Local application of engineered insulin-like growth factor I mRNA demonstrates regenerative therapeutic potential in vivo.

Insulin-like growth factor I (IGF-I) is a growth-promoting anabolic hormone that fosters cell growth and tissue homeostasis. IGF-I deficiency is associated with several diseases, including growth disorders and neurological and musculoskeletal diseases due to impaired regeneration. Despite the vast regenerative potential of IGF-I, its unfavorable pharmacokinetic profile has prevented it from being used therapeutically. In this study, we resolved these challenges by the local administration of IGF-I mRNA, which ensures desirable homeostatic kinetics and non-systemic, local dose-dependent expression of IGF-I protein. Furthermore, IGF-I mRNA constructs were sequence engineered with heterologous signal peptides, which improved in vitro protein secretion (2- to 6-fold) and accelerated in vivo functional regeneration (16-fold) over endogenous IGF-I mRNA. The regenerative potential of engineered IGF-I mRNA was validated in a mouse myotoxic muscle injury and rabbit spinal disc herniation models. Engineered IGF-I mRNA had a half-life of 17-25 h in muscle tissue and showed dose-dependent expression of IGF-I over 2-3 days. Animal models confirm that locally administered IGF-I mRNA remained at the site of injection, contributing to the safety profile of mRNA-based treatment in regenerative medicine. In summary, we demonstrate that engineered IGF-I mRNA holds therapeutic potential with high clinical translatability in different diseases.

A high quality, industrial data set for binding affinity prediction: performance comparison in different early drug discovery scenarios.

We release a new, high quality data set of 1162 PDE10A inhibitors with experimentally determined binding affinities together with 77 PDE10A X-ray co-crystal structures from a Roche legacy project. This data set is used to compare the performance of different 2D- and 3D-machine learning (ML) as well as empirical scoring functions for predicting binding affinities with high throughput. We simulate use cases that are relevant in the lead optimization phase of early drug discovery. ML methods perform well at interpolation, but poorly in extrapolation scenarios-which are most relevant to a real-world application. Moreover, we find that investing into the docking workflow for binding pose generation using multi-template docking is rewarded with an improved scoring performance. A combination of 2D-ML and 3D scoring using a modified piecewise linear potential shows best overall performance, combining information on the protein environment with learning from existing SAR data.

Discovery of the first low-shift positive allosteric modulators for the muscarinic M1 receptor.

Positive modulation of the muscarinic M1-receptor has for a long time attracted scientists and drug developers for the potential treatment of Alzheimer's disease or Schizophrenia. The precognitive potential of M1 activation has however not been clinically demonstrated as a result of side effects associated both with agonists and positive allosteric modulators (PAM's) of the M1-receptor. To avoid excessive activation of the M1-receptor we have designed a new screening format and developed the first low-shift positive allosteric modulators for the M1 receptor. Low-shift PAM's offer the potential of "use-dependent" attenuation of transmitter-signaling while avoiding pseudo-agonistic behavior in vivo as a common limitation of the so far described high-shift PAM's. With these novel M1-PAM's, the M1 receptor is potentially the first GPCR for which both, high- and low shift PAM's have become available.

Discovery of 7-arylsulfonyl-1,2,3,4, 4a,9a-hexahydro-benzo[4,5]furo[2,3-c]pyridines: identification of a potent and selective 5-HT6 receptor antagonist showing activity in rat social recognition test.

Serotoninergic neurotransmission has been implicated in modulation of learning and memory. It has been demonstrated that 5-hydroxytryptamine(6) (5-HT(6)) receptor antagonists show beneficial effect on cognition in several animal models. Based on a pharmacophore model reported in the literature, we have designed and successfully identified a 7-benzenesulfonyl-1,2,3,4-tetrahydro-benzo[4,5]furo[2,3-c]pyridine (3a) scaffold as a novel class of 5-HT(6) receptor antagonists. Despite good activity against 5-HT(6) receptor, 3a exhibited poor liver microsome stability in mouse, rat and dog. It was demonstrated that the saturation of the double bond of the tetrahydropyridine ring of 3a enhanced metabolic stability. However the resulting compound, 4a (7-phenylsulfonyl-1,2,3,4,4a,9a-hexahydro-benzo[4,5]furo[2,3-c] pyridine-HCl salt) exhibited ∼30-fold loss in potency along with introduction of two chiral centers. In our optimization process for this series, we found that substituents at the 2 or 3 positions on the distal aryl group are important for enhancing activity against 5-HT(6). Separation of enantiomers and subsequent optimization and SAR with bis substituted phenyl sulfone provided potent 5-HT(6) antagonists with improved PK profiles in rat. A potent, selective 5-HT(6)R antagonist (15k) was identified from this study which showed good oral bioavailability (F=39%) in rat with brain penetration (B/P=2.76) and in vivo activity in a rat social recognition test.

