Development of Orally Efficacious Allosteric Inhibitors of TNFα via Fragment-Based Drug Design.

Tumor necrosis factor α (TNFα) is a soluble cytokine that is directly involved in systemic inflammation through the regulation of the intracellular NF-κB and MAPK signaling pathways. The development of biologic drugs that inhibit TNFα has led to improved clinical outcomes for patients with rheumatoid arthritis and other chronic autoimmune diseases; however, TNFα has proven to be difficult to drug with small molecules. Herein, we present a two-phase, fragment-based drug discovery (FBDD) effort in which we first identified isoquinoline fragments that disrupt TNFα ligand-receptor binding through an allosteric desymmetrization mechanism as observed in high-resolution crystal structures. The second phase of discovery focused on the de novo design and optimization of fragments with improved binding efficiency and drug-like properties. The 3-indolinone-based lead presented here displays oral, in vivo efficacy in a mouse glucose-6-phosphate isomerase (GPI)-induced paw swelling model comparable to that seen with a TNFα antibody.

eIF2B activator prevents neurological defects caused by a chronic integrated stress response.

The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts.

Short-term oral gavage administration of adenine induces a model of fibrotic kidney disease in rats.

INTRODUCTION: The adenine model of kidney disease typically involves dietary delivery of adenine over several weeks. This model can be variable in its disease progression and can result in significant mortality. In the current study, the amount of adenine delivered to rats was controlled by utilizing oral gavage administration over a short period in an attempt to induce robust renal pathology while addressing variability and viability of the animals. METHODS: Adenine (150 or 200 mg/kg) was administered via oral gavage for 10 consecutive days, and assessed over a total of 20 days. RESULTS: Both adenine dose groups manifested pathophysiological features of kidney disease such as proteinuria, elevated serum creatinine and BUN, and tubulointerstitial fibrosis. The animals also displayed a decline in glomerular filtration rate. Renal mRNA expression of genes associated with injury, inflammation, and fibrosis (i.e., Col1a1, Acta2, Serpine1, Timp1, Fn-Eda, Tgfb1, Ccl2, Nlrp3, Aqp1 and Ccnd1) were elevated as were urinary biomarkers that have translational utility (i.e., clusterin, KIM-1, MCP-1, OPN, NGAL, B2M, calbindin, and cystatin C). All disease endpoints were more pronounced in the 200 mg/kg group, however, while measures of tissue fibrosis were sustained, there was partial recovery by day 20 in functional readouts. No mortality was observed in either dose group. DISCUSSION: Short-term delivery of adenine via precise gavage delivery induced a robust model with hallmarks of fibrotic kidney disease, had limited variance between animals, and no animal morbidity within the 20 days studied. This model represents a methodical alternative to long-term dietary dosing of adenine.

Synthesis and in vitro activity of N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine P2X(7) antagonists.

Synthesis and biological evaluation of a novel class of substituted N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine derivatives resulted in the identification of potent P2X(7) antagonists. These compounds were assayed for activity at both the human and rat P2X(7) receptors. On the benzyl moiety, a variety of functional groups were tolerated, including both electron-withdrawing and electron-donating substituents. Ortho-substitution on the benzyl group provided the greatest potency. The ortho-substituted analogs showed approximately 2.5-fold greater potency at human compared to rat P2X(7) receptors. Compounds 12 and 38 displayed hP2X(7)pIC(50)s>7.8 with less than 2-fold difference in potency at the rP2X(7).

Comparative analysis of inactivated-state block of N-type (Ca(v)2.2) calcium channels.

OBJECTIVE: The aim of this study was to compare a diverse set of peptide and small-molecule calcium channel blockers for inactivated-state block of native and recombinant N-type calcium channels using fluorescence-based and automated patch-clamp electrophysiology assays. METHODS: The pharmacology of calcium channel blockers was determined at N-type channels in IMR-32 cells and in HEK cells overexpressing the inward rectifying K(+) channel Kir2.1. N-type channels were opened by increasing extracellular KCl. In the Kir2.1/N-type cell line the membrane potential could be modulated by adjusting the extracellular KCl, allowing determination of resting and inactivated-state block of N-type calcium channels. The potency and degree of state-dependent inhibition of these blockers were also determined by automated patch-clamp electrophysiology. RESULTS: N-type-mediated calcium influx in IMR-32 cells was determined for a panel of blockers with IC(50) values of 0.001-7 µM and this positively correlated with inactivated-state block of recombinant channels measured using electrophysiology. The potency of several compounds was markedly weaker in the state-dependent fluorescence-based assay compared to the electrophysiology assay, although the degree of state-dependent blockade was comparable. CONCLUSIONS: The present data demonstrate that fluorescence-based assays are suitable for assessing the ability of blockers to selectively interact with the inactivated state of the N-type channel.

