Development of an oral treatment that rescues gait ataxia and retinal degeneration in a phenotypic mouse model of familial dysautonomia.

Familial dysautonomia (FD) is a rare neurodegenerative disease caused by a splicing mutation in elongator acetyltransferase complex subunit 1 (ELP1). This mutation leads to the skipping of exon 20 and a tissue-specific reduction of ELP1, mainly in the central and peripheral nervous systems. FD is a complex neurological disorder accompanied by severe gait ataxia and retinal degeneration. There is currently no effective treatment to restore ELP1 production in individuals with FD, and the disease is ultimately fatal. After identifying kinetin as a small molecule able to correct the ELP1 splicing defect, we worked on its optimization to generate novel splicing modulator compounds (SMCs) that can be used in individuals with FD. Here, we optimize the potency, efficacy, and bio-distribution of second-generation kinetin derivatives to develop an oral treatment for FD that can efficiently pass the blood-brain barrier and correct the ELP1 splicing defect in the nervous system. We demonstrate that the novel compound PTC258 efficiently restores correct ELP1 splicing in mouse tissues, including brain, and most importantly, prevents the progressive neuronal degeneration that is characteristic of FD. Postnatal oral administration of PTC258 to the phenotypic mouse model TgFD9;Elp1Δ20/flox increases full-length ELP1 transcript in a dose-dependent manner and leads to a 2-fold increase in functional ELP1 in the brain. Remarkably, PTC258 treatment improves survival, gait ataxia, and retinal degeneration in the phenotypic FD mice. Our findings highlight the great therapeutic potential of this novel class of small molecules as an oral treatment for FD.

Discovery of 2-(4-sulfonamidophenyl)-indole 3-carboxamides as potent and selective inhibitors with broad hepatitis C virus genotype activity targeting HCV NS4B.

A novel series of 2-(4-sulfonamidophenyl)-indole 3-carboxamides was identified and optimized for activity against the HCV genotype 1b replicon resulting in compounds with potent and selective activity. Further evaluation of this series demonstrated potent activity across HCV genotypes 1a, 2a and 3a. Compound 4z had reduced activity against HCV genotype 1b replicons containing single mutations in the NS4B coding sequence (F98C and V105M) indicating that NS4B is the target. This novel series of 2-(4-sulfonamidophenyl)-indole 3-carboxamides serves as a promising starting point for a pan-genotype HCV discovery program.

6-(Azaindol-2-yl)pyridine-3-sulfonamides as potent and selective inhibitors targeting hepatitis C virus NS4B.

A structure-activity relationship investigation of various 6-(azaindol-2-yl)pyridine-3-sulfonamides using the HCV replicon cell culture assay led to the identification of a potent series of 7-azaindoles that target the hepatitis C virus NS4B. Compound 2ac, identified via further optimization of the series, has excellent potency against the HCV 1b replicon with an EC50 of 2nM and a selectivity index of >5000 with respect to cellular GAPDH RNA. Compound 2ac also has excellent oral plasma exposure levels in rats, dogs and monkeys and has a favorable liver to plasma distribution profile in rats.

Discovery of novel HCV inhibitors: synthesis and biological activity of 6-(indol-2-yl)pyridine-3-sulfonamides targeting hepatitis C virus NS4B.

A novel series of 6-(indol-2-yl)pyridine-3-sulfonamides was prepared and evaluated for their ability to inhibit HCV RNA replication in the HCV replicon cell culture assay. Preliminary optimization of this series furnished compounds with low nanomolar potency against the HCV genotype 1b replicon. Among these, compound 8c has identified as a potent HCV replicon inhibitor (EC50=4 nM) with a selectivity index with respect to cellular GAPDH of more than 2500. Further, compound 8c had a good pharmacokinetic profile in rats with an IV half-life of 6h and oral bioavailability (F) of 62%. Selection of HCV replicon resistance identified an amino acid substitution in HCV NS4B that confers resistance to these compounds. These compounds hold promise as a new chemotype with anti-HCV activity mediated through an underexploited viral target.

Discovery and Optimization of Indolyl-Containing 4-Hydroxy-2-Pyridone Type II DNA Topoisomerase Inhibitors Active against Multidrug Resistant Gram-negative Bacteria.