Novel brain penetrant benzofuropiperidine 5-HT6 receptor antagonists.

7-Arylsulfonyl substituted benzofuropiperidine was discovered as a novel scaffold for 5HT(6) receptor antagonists. Optimization by substitution at C-1 position led to identification of selective, orally bioavailable, brain penetrant antagonists with reduced hERG liability. An advanced analog tested in rat social recognition model showed significant activity suggesting potential utility in the enhancement of short-term memory.

CEP-26401 (irdabisant), a potent and selective histamine H3 receptor antagonist/inverse agonist with cognition-enhancing and wake-promoting activities.

CEP-26401 [irdabisant; 6-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)-propoxy]-phenyl}-2H-pyridazin-3-one HCl] is a novel, potent histamine H3 receptor (H3R) antagonist/inverse agonist with drug-like properties. High affinity of CEP-26401 for H3R was demonstrated in radioligand binding displacement assays in rat brain membranes (K(i) = 2.7 ± 0.3 nM) and recombinant rat and human H3R-expressing systems (K(i) = 7.2 ± 0.4 and 2.0 ± 1.0 nM, respectively). CEP-26401 displayed potent antagonist and inverse agonist activities in [³5S]guanosine 5'-O-(γ-thio)triphosphate binding assays. After oral dosing of CEP-26401, occupancy of H3R was estimated by the inhibition of ex vivo binding in rat cortical slices (OCC50 = 0.1 ± 0.003 mg/kg), and antagonism of the H3R agonist R-α-methylhistamine- induced drinking response in the rat dipsogenia model was demonstrated in a similar dose range (ED50 = 0.06 mg/kg). CEP-26401 improved performance in the rat social recognition model of short-term memory at doses of 0.01 to 0.1 mg/kg p.o. and was wake-promoting at 3 to 30 mg/kg p.o. In DBA/2NCrl mice, CEP-26401 at 10 and 30 mg/kg i.p. increased prepulse inhibition (PPI), whereas the antipsychotic risperidone was effective at 0.3 and 1 mg/kg i.p. Coadministration of CEP-26401 and risperidone at subefficacious doses (3 and 0.1 mg/kg i.p., respectively) increased PPI. These results demonstrate potent behavioral effects of CEP-26401 in rodent models and suggest that this novel H3R antagonist may have therapeutic utility in the treatment of cognitive and attentional disorders. CEP-26401 may also have therapeutic utility in treating schizophrenia or as adjunctive therapy to approved antipsychotics.

5-HT6 receptor antagonists as potential therapeutics for cognitive impairment.

Cognitive impairment (CI) has been recognized as a core feature of Alzheimer's disease (AD) and schizophrenia. The 5-HT(6) receptor is an attractive target for the development of cognitive enhancers due to its unique localization and pharmacology. 5-HT(6) receptor antagonists have been shown to modulate multiple neurotransmitter systems and therefore enhance cognition in preclinical studies. This premise translated into the clinical efficacy of the 5-HT(6) receptor antagonist SB-742457 in mild-to-moderate AD patients. Advances in the understanding of the structure-activity-relationship, the design of novel 5-HT(6) receptor ligands and their potential application for the treatment of CI are reviewed.

Detection of low level histamine H3 receptor occupancy by autoradiography.

Histamine H(3) receptor antagonists have been proposed as a novel approach to the treatment of cognitive, attentional, and sleep disorders. It is apparent that H(3) receptor antagonists produce in vivo effects in preclinical animal models of central diseases across a wide dose range. In order to characterize the relationship between efficacy in the preclinical models and H(3) receptor occupancy, a brain slice receptor autoradiography method was used. Brain slice receptor autoradiography requires less in vitro tissue processing, preserves brain structure, and provides anatomical localization of compound in the brain. Consistent with H(3) receptor distribution, in vitro autoradiography experiments demonstrated specific binding of [(3)H]NAMH (N-alpha-methylhistamine) in rat cortex, and other brain regions, but not in cerebellum. Ex vivo H(3)R brain slice autoradiography was able to detect H(3) receptor occupancy by reference antagonists at doses lower than previously found using a homogenate assay format. The method is relatively quick with image acquisition on a beta-imager and is capable of detecting receptor occupancy in different brain regions simultaneously. Furthermore, the increased sensitivity should be useful in providing dosing guidelines for H(3) antagonists in both preclinical and clinical settings.