Mammalian P2X7 receptor pharmacology: comparison of recombinant mouse, rat and human P2X7 receptors.

BACKGROUND AND PURPOSE: Acute activation of P2X7 receptors rapidly opens a non-selective cation channel. Sustained P2X7 receptor activation leads to the formation of cytolytic pores, mediated by downstream recruitment of hemichannels to the cell surface. Species- and single-nucleotide polymorphism-mediated differences in P2X7 receptor activation have been reported that complicate understanding of the physiological role of P2X7 receptors. Studies were conducted to determine pharmacological differences between human, rat and mouse P2X7 receptors. EXPERIMENTAL APPROACH: Receptor-mediated changes in calcium influx and Yo-Pro uptake were compared between recombinant mouse, rat and human P2X7 receptors. For mouse P2X7 receptors, wild-type (BALB/c) and a reported loss of function (C57BL/6) P2X7 receptor were also compared. KEY RESULTS: BzATP [2,3-O-(4-benzoylbenzoyl)-ATP] was more potent than ATP in stimulating calcium influx and Yo-Pro uptake at rat, human, BALB/c and C57BL/6 mouse P2X7 receptors. Two selective P2X7 receptor antagonists, A-740003 and A-438079, potently blocked P2X7 receptor activation across mammalian species. Several reported P2X1 receptor antagonists [e.g. MRS 2159 (4-[(4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl}-2-pyridinyl)azo]-benzoic acid), PPNDS and NF279] blocked P2X7 receptors. NF279 fully blocked human P2X7 receptors, but only partially blocked BALB/c P2X7 receptors and was inactive at C57BL/6 P2X7 receptors. CONCLUSIONS AND IMPLICATIONS: These data provide new insights into P2X7 receptor antagonist pharmacology across mammalian species. P2X7 receptor pharmacology in a widely used knockout background mouse strain (C57BL/6) was similar to wild-type mouse P2X7 receptors. Several structurally novel, selective and competitive P2X7 receptor antagonists show less species differences compared with earlier non-selective antagonists.

Discovery and biological evaluation of novel cyanoguanidine P2X(7) antagonists with analgesic activity in a rat model of neuropathic pain.

We disclose the design of a novel series of cyanoguanidines that are potent (IC(50) approximately 10-100 nM) and selective (> or = 100-fold) P2X(7) receptor antagonists against the other P2 receptor subtypes such as the P2Y(2), P2X(4), and P2X(3). We also found that these P2X(7) antagonists effectively reduced nociception in a rat model of neuropathic pain (Chung model). Particularly, analogue 53 proved to be effective in the Chung model, with an ED(50) of 38 micromol/kg after intraperitoneal administration. In addition compound 53 exhibited antiallodynic effects following oral administration and maintained its efficacy following repeated administration in the Chung model. These results suggest an important role of P2X(7) receptors in neuropathic pain and therefore a potential use of P2X(7) antagonists as novel therapeutic tools for the treatment of this type of pain.

[3H]A-804598 ([3H]2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine) is a novel, potent, and selective antagonist radioligand for P2X7 receptors.

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1beta, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC50 = 10 nM), mouse (IC50 = 9 nM) and human (IC50 = 11 nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1beta and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated ([3H]A-804598; 8.1Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. [3H]A-804598 labeled a single class of high affinity binding sites (Kd=2.4 nM and apparent Bmax=0.56 pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit [3H]A-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r=0.95, P<0.05). These data demonstrate that A-804598 is one of the most potent and selective antagonists for mammalian P2X7 receptors described to date and [3H]A-804598 is a high affinity antagonist radioligand that specifically labels rat P2X7 receptors.

Structure-activity relationship studies on N'-aryl carbohydrazide P2X7 antagonists.

