Timothy Bailey

ProTx-II, a Selective Inhibitor of NaV1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors

Voltage-gated sodium (NaV1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to NaV1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding NaV1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two inherited painful neuropathies. Although the human genetic data make NaV1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires NaV1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with NaV1.7 channels, inhibiting NaV1.7 with an IC50 value of 0.3 nM, compared with IC50 values of 30 to 150 nM for other heterologously expressed NaV1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little effect on Aβ-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve, which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II (125I-ProTx-II) binds with high affinity (Kd = 0.3 nM) to recombinant hNaV1.7 channels. Binding of 125I-ProTx-II is insensitive to the presence of other well characterized NaV1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the 125I-ProTx-II binding assay, described here, offers a new tool in the search for novel NaV1.7-selective blockers.

Pharmacologic Inhibition of the Renal Outer Medullary Potassium Channel Causes Diuresis and Natriuresis in the Absence of Kaliuresis

The renal outer medullary potassium (ROMK) channel, which is located at the apical membrane of epithelial cells lining the thick ascending loop of Henle and cortical collecting duct, plays an important role in kidney physiology by regulating salt reabsorption. Loss-of-function mutations in the human ROMK channel are associated with antenatal type II Bartter’s syndrome, an autosomal recessive life-threatening salt-wasting disorder with mild hypokalemia. Similar observations have been reported from studies with ROMK knockout mice and rats. It is noteworthy that heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter’s syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. Although selective ROMK inhibitors would be expected to represent a new class of diuretics, this hypothesis has not been pharmacologically tested. Compound A [5-(2-(4-(2-(4-(1H-tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one)], a potent ROMK inhibitor with appropriate selectivity and characteristics for in vivo testing, has been identified. Compound A accesses the channel through the cytoplasmic side and binds to residues lining the pore within the transmembrane region below the selectivity filter. In normotensive rats and dogs, short-term oral administration of compound A caused concentration-dependent diuresis and natriuresis that were comparable to hydrochlorothiazide. Unlike hydrochlorothiazide, however, compound A did not cause any significant urinary potassium losses or changes in plasma electrolyte levels. These data indicate that pharmacologic inhibition of ROMK has the potential for affording diuretic/natriuretic efficacy similar to that of clinically used diuretics but without the dose-limiting hypokalemia associated with the use of loop and thiazide-like diuretics.

Stromal platelet-derived growth factor receptor α (PDGFRα) provides a therapeutic target independent of tumor cell PDGFRα expression in lung cancer xenografts

In lung cancer, platelet-derived growth factor receptor α (PDGFRα) is expressed frequently by tumor-associated stromal cells and by cancer cells in a subset of tumors. We sought to determine the effect of targeting stromal PDGFRα in preclinical lung tumor xenograft models (human tumor, mouse stroma). Effects of anti-human (IMC-3G3) and anti-mouse (1E10) PDGFRα monoclonal antibodies (mAb) on proliferation and PDGFRα signaling were evaluated in lung cancer cell lines and mouse fibroblasts. Therapy studies were conducted using established PDGFRα-positive H1703 cells and PDGFRα-negative Calu-6, H1993, and A549 subcutaneous tumors in immunocompromised mice treated with vehicle, anti-PDGFRα mAbs, chemotherapy, or combination therapy. Tumors were analyzed for growth and levels of growth factors. IMC-3G3 inhibited PDGFRα activation and the growth of H1703 cells in vitro and tumor growth in vivo, but had no effect on PDGFRα-negative cell lines or mouse fibroblasts. 1E10 inhibited growth and PDGFRα activation of mouse fibroblasts, but had no effect on human cancer cell lines in vitro. In vivo, 1E10-targeted inhibition of murine PDGFRα reduced tumor growth as single-agent therapy in Calu-6 cells and enhanced the effect of chemotherapy in xenografts derived from A549 cells. We also identified that low expression cancer cell expression of VEGF-A and elevated expression of PDGF-AA were associated with response to stromal PDGFRα targeting. We conclude that stromal PDGFRα inhibition represents a means for enhancing control of lung cancer growth in some cases, independent of tumor cell PDGFRα expression. Mol Cancer Ther; 11(11); 2473–82. ©2012 AACR.

Discovery of Selective Small Molecule ROMK Inhibitors as Potential New Mechanism Diuretics.

The renal outer medullary potassium channel (ROMK or Kir1.1) is a putative drug target for a novel class of diuretics that could be used for the treatment of hypertension and edematous states such as heart failure. An internal high-throughput screening campaign identified 1,4-bis(4-nitrophenethyl)piperazine (5) as a potent ROMK inhibitor. It is worth noting that this compound was identified as a minor impurity in a screening hit that was responsible for all of the initially observed ROMK activity. Structure-activity studies resulted in analogues with improved rat pharmacokinetic properties and selectivity over the hERG channel, providing tool compounds that can be used for in vivo pharmacological assessment. The featured ROMK inhibitors were also selective against other members of the inward rectifier family of potassium channels.