Dimebolin is a 5-HT6 antagonist with acute cognition enhancing activities.

Dimebolin (Dimebon), is a non-selective antihistamine approved in Russia for the treatment of allergy. Recently, this drug has been shown to be neuroprotective in cellular models of Alzheimer's disease and Huntington's disease, and to preserve cognitive function when chronically administered to AF64A lesioned rats. Interests in identifying the molecular targets of dimebolin have intensified with reports of efficacy in clinical trials with Alzheimer's patients. Dimebolin has been found to interact with a number of molecular targets including acetylcholinesterases, N-methyl-d-aspartate receptors, and voltage-gated calcium channels, with potencies in the range of 5-50 microM. In the present study, the action of dimebolin at the serotonin 5-HT(6) receptor was investigated. Dimebolin binds with moderate affinity to both the human and rat recombinant 5-HT(6) receptor (K(i)=26.0+/-2.5 nM and 119.0+/-14.0 nM respectively) as well as the native rat 5-HT(6) receptor, and acts as an antagonist in functional cAMP assays. Furthermore, dimebolin occupies the 5-HT(6) receptor in vivo as assessed by ex vivo autoradiography, with a dose-occupancy relationship similar to that of the selective 5-HT(6) antagonist SB-399885. Finally, both SB-399885 and dimebolin produce an acute enhancement of short-term social recognition memory, although dimebolin is approximately 10-fold less potent than SB-399885. Taken together, these studies demonstrate that dimebolin antagonizes the 5-HT(6) receptor with higher affinity than other targets characterized to date, and suggest that this activity may play a role in the acute cognition enhancing effects of this compound in preclinical models and in the clinic.

The phosphatidylinositol 3-kinase inhibitor LY 294002 inhibits GlyT1-mediated glycine uptake.

The actions of neurotransmitter glycine are regulated by the Na+/Cl(-) dependent high-affinity glycine transporters, GlyT1 and GlyT2. These two members of the SLC6 transport family have been cloned and extensively characterized, however relatively little is known regarding their modulation. In the present study, glycine uptake in primary cultures of rat embryonic cortex has been characterized and the effects of the phosphatidylinositol 3 (PI3) kinase inhibitors LY 294002 and wortmannin on GlyT1- and GlyT2-mediated glycine uptake were investigated. GlyT1 inhibitors ALX 5407 and sarcosine reduced total glycine uptake to 80% whereas the specific GlyT2 inhibitor Org 25543 had no effect. In the presence of alanine, glycine uptake was completely blocked by the GlyT1 inhibitors ALX 5407 and sarcosine, suggesting that the high-affinity glycine uptake occurs predominantly via GlyT1. Kinetic analysis of GlyT1 revealed the Km value of 27+/-1.5 microM and Vmax value of 157+/-14 pmol/mg/min. LY 294002, a PI3 kinase inhibitor, blocked the GlyT1-mediated glycine uptake with an IC50 value of 81+/-2 microM, whereas another inhibitor wortmannin did not show any effect. In human placental choriocarcinoma (JAR) cells, which have been previously shown to predominantly express GlyT1a, LY 294002 showed a similar potency with an IC50 value of 86+/-3 microM. Immunoblots demonstrated that LY 294002 and wortmannin inhibited PI3 kinase-dependent Akt phosphorylation in the primary cultures with IC50 values of 10+/-4 microM and 7+/-1 nM, respectively. These results suggest that the commonly used PI3 kinase blocker LY 294002 may modulate GlyT1 function independent of PI3 kinase inhibition. Kinetic analysis in the presence of LY 294002 demonstrated significant decreases of both Km and Vmax values, suggesting a mechanism of uncompetitive inhibition on GlyT1-mediated glycine uptake. In addition, glycine release was blocked by LY 294002. These results raised a possibility that LY 294002 might interact with GlyT1.

Transposition of three amino acids transforms the human metabotropic glutamate receptor (mGluR)-3-positive allosteric modulation site to mGluR2, and additional characterization of the mGluR2-positive allosteric modulation site.

