr-Abl and PDGFRbeta. SU11248 treatment resulted in significant tumor growth inhibition, whereas inhibition from STI571 treatment was less dramatic. Both compounds reduced phospho-KIT levels in NCI-H526 tumors, with a greater reduction by SU11248, correlating with efficacy. Likewise, phospho-PDGFRbeta levels contributed by tumor stroma and with known involvement in angiogenesis were strongly inhibited by SU11248 and less so by STI571. Because platinum-based chemotherapy is part of the standard of care for SCLC, SU11248 was combined with cisplatin, and significant tumor growth delay was measured compared with either agent alone. These results expand the profile of SU11248 as a KIT signaling inhibitor and suggest that SU11248 may have clinical potential in the treatment of SCLC via direct antitumor activity mediated via KIT as well as tumor angiogenesis via vascular endothelial growth factor receptor FLK1/KDR and PDGFRbeta.