There exists an urgent medical need to identify new chemical entities (NCEs) targeting multidrug resistant (MDR) bacterial infections, particularly those caused by Gram-negative pathogens. 4-Hydroxy-2-pyridones represent a novel class of nonfluoroquinolone inhibitors of bacterial type II topoisomerases active against MDR Gram-negative bacteria. Herein, we report on the discovery and structure-activity relationships of a series of fused indolyl-containing 4-hydroxy-2-pyridones with improved in vitro antibacterial activity against fluoroquinolone resistant strains. Compounds 6o and 6v are representative of this class, targeting both bacterial DNA gyrase and topoisomerase IV (Topo IV). In an abbreviated susceptibility screen, compounds 6o and 6v showed improved MIC90 values against Escherichia coli (0.5-1 µg/mL) and Acinetobacter baumannii (8-16 µg/mL) compared to the precursor compounds. In a murine septicemia model, both compounds showed complete protection in mice infected with a lethal dose of E. coli.

Specific Correction of Alternative Survival Motor Neuron 2 Splicing by Small Molecules: Discovery of a Potential Novel Medicine To Treat Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant and toddler mortality, and there is currently no approved therapy available. SMA is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. These mutations or deletions result in low levels of functional SMN protein. SMN2, a paralogous gene to SMN1, undergoes alternative splicing and exclusion of exon 7, producing an unstable, truncated SMNΔ7 protein. Herein, we report the identification of a pyridopyrimidinone series of small molecules that modify the alternative splicing of SMN2, increasing the production of full-length SMN2 mRNA. Upon oral administration of our small molecules, the levels of full-length SMN protein were restored in two mouse models of SMA. In-depth lead optimization in the pyridopyrimidinone series culminated in the selection of compound 3 (RG7800), the first small molecule SMN2 splicing modifier to enter human clinical trials.

Discovery and Optimization of Small Molecule Splicing Modifiers of Survival Motor Neuron 2 as a Treatment for Spinal Muscular Atrophy.

The underlying cause of spinal muscular atrophy (SMA) is a deficiency of the survival motor neuron (SMN) protein. Starting from hits identified in a high-throughput screening campaign and through structure-activity relationship investigations, we have developed small molecules that potently shift the alternative splicing of the SMN2 exon 7, resulting in increased production of the full-length SMN mRNA and protein. Three novel chemical series, represented by compounds 9, 14, and 20, have been optimized to increase the level of SMN protein by >50% in SMA patient-derived fibroblasts at concentrations of <160 nM. Daily administration of these compounds to severe SMA Δ7 mice results in an increased production of SMN protein in disease-relevant tissues and a significant increase in median survival time in a dose-dependent manner. Our work supports the development of an orally administered small molecule for the treatment of patients with SMA.

Structural features of azidopyridinyl neonicotinoid probes conferring high affinity and selectivity for mammalian alpha4beta2 and Drosophila nicotinic receptors.

The higher toxicity of neonicotinoid insecticides such as N-(6-chloropyridin-3-ylmethyl)-2-nitroiminoimidazolidine (imidacloprid) to insects than mammals is due in large part to target site specificity at the corresponding nicotinic acetylcholine receptors (nAChRs). We propose that neonicotinoids with a protonated N-unsubstituted imine or equivalent substituent recognize the anionic subsite of the mammalian alpha4beta2 nAChR whereas the negatively charged (delta(-)) tip of the neonicotinoid insecticides interacts with a putative cationic subsite of the insect nAChR. This hypothesis can be tested by using two photoaffinity probes that differ only in the N-unsubstituted imine vs negatively charged (delta(-)) tip. Synthesis methodology was developed for compounds combining three moieties: pyridin-3-ylmethyl or 6-chloropyridin-3-ylmethyl and their 4- and 5-azido analogues; imidazolidine, 4-imidazoline or 4-thiazoline; and N-unsubstituted imine, nitroimine, cyanoimine, or nitromethylene. Structure-activity studies compared displacement of [(3)H]nicotine binding in mammalian alpha4beta2 nAChR and [(3)H]imidacloprid binding in Drosophila nAChR. Preferred compounds are N-(5-azido-6-chloropyridin-3-ylmethyl) with 2-iminothiazoline for alpha4beta2 (K(i) = 0.47 nM) and with 2-nitroiminothiazoline or 2-nitromethyleneimidazolidine for Drosophila (K(i) = 0.72-3.9 nM).