Glutamate is a major neurotransmitter in the central nervous system, and abnormal glutamate neurotransmission has been implicated in many neurological disorders, including schizophrenia, Alzheimer's disease, Parkinson's disease, addiction, anxiety, depression, epilepsy, and pain. Metabotropic glutamate receptors (mGluRs) activate intracellular signaling cascades in a G protein-dependent manner, which offer the opportunity for developing drugs that regulate glutamate neurotransmission in a functionally selective manner. In the present study, we further characterize the human mGluR2 (hmGluR2) potentiator binding site by showing that the substitution of the three amino acids found to be required for hmGluR2 potentiation, specifically Ser(688), Gly(689), and Asn(735), with the homologous hmGluR3 amino acids, inactivates the positive allosteric modulator activity of several structurally unique mGluR2 potentiators. Based on the characterization of the hmGluR2 potentiator binding site, we developed a novel scintillation proximity assay that was able to discriminate between compounds that were hmGluR2-specific potentiators, and those that were active on both hmGluR2 and hmGluR3. In addition, we substituted Ser(688), Gly(689), and Asn(735) into hmGluR3 and created an active hmGluR2 allosteric modulation site on the hmGluR3 receptor.

Distribution of metabotropic glutamate 2 and 3 receptors in the rat forebrain: Implication in emotional responses and central disinhibition.

The receptor localization of metabotropic glutamate receptors (mGlu) 2 and 3 was examined by using in situ hybridization and a well-characterized mGlu2-selective antibody in the rat forebrain. mGlu2 was highly and discretely expressed in cell bodies in almost all of the key regions of the limbic system in the forebrain, including the midline and intralaminar structures of the thalamus, the association cortices, the dentate gyrus of the hippocampus, the medial mammillary nucleus, and the lateral and basolateral nuclei of the amygdala. Moreover, presynaptic mGlu2 terminals were found in most of the forebrain structures, especially in the lateral part of the central nucleus of the amygdala, and the CA1 region of the hippocampus. Although some overlaps exist, such as in the hippocampus and the amygdala, the expression of mGlu3 mRNA, however, appeared to be more disperse, compared with that of mGlu2 mRNA. These distribution results support previous behavioral studies that the mGlu2 and 3 receptors may play important roles in emotional responses. In addition to its expression in glia, mGlu3 was distinctively expressed in cells in the GABAergic reticular nucleus of the thalamus. Local infusion of a non-selective mGlu2/3 agonist, LY379268, in the reticular nucleus of the thalamus, significantly reduced GABA release, suggesting that mGlu3 may also play a role in central disinhibition.

Correlation between ex vivo receptor occupancy and wake-promoting activity of selective H3 receptor antagonists.

The histamine H3 receptor (H3R) modulates the release of neurotransmitters that are involved in vigilance, cognition, and sleep-wake regulation. H3R antagonism has been proposed as a novel approach to the treatment of cognitive and attention deficit as well as sleep disorders. It is apparent that H3R antagonists produce pharmacological effects in preclinical animal models across a wide dose range. Several H3R antagonists were reported to be effective at producing cognitive enhancing effects at low doses, while producing robust wake enhancement at higher doses. To better understand the effect of H3R antagonists across a broad dose range, an ex vivo receptor binding assay has been used to estimate the degree of H3R occupancy in vivo. The H3R antagonists ciproxifan, thioperamide, GSK189254 (6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride), and ABT-239 ([4-(2-{2-[(2R)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile) produced wake-promoting activity in vivo and a dose-dependent inhibition of H3R binding ex vivo. For ciproxifan, thioperamide, and GSK189254, a relatively low level of cumulative wake activity was linearly correlated with up to 80% of the receptor occupancy. In contrast, an abrupt break from linearity and a robust increase of waking activity was observed at doses that produce greater than 80% occupancy. Our results suggest a relatively small increase of waking activity at low levels of receptor occupancy that may be consistent with reported enhancement of attention and cognitive function. Robust waking activity at higher levels of H3R occupancy may be mechanistically different from activities at low levels of H3R occupancy.

Allosteric approaches to the targeting of G-protein-coupled receptors for novel drug discovery: a critical assessment.

In recent years, the concept of allosteric modulation of G-protein-coupled receptors (GPCRs) has matured and now represents an increasingly viable approach to drug discovery. This is evident in the fact that allosteric modulators have been reported for every class of GPCR, and several are currently in clinical trials with one drug example approved and launched. The allosteric approach has been highlighted for the potential of identifying highly selective compounds with a minimal propensity to produce adverse effect. While much has been written regarding the promises of this approach, important challenges, caveats, and pitfalls exist that are often overlooked. Therefore, a balanced overview of the field that describes both the promises and the challenges of discovering allosteric modulators of GPCRs as novel drugs is presented.