N'-aryl acyl hydrazides were identified as P2X7 receptor antagonists. Structure-activity relationship (SAR) studies evaluated functional activity by monitoring calcium flux inhibition in cell lines expressing recombinant human and rat P2X7 receptors. Selected analogs were assayed in vitro for their capacity to inhibit release of cytokine IL-1beta. Compounds with potent antagonist function were evaluated in vivo using the zymosan-induced peritonitis model. A representative compound effectively attenuated mechanical allodynia in a rat model of neuropathic pain.

Synthesis and in vitro activity of 1-(2,3-dichlorophenyl)-N-(pyridin-3-ylmethyl)-1H-1,2,4-triazol-5-amine and 4-(2,3-dichlorophenyl)-N-(pyridin-3-ylmethyl)-4H-1,2,4-triazol-3-amine P2X7 antagonists.

A novel series of aminotriazole-based P2X(7) antagonists was synthesized, and their structure-activity relationships (SAR) were investigated for activity at both human and rat P2X(7) receptors. Most compounds showed greater potency at the human receptor although several analogs were discovered with potent activity (pIC(50) > or = 7.5) at both human and rat P2X(7).

Synthesis and in vitro activity of N'-cyano-4-(2-phenylacetyl)-N-o-tolylpiperazine-1-carboximidamide P2X7 antagonists.

A novel series of cyanoguanidine-piperazine P2X(7) antagonists was designed based upon the structure of A-740003. Structure-activity relationship (SAR) studies focused on the piperazine moiety and the right hand side substitution. Compounds were assayed for activity at human and rat P2X(7) receptors and compound 29 was found to possess potent activity (IC(50)=30-60 nM) at both species.

Novel and potent 3-(2,3-dichlorophenyl)-4-(benzyl)-4H-1,2,4-triazole P2X7 antagonists.

Structure-activity relationship (SAR) studies were conducted around early tetrazole-based leads 3 and 4. Replacements for the tetrazole core were investigated and the pendant benzyl substitution was reoptimized with a triazole isostere. Triazole-based P2X(7) antagonists were identified with similar potency to the lead compound 4 but with improved physiochemical properties. Compound 12 was active in a rat model of neuropathic pain.

Discovery of 3-methyl-N-(1-oxy-3',4',5',6'-tetrahydro-2'H-[2,4'-bipyridine]-1'-ylmethyl)benzamide (ABT-670), an orally bioavailable dopamine D4 agonist for the treatment of erectile dysfunction.

The goal of this study was to identify a structurally distinct D(4)-selective agonist with superior oral bioavailability to our first-generation clinical candidate 1a (ABT-724) for the potential treatment of erectile dysfunction. Arylpiperazines such as (heteroarylmethyl)piperazine 1a, benzamide 2, and acetamides such as 3a,b exhibit poor oral bioavailability. Structure-activity relationship (SAR) studies with the arylpiperidine template provided potent partial agonists such as 4d and 5k that demonstrated no improvement in oral bioavailability. Further optimization with the (N-oxy-2-pyridinyl)piperidine template led to the discovery of compound 6b (ABT-670), which exhibited excellent oral bioavailability in rat, dog, and monkey (68%, 85%, and 91%, respectively) with comparable efficacy, safety, and tolerability to 1a. The N-oxy-2-pyridinyl moiety not only provided the structural motif required for agonist function but also reduced metabolism rates. The SAR study leading to the discovery of 6b is described herein.

A-85380: a pharmacological probe for the preclinical and clinical investigation of the alphabeta neuronal nicotinic acetylcholine receptor.

A-85380 [3-(2(s)-azetidinylmethoxy) pyridine] is a neuronal nicotinic acetylcholine receptor (nAChR) agonist that has been a useful tool in the investigation of the function of nAChRs in both preclinical and clinical studies. Amongst nAChR subtypes, A-85380 shows selectivity for the alpha(4)beta(2) vs. the alpha(7) or alpha(1)beta(1)deltagamma nAChRs. In functional in vitro cation flux assays, A-85380 is a potent and full agonist. A-85380 has a broad-spectrum analgesic profile with efficacy in acute, persistent, and neuropathic pain models. As demonstrated using selective nAChR antagonists or alpha(4) antisense, the alpha(4)beta(2) nAChR mediates the analgesic effects of A-85380. Interestingly, the site of action depends upon the type of pain as antinociception is mediated by descending inhibition into the spinal cord whereas anti-allodynia in neuropathic pain is mediated at both central and peripheral sites. Radiolabelled forms of A-85380 have been developed and shown to be safe for use in vivo in humans. In clinical studies using positron and photon emission tomography, marked decreases in alpha(4)beta(2) nAChRs have been seen in patients with Parkinson's and Alzheimer's disease. Although not developed as a therapeutic agent, A-85380 has proven to be an important component in the development of novel nAChR ligands for the treatment of pain and other disorders.