Discovery of a novel sub-class of ROMK channel inhibitors typified by 5-(2-(4-(2-(4-(1H-Tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one.

A sub-class of distinct small molecule ROMK inhibitors were developed from the original lead 1. Medicinal chemistry endeavors led to novel ROMK inhibitors with good ROMK functional potency and improved hERG selectivity. Two of the described ROMK inhibitors were characterized for the first in vivo proof-of-concept biology studies, and results from an acute rat diuresis model confirmed the hypothesis that ROMK inhibitors represent new mechanism diuretic and natriuretic agents.

The Inwardly Rectifying Potassium Channel Kir1.1: Development of Functional Assays to Identify and Characterize Channel Inhibitors

The renal outer medullary potassium (ROMK) channel is a member of the inwardly rectifying family of potassium (Kir) channels. ROMK (Kir1.1) is predominantly expressed in kidney where it plays a major role in the salt reabsorption process. Loss-of-function mutations in the human Kir1.1 channel are associated with antenatal Bartters syndrome type II, a life-threatening salt and water balance disorder. Heterozygous carriers of Kir1.1 mutations associated with antenatal Bartters syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. These data suggest that Kir1.1 inhibitors could represent novel diuretics for the treatment of hypertension. Because little is known about the molecular pharmacology of Kir1.1 channels, assays that provide a robust, reliable readout of channel activity-while operating in high-capacity mode-are needed. In the present study, we describe high-capacity, 384- and 1,536-well plate, functional thallium flux, and IonWorks electrophysiology assays for the Kir1.1 channel that fulfill these criteria. In addition, 96-well (86)Rb(+) flux assays were established that can operate in the presence of 100% serum, and can provide an indication of the effect of a serum shift on compound potencies. The ability to grow Madin-Darby canine kidney cells expressing Kir1.1 in Transwell supports provides a polarized cell system that can be used to study the mechanism of Kir1.1 inhibition by different agents. All these functional Kir1.1 assays together can play an important role in supporting different aspects of drug development efforts during lead identification and/or optimization.

Discovery of a Potent and Selective ROMK Inhibitor with Pharmacokinetic Properties Suitable for Preclinical Evaluation

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.

Discovery of MK-7145, an Oral Small Molecule ROMK Inhibitor for the Treatment of Hypertension and Heart Failure

ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.

Abstract C54: In vitro and in vivo anti-tumor activity of the antimacrophage stimulating 1-receptor antibody IMC-RON8 in breast and bladder cancer models.

Macrophage stimulating 1-receptor (RON) is a member of the c-Met receptor tyrosine kinase family. RON is normally expressed on macrophages and epithelial cells. However, it is overexpressed and activated in a large number of human tumors. The fully human anti-RON antibody, IMC-RON8, blocks the RON ligand, macrophage-stimulating protein (MSP), from binding to RON and has been demonstrated to have antitumor activity against human colon, lung, and pancreatic xenografts in mice. Overexpression of RON correlates with a worse clinical outcome for patients in at least two human cancer indications, namely breast and bladder. Given this correlation, we investigated the effect of IMC-RON8 in several in vitro and in vivo systems with the RON-positive breast and bladder cancer cell lines JIMT-1 and BFTC-905, respectively. Both of these were found from a screen of breast and bladder cancer cell lines designed to identify those that are RON positive and responsive to MSP through activation of the MAP Kinase signaling pathway. IMC-RON8 inhibited the phosphorylation of MAP Kinase in response to 5nM MSP stimulation for JIMT-1 and BFTC-905. IMC-RON8 also inhibited the MSP-induced cellular migration of JIMT-1 in a wound healing assay, completely preventing progression towards closing of the wound. As measured by flow cytometry, IMC-RON8 at 33.3nM induced the internalization of 38% and 31% cell-surface RON expressed on JIMT-1 and BFTC-905 cells, respectively, following 24 hours of treatment at 37°C. To investigate IMC-RON8-induced RON downmodulation, clonal Hela cells stably expressing a RON-GFP protein were generated. These cells were treated with MSP, a RON agonist antibody (RON2), or IMC-RON8 and the level of RON-GFP was then measured by confocal microscopy in a live-cell time lapse experiment. IMC-RON8 treatment actively induced receptor interalization and caused RON-GFP degradation (reduction of the GFP signal) relative to IgG controls, although to a lesser extent than MSP and RON2. In an in vivo JIMT-1 xenograft model, IMC-RON8 significantly inhibited tumor growth with a %T/C value of 59 when administered at 60 mg/kg twice a week. Combination of IMC-RON8 with the chemotherapeutic agent, docetaxel at 12 mg/kg once per week, significantly improved the anti-tumor effects compared to either monotherapy with a %T/C value of 20. In an in vivo BFTC-905 xenograft model, IMC-RON8 significantly inhibited tumor growth with a %T/C value of 58 when administered at 60 mg/kg twice a week. BFTC-905 tumors removed after the last IMC-RON8 dose (6 total doses over a 3 week study period), showed a decrease in the total level of RON receptor present in tumors relative to controls (p-value of Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C54.