Challenges in the development of mGluR5 positive allosteric modulators: the discovery of CPPHA.

This Letter describes, for the first time, the synthesis and SAR, developed through an iterative analog library approach, that led to the discovery of the positive allosteric modulator (PAM) of the metabotropic glutamate receptor mGluR5 CPPHA. Binding to a unique allosteric binding site distinct from other mGluR5 PAMs, CPPHA has been the focus of numerous pharmacology studies by several laboratories.

Biphenyl-indanones: allosteric potentiators of the metabotropic glutamate subtype 2 receptor.

We have identified and synthesized a series of biphenyl-carboxylic acid indanones as allosteric potentiators of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward improving the potency and the brain to plasma ratio of the initial lead led to the discovery of 5 and 23 (EC50=111 and 5 nM, respectively).

Benzazoles as allosteric potentiators of metabotropic glutamate receptor 2 (mGluR2): efficacy in an animal model for schizophrenia.

Metabotropic glutamate receptor 2 (mGluR2) has been implicated in a variety of CNS disorders, including schizophrenia. Disclosed herein is the development of a new series of allosteric potentiators of mGluR2. Structure-activity relationship studies in conjunction with pharmacokinetic data led to the discovery of indole 5, which is active in an animal model for schizophrenia.

Distribution and abundance of metabotropic glutamate receptor subtype 2 in rat brain revealed by [3H]LY354740 binding in vitro and quantitative radioautography: correlation with the sites of synthesis, expression, and agonist stimulation of [35S]GTPgammas binding.

Until recently, there was a lack of selective radioligands for the subtypes of metabotropic glutamate (mGlu) receptors. [(3)H]LY354740 ((+)-2-aminobicyclo[3,1,0]hexane-2,6-dicarboxylic acid), a selective agonist for group II receptors (mGlu2 and -3, which are negatively coupled to cAMP production), has now been used to map their brain distribution and abundance by in vitro binding and quantitative radioautography. The selective cation dependence of its binding allowed the discrimination between mGlu2 and mGlu3 receptor labeling. Thus, in the presence of Ca(2+) and Mg(2+) ions, the agonist bound selectively to mGlu2 receptors as evidenced by: 1) the correlative distribution and abundance of binding sites (highest in the lacunosum moleculare of the hippocampus and lowest in white matter) with mGlu2 receptor mRNA and protein revealed by in situ hybridization histochemistry and immunohistochemistry, respectively; 2) its selective pharmacology; and 3) the distribution of LY354740-stimulated [(35)S]GTPgammaS binding (25-97% above basal, according to the brain region), revealing G protein-coupled receptor coupling to G(i) proteins. Nonspecific binding (in the presence of 10 muM DCG-IV, a group II-selective, mGlu2-preferring, receptor agonist) was <10% of total. In adjacent sections, the distribution of binding sites for [(3)H]DCG-IV was very similar. This extensive study paves the way for investigations of the regional expression and regulation of mGlu2 receptors in human CNS diseases, such as Alzheimer's disease, which may reveal their functional roles and identify potential therapeutic drug targets. Indeed, it has recently been demonstrated (Higgins et al. [2004] Neuropharmacology 46:907-917) that pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent.

3-(2-Ethoxy-4-{4-[3-hydroxy-2-methyl-4-(3-methylbutanoyl)phenoxy]butoxy}phenyl)propanoic acid: a brain penetrant allosteric potentiator at the metabotropic glutamate receptor 2 (mGluR2).

We have identified and synthesized a brain penetrant propanoic acid as an allosteric potentiator of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward improving the potency, level of potentiation and brain penetration led to the discovery of 8 (EC50=1200 nM, 77% potentiation, 119% brain/plasma in rat, 20 mpk i.p., brain level of 5700 nM).

Allosteric potentiators of the metabotropic glutamate receptor 2 (mGlu2). Part 3: Identification and biological activity of indanone containing mGlu2 receptor potentiators.

We have identified and synthesized a series of phenyl-tetrazolyl and 4-thiopyridyl indanones as allosteric potentiators of the metabotropic glutamate receptor 2. Structure activity relationship studies directed toward improving the potency and level of potentiation, as well as PK properties, led to the discovery of 28 (EC50=186 nM), which displayed activity in a rodent model for schizophrenia.