1-aryl-3-(4-pyridine-2-ylpiperazin-1-yl)propan-1-one oximes as potent dopamine D4 receptor agonists for the treatment of erectile dysfunction.

A new series of dopamine D4 receptor agonists, 1-aryl-3-(4-pyridinepiperazin-1-yl)propanone oximes, was designed through the modification of known dopamine D4 receptor agonist PD 168077. Replacement of the amide group with a methylene-oxime moiety produced compounds with improved stability and efficacy. Structure-activity relationsips (SAR) of the aromatic ring linked to the N-4-piperazine ring confirmed the superiority of 2-pyridine as a core for D4 agonist activity. A two-methylene linker between the oxime group and the N-1-piperazine ring displayed the best profile. New dopamine D4 receptor agonists, exemplified by (E)-1-(4-chlorophenyl)-3-(4-pyridin-2-ylpiperazin-1-yl)propan-1-one O-methyloxime (59a) and (E)-1-(3-chloro-4-fluorophenyl)-3-(4-pyridin-2-ylpiperazin-1-yl)propan-1-one O-methyloxime (64a), exhibited favorable pharmacokinetic profiles and showed oral bioavailability in rat and dog. Subsequent evaluation of 59a in the rat penile erection model revealed in vivo activity, comparable in efficacy to apomorphine. Our results suggest that the oximes provide a novel structural linker for 4-arylpiperazine-based D4 agonists, possessing leadlike quality and with potential to develop a new class of potent and selective dopamine D4 receptor agonists.

Structure-activity relationship studies on a series of novel, substituted 1-benzyl-5-phenyltetrazole P2X7 antagonists.

1-Benzyl-5-aryltetrazoles were discovered to be novel antagonists for the P2X(7) receptor. Structure-activity relationship (SAR) studies were conducted around both the benzyl and phenyl moieties. In addition, the importance of the regiochemical substitution on the tetrazole was examined. Compounds were evaluated for activity to inhibit calcium flux in both human and rat recombinant P2X(7) cell lines using fluorometric imaging plate reader technology. Analogues were also assayed for their ability to inhibit IL-1beta release and to inhibit P2X(7)-mediated pore formation in human THP-1 cells. Compound 15d was advanced to efficacy studies in a model of neuropathic pain where significant reversal of mechanical allodynia was observed at doses that did not affect motor coordination.

2-[4-(3,4-Dimethylphenyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393), a selective dopamine D4 receptor antagonist.

2-[4-(3,4-Dimethylphenlyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393) was identified as a potent dopamine D4 receptor antagonist with excellent receptor selectivity. [3H]-spiperone competition binding assays showed that A-381393 potently bound to membrane from cells expressing recombinant human dopamine D4.4 receptor (Ki=1.5 nM), which was 20-fold higher than that of clozapine (Ki=30.4 nM). A-381393 exhibited highly selective binding for the dopamine D4.4 receptor (>2700-fold) when compared to D1, D2, D3 and D5 dopamine receptors. Furthermore, in comparison to clozapine and L-745870, A-381393 exhibits better receptor selectivity, showing no affinity up to 10 microM for a panel of more than 70 receptors and channels, with the exception of moderate affinity for 5-HT2A (Ki=370 nM). A-381393 potently inhibited the functional activity of agonist-induced GTP-gamma-S binding assay and 1 microM dopamine induced-Ca2+ flux in human dopamine D4.4 receptor expressing cells, but not in human dopamine D2L or D3 receptor cells. In contrast to L-745870, A-381393 did not exhibit any significant intrinsic activity in a D4.4 receptor. In vivo, A-381393 has good brain penetration after subcutaneous administration. A-381393 inhibited penile erection induced by the selective D4 agonist PD168077 in conscious rats. Thus, A-381393 is a novel selective D4 antagonist that will enhance the ability to study dopamine D4 receptors both in vitro and in vivo.