Drosophila nicotinic receptors: evidence for imidacloprid insecticide and alpha-bungarotoxin binding to distinct sites.

The principal mammalian brain nicotinic acetylcholine receptors (nAChRs) are the alpha-bungarotoxin (alpha-BGT)-insensitive alpha 4 beta 2 and the alpha-BGT-sensitive alpha 7 subtypes assayed with radiolabeled nicotinoids and alpha-BGT, respectively. Drosophila head membranes bind the insecticide radioligand [(3)H]imidacloprid ([(3)H]IMI) and [(3)H]alpha-BGT with K(D) 5.7 and 2.7 nM and B(max) 980 and 1400 fmol/mg protein, respectively. The hypothesis that [(3)H]IMI at 2.5 or 20 nM and [(3)H]alpha-BGT at 1 or 10 nM bind to distinct sites or subtypes is tested by using these radioligands alone and together in simultaneous dual binding experiments. These studies show no interference by one radioligand in the binding of the other one, i.e., independent binding, and that both unlabeled IMI and alpha-BGT give biphasic displacement curves. The pharmacological profiles of [(3)H]IMI and [(3)H]alpha-BGT suggest distinct binding sites for the two radioligands. These findings are consistent with those obtained with hybrid receptors assembled from Drosophila alpha subunits and a vertebrate beta subunit and with immunological and protein biochemical approaches. This study, therefore, provides direct evidence for distinct IMI- and alpha-BGT-sensitive sites or subtypes in Drosophila brain.

Insecticides in Chinese medicinal plants: survey leading to jacaranone, a neurotoxicant and glutathione-reactive quinol.

Sixty-two plant species from central China were purported to have insecticidal activity. Adult house fly (Musca domestica) toxicity assays were used to identify the two most active plants and guide the chromatographic isolation of the insecticidal components. The active ingredient of Senecio palmatusPall. (Asteraceae) was characterized as methyl (1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl)acetate (jacaranone) (1), previously known to have insect antifeedant activity. Mono- and bisglutathione (GSH) adducts are formed on incubation of 1 with GSH and rat liver GSH S-transferase. The toxic action of 1 in mice (intraperitoneal LD(50) = 150-200 mg/kg) is associated with both neurological signs and GSH depletion in liver 90 min after treatment. Paeonia suffruticosa var. papaveracea (Andr.) Kerner (Paeoniaceae) was the other active plant found here to have 2'-hydroxy-4'-methoxyacetophenone (paeonol) (2) as an insecticidal ingredient in the root and in one case, for the whole plant, contaminated with S-tert-butylthiomethyl O,O-diethyl phosphorodithioate (terbufos) (3), a synthetic anticholinesterase insecticide.

Structure-activity relationship (SAR) optimization of 6-(indol-2-yl)pyridine-3-sulfonamides: identification of potent, selective, and orally bioavailable small molecules targeting hepatitis C (HCV) NS4B.

A novel, potent, and orally bioavailable inhibitor of hepatitis C RNA replication targeting NS4B, compound 4t (PTC725), has been identified through chemical optimization of the 6-(indol-2-yl)pyridine-3-sulfonamide 2 to improve DMPK and safety properties. The focus of the SAR investigations has been to identify the optimal combination of substituents at the indole N-1, C-5, and C-6 positions and the sulfonamide group to limit the potential for in vivo oxidative metabolism and to achieve an acceptable pharmacokinetic profile. Compound 4t has excellent potency against the HCV 1b replicon, with an EC50 = 2 nM and a selectivity index of >5000 with respect to cellular GAPDH. Compound 4t has an overall favorable pharmacokinetic profile with oral bioavailability values of 62%, 78%, and 18% in rats, dogs, and monkeys, respectively, as well as favorable tissue distribution properties with a liver to plasma exposure ratio of 25 in rats.

Motor neuron disease. SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA.

Defining nicotinic agonist binding surfaces through photoaffinity labeling.

