Rapid sp3-Enriched Scaffold Generation via a Selective Aziridine Amide Ring-Opening Reaction.

Sp3-enriched small molecules play a critical role in developing drug candidates. While designing analogues with greater sp3 character, a methodology utilizing a less explored cyclic-aziridine amide ring-opening reaction to generate sp3-enriched scaffolds has been developed and reported. This methodology enables rapid access to substructures with higher fsp3 values, attracting greater attention within the past few decades. The reaction exhibits a wide reaction scope, featuring a highly sterically hindered phenolic ether, thiophenolic ethers, protected aniline formations, and aliphatic/heteroaromatic ring-containing aziridine amides as substrates. Additionally, this reaction provides access to congested tertiary ether formations through regioselective transformation, applicable to an extensive range of drug discovery targets, construction of complex small molecules, and natural product syntheses. The scaffolds developed show improved physicochemical properties.

Design, Optimization, and Study of Small Molecules That Target Tau Pre-mRNA and Affect Splicing.

Approximately 95% of human genes are alternatively spliced, and aberrant splicing events can cause disease. One pre-mRNA that is alternatively spliced and linked to neurodegenerative diseases is tau (microtubule-associated protein tau), which can cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and can contribute to Alzheimer's disease. Here, we describe the design of structure-specific lead small molecules that directly target tau pre-mRNA from sequence. This was followed by hit expansion and analogue synthesis to further improve upon these initial lead molecules. The emergent compounds were assessed for functional activity in a battery of assays, including binding assays and an assay that mimics molecular recognition of tau pre-mRNA by a U1 small nuclear ribonucleoprotein (snRNP) splicing factor. Compounds that emerged from these studies had enhanced potency and selectivity for the target RNA relative to the initial hits, while also having significantly improved drug-like properties. The compounds are shown to directly target tau pre-mRNA in cells, via chemical cross-linking and isolation by pull-down target profiling, and to rescue disease-relevant splicing of tau pre-mRNA in a variety of cellular systems, including primary neurons. More broadly, this study shows that lead, structure-specific compounds can be designed from sequence and then further optimized for their physicochemical properties while at the same time enhancing their activity.

Discovery and in vitro and in vivo profiles of N-ethyl-N-[2-[3-(5-fluoro-2-pyridinyl)-1H-pyrazol-1-yl]ethyl]-2-(2H-1,2,3-triazol-2-yl)-benzamide as a novel class of dual orexin receptor antagonist.

Orexins play an important role in sleep/wake regulation, and orexin receptor antagonists are a focus of novel therapy for the treatment of insomnia. We identified 27e (TASP0428980) as a potent dual orexin receptor antagonist through the systematic modification of our original designed lead A. We demonstrated the potent sleep-promoting effects of 27e at ip dose of 3mg/kg in a rat polysomnogram study. 27e exhibited relatively short half-life profiles in rats and dogs. Furthermore, accumulating evidence regarding ADME profiles indicates that the predicted human half-life of 27e should be 1.2-1.4h. These data indicated that 27e has a short-acting hypnotic property, suggesting that 27e might be useful for treating primary insomnia while exhibiting a low risk of next-day residual somnolence. Thus, 27e and its related compounds should be further evaluated to enable advancement to clinical trials.

[Electrophoretic Analysis of Abnormal Data on Serum Protein Electrophoresis].

We recently demonstrated glycation of monoclonal IgA and the presence of IgA-albumin complexes, but the significance of the complexes was not clear. We describe a non-diabetic patient with IgA type M-protein whose serum fructosamine and glycoalbumin levels were elevated. On electrophoresis of the serum protein of the patient, the albumin band shifted to the cathode side. The abnormal precipitin arc of IgA-albumin complexes was detected by immunoelectrophoresis. To elucidate the mechanism of IgA-albumin complexes, we analyzed their properties using immunoelectrophoresis, Western blotting, and two-dimensional gel electrophoresis. The macromolecularized albumin spots were demonstrated by two-dimensional Western blotting with antiserum to human albumin of the patient's serum. Moreover, the IgA-albumin complexes were dissociated on treatment with 2-mercaptoethanol. It can be considered that albumin is bound to the monoclonal IgA molecule by covalent disulfide bonds, and that the albumin binding site is located near the hinge region (311Cys) of the IgA molecule and involves the free SH group, thought to be present in the α-chain.

A Druglike Small Molecule that Targets r(CCUG) Repeats in Myotonic Dystrophy Type 2 Facilitates Degradation by RNA Quality Control Pathways.

Myotonic dystrophy type 2 (DM2) is one of >40 microsatellite disorders caused by RNA repeat expansions. The DM2 repeat expansion, r(CCUG)exp (where "exp" denotes expanded repeating nucleotides), is harbored in intron 1 of the CCHC-type zinc finger nucleic acid binding protein (CNBP). The expanded RNA repeat causes disease by a gain-of-function mechanism, sequestering various RNA-binding proteins including the pre-mRNA splicing regulator MBNL1. Sequestration of MBNL1 results in its loss-of-function and concomitant deregulation of the alternative splicing of its native substrates. Notably, this r(CCUG)exp causes retention of intron 1 in the mature CNBP mRNA. Herein, we report druglike small molecules that bind the structure adopted by r(CCUG)exp and improve DM2-associated defects. These small molecules were optimized from screening hits from an RNA-focused small-molecule library to afford a compound that binds r(CCUG)exp specifically and with nanomolar affinity, facilitates endogenous degradation of the aberrantly retained intron in which it is harbored, and rescues alternative splicing defects.

Optimization of the Linker Domain in a Dimeric Compound that Degrades an r(CUG) Repeat Expansion in Cells.

RNA repeat expansions are responsible for more than 30 incurable diseases. Among them is myotonic dystrophy type 1 (DM1), the most common form of adult on-set muscular dystrophy. DM1 is caused by an r(CUG) repeat expansion [r(CUG)exp] located in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase gene. This repeat expansion is highly structured, forming a periodic array of 5'CUG/3'GUC internal loop motifs. We therefore designed dimeric compounds that simultaneously bind two of these motifs by connecting two RNA-binding modules with peptoid linkers of different geometries and lengths. The optimal linker contains two proline residues and enhances compound affinity. Equipping this molecule with a bleomycin A5 cleaving module converts the simple binding compound into a potent allele-selective cleaver of r(CUG)exp. This study shows that the linker in modularly assembled ligands targeting RNA can be optimized to afford potent biological activity.