In vitro and in vivo effects of an alpha3 neuronal nicotinic acetylcholine receptor antisense oligonucleotide.

In the mammalian central nervous system (CNS), a family of alpha and beta subunits (alpha2-7, beta2-4) assemble to form both hetero- and homopentameric neuronal nicotinic acetylcholine receptors (nAChRs). In contrast to alpha4beta2 and alpha7, the predominant brain subtypes, far less is known regarding the functional expression and significance of alpha3-containing nAChRs in the CNS. In trying to better understand the role alpha3 in the CNS, an antisense knockdown strategy was utilized in the present studies. Specifically, Isis 106567 was identified out of 80 antisense oligonucleotides (aONs) designed and screened for their ability to reduce alpha3 mRNA expression in PC-12 cells. In addition to reducing alpha3 mRNA by greater than 75%, Isis 106567 attenuated nicotine-induced calcium influx in alpha3-expressing F11 cells. In vivo studies revealed significant reduction of alpha3 mRNA levels in both thalamus and medial habenula, regions known to express alpha3, following continuous (7 days) intracerebroventricular (i.c.v.) infusion of Isis 106567 in rats. Consistent with functional alpha3 knockdown, epibatidine-induced c-Fos expression in the medial habenula was attenuated in aON-treated rats. Known physiological responses elicited by epibatidine, such as hypothermia and micturition, were not affected by alpha3 aON treatment. However, the incidence of epibatidine-induced seizures was reduced in alpha3-antisense aON-treated rats, suggesting that alpha3 may be involved in mediating seizures produced by the nAChR agonist. Results of our studies suggest that Isis 106567 may be a useful in vivo tool for characterizing the functional significance of alpha3 expression in the CNS.

Synthesis and activity of 2-[4-(4-[3H]-2-cyanophenyl)piperazinyl]-N-(2,4,6-[3H]3-3-methylphenyl)acetamide: a selective dopamine D4 receptor agonist and radioligand.

The first selective dopamine D4 agonist radioligand is described. The synthesis of these piperazine radioligands relied on the transformation of brominated precursors 4a and 4b with tritium gas in the presence of a sensitive cyano functional group. The specific activity of these two radioligands was measured and [3H]6b found to be suitable for use in D4 saturation and competition binding studies. The synthesis, biological, and radioactivity of this new agonist radioligand as well as preliminary SAR will be discussed.

Comparative pharmacology of human dopamine D(2)-like receptor stable cell lines coupled to calcium flux through Galpha(qo5).

The goal of this study was to develop a new approach to study the pharmacology of the dopamine D(4) receptor that could be used in comparative studies with dopamine D(2) and D(3) receptors. Stable HEK-293 cell lines co-expressing recombinant human D(2L), D(3) or D(4) receptors along with Galpha(qo5) cDNA were prepared. Dopamine induced a robust, transient calcium signal in these cell lines with EC(50)s for D(2L), D(3) and D(4) of 18.0, 11.9 and 2.2 nM, respectively. Reported D(4)-selective agonists CP226269 and PD168077 were potent, partial D(4) agonists exhibiting 31-1700-fold selectivity for D(4) over D(3) or D(2). Non-selective D(2)-like agonists apomorphine and quinpirole showed full efficacy but did not discriminate across the three receptors. D(3)-selective agonists 7-hydroxy-DPAT and PD128907 were potent but non-selective D(2)-like agonists. The reported D(3) partial agonist BP-897 exhibited minimal agonist activity at D(3) but was a potent D(3) antagonist and a partial D(4) agonist. Other D(2)-like antagonists, haloperidol, clozapine, and domperidone showed concentration-dependent inhibition of dopamine responses at all three receptors with K(i) ranging from 0.05 to 48.3 nM. The D(3) selective antagonist S33084 and D(4)-selective antagonist L-745870 were highly selective for D(3) and D(4) receptors with K(b) of 0.7 and 0.1 nM, respectively. Stable co-expression of D(2)-like receptors with chimeric Galpha(qo5) proteins in HEK-293 cells is an efficient method to study receptor activation in a common cellular background and an efficient method for direct comparison of ligand affinity and efficacy across human D(2L), D(3) and D(4) receptors.