Nicotinic acetylcholine (ACh) receptor (nAChR) agonists are potential therapeutic agents for neurological dysfunction. In the present study, the homopentameric mollusk ACh binding protein (AChBP), used as a surrogate for the extracellular ligand-binding domain of the nAChR, was specifically derivatized by the highly potent agonist azidoepibatidine (AzEPI) prepared as a photoaffinity probe and radioligand. One EPI-nitrene photoactivated molecule was incorporated in each subunit interface binding site based on analysis of the intact derivatized protein. Tryptic fragments of the modified AChBP were analyzed by collision-induced dissociation and Edman sequencing of radiolabeled peptides. Each specific EPI-nitrene-modified site involved either Tyr195 of loop C on the principal or (+)-face or Met116 of loop E on the complementary or (-)-face. The two derivatization sites were observed in similar frequency, providing evidence of the reactivity of the azido/nitrene probe substituent and close proximity to both residues. [3H]AzEPI binds to the alpha4beta2 nAChR at a single high-affinity site and photoaffinity-labels only the alpha4 subunit, presumably modifying Tyr225 spatially corresponding to Tyr195 of AChBP. Phe137 of the beta2 nAChR subunit, equivalent to Met116 of AChBP, conceivably lacks sufficient reactivity with the nitrene generated from the probe. The present photoaffinity labeling in a physiologically relevant condition combined with the crystal structure of AChBP allows development of precise structural models for the AzEPI interactions with AChBP and alpha4beta2 nAChR. These findings enabled us to use AChBP as a structural surrogate to define the nAChR agonist site.

Novel irreversible butyrylcholinesterase inhibitors: 2-chloro-1-(substituted-phenyl)ethylphosphonic acids.

2-Chloroethylphosphonic acid (ethephon) as the dianion phosphorylates butyrylcholinesterase (BChE) at its active site. In contrast, the classical organophosphorus esterase inhibitors include substituted-phenyl dialkylphosphates (e.g., paraoxon) with electron-withdrawing aryl substituents. The chloroethyl and substituted-phenyl moieties are combined in this study as 2-chloro-1-(substituted-phenyl)ethylphosphonic acids (1) to define the structure--activity relationships and mechanism of BChE inhibition by ethephon and its analogues. Phenyl substituents considered are 3- and 4-nitro, 3- and 4-dimethylamino, and 3- and 4-trimethylammonium. Phosphonic acids were synthesized via the corresponding O,O-diethyl phosphonate precursors followed by deprotection with trimethylsilyl bromide. They decompose under basic conditions about 100-fold faster than ethephon to yield the corresponding styrene derivatives. Electron-withdrawing substituents on the phenyl ring decrease the hydrolysis rate while electron-donating substituents increase the rate. The 4-trimethylammonium analogue has the highest affinity (K(i)=180 microM) and potency (IC(50)=19 microM) in first binding reversibly at the substrate site (possibly with stabilization in a dianion--monoanion environment) and then progressively and irreversibly inhibiting the enzyme activity. These observations suggest dissociation of chloride as the first and rate-limiting step both in the hydrolysis and by analogy in phosphorylation of BChE by bound at the active site.

5-Azidoepibatidine: an exceptionally potent photoaffinity ligand for neuronal alpha 4 beta 2 and alpha 7 nicotinic acetylcholine receptors.

Racemic 5-azidoepibatidine [(+/-)-1] was synthesized via 5-aminoepibatidine as a candidate photoaffinity ligand with exceptionally high affinity at the mammalian neuronal nicotinic receptors (K(i) values of 0.027 nM for alpha 4 beta 2 and 9.7 nM for alpha 7) and excellent photoreactivity.

Alpha-nitro ketone as an electrophile and nucleophile: synthesis of 3-substituted 2-nitromethylenetetrahydrothiophene and -tetrahydrofuran as drosophila nicotinic receptor probes.

3-(6-Chloropyridin-3-yl)methyl-2-nitromethylenetetrahydrothiophene 2 and -tetrahydrofuran 3 were synthesized through novel approaches using alpha-nitro ketone intermediates as an electrophile and nucleophile, respectively. The 2-nitromethylenetetrahydrothiophene 2 was formed exclusively as the Z-isomer through intramolecular attack by a thiol substituent at the carbonyl group of an alpha-nitro ketone, in which the alpha-nitro ketone served as an electrophile. In contrast, the corresponding 2-nitromethylenetetrahydrofuran 3, not accessible by the above route due to limited stability, was prepared as a mixture of E- and Z-isomers by intramolecular attack of the alpha-nitro ketone enol anion in which the deprotonated alpha-nitro ketone served as a nucleophile. These compounds, together with the corresponding 2-nitromethylenepyrrolidine (1), were used to probe the Drosophila neonicotinoid-nicotinic acetylcholine receptor interaction.

The neonicotinoid electronegative pharmacophore plays the crucial role in the high affinity and selectivity for the Drosophila nicotinic receptor: an anomaly for the nicotinoid cation--pi interaction model.

Cation-pi interaction, a prominent feature in agonist recognition by neurotransmitter-gated ion channels, does not apply to the anomalous action of neonicotinoids at the insect nicotinic acetylcholine receptor (nAChR). Insect-selective neonicotinoids have an electronegative pharmacophore (tip) in place of the ammonium or iminium cation of the vertebrate-selective nicotinoids, suggesting topological divergence of the agonist-binding sites in insect and vertebrate nAChRs. This study defines the molecular and electronic basis for the potent and selective interaction of the neonicotinoid electronegative pharmacophore with a unique subsite of the Drosophila but not of the vertebrate alpha4beta2 nAChR. Target site potency and selectivity are retained when the usual neonicotinoid N-nitroimine (=NNO(2)) electronegative tip is replaced with N-nitrosoimine (=NNO) or N-(trifluoroacetyl)imine (=NCOCF(3)) in combination with an imidazolidine, imidazoline, thiazolidine, or thiazoline heterocycle. X-ray crystallography establishes coplanarity between the heterocyclic and imine planes, including the electronegative substituent in the trans configuration. The functional tip is the coplanar oxygen atom of the N-nitrosoimine or the equivalent oxygen of the N-nitroimine. Quantum mechanics in the gas and aqueous phases fully support the conserved coplanarity and projection of the strongly electronegative tip. Further, a bicyclic analogue with a nitro tip in the cis configuration but retaining coplanarity has a high potency, whereas the N-trifluoromethanesulfonylimine (=NSO(2)CF(3)) moiety lacking coplanarity confers very low activity. The coplanar system between the electronegative tip and guanidine-amidine moiety extends the conjugation and facilitates negative charge (delta(-)) flow toward the tip, thereby enhancing interaction with the proposed cationic subsite such as lysine or arginine in the Drosophila nAChR.

Rotenone, deguelin, their metabolites, and the rat model of Parkinson's disease.

Rotenone and deguelin are the major active ingredients and principal components of cuberesin from Lonchocarpus utilis used as a botanical insecticide and piscicide. They are also potent complex I (NADH:ubiquinone oxidoreductase) inhibitors. Rotenone was known earlier, and deguelin is shown here to induce a Parkinson's disease (PD)-like syndrome after subcutaneous treatment of rats by osmotic minipump. Rotenone at 3 mg/kg/day or deguelin at 6 but not 3 mg/kg/day induces degeneration of the nigrostriatal dopaminergic pathway, as shown by reduced tyrosine hydroxylase immunoreactivity with treatments for 5 or 6 days. The neuropathological lesions are associated with a brain level of parent rotenoid of 0.4-1.3 ppm but not with the much smaller brain level of 12abeta-hydroxyrotenoids or other metabolites analyzed by HPLC and LC/MS. We previously established that the hydroxylated metabolites and derivatives of rotenone and deguelin are all less active (i.e., detoxified) as complex I inhibitors relative to the parent rotenoids. The PD-like syndrome induced in rats by rotenone and deguelin is therefore due to the parent compounds rather than metabolites. Deguelin is about half as active as rotenone in inducing the PD-like syndrome in rats and in acute ip LD50 in mice. Rotenone and deguelin are metabolized by human recombinant 3A4 and 2C19 but not five other P450 enzymes. 2C19 is more selective than 3A4 in forming the 12abeta-hydroxyrotenoids. Identified sites of metabolic attack individually or in combination are as follows: 12abeta hydroxylation and 2-O-demethylation of both compounds, oxidation of the rotenone isopropenyl substituent to mono and diol derivatives, and probable oxidation of the deguelin dimethylchromene double bond. These toxicological features must be considered in using rotenone, deguelin, and their analogues as pesticides, candidate radioimaging and cancer chemopreventive agents, and models of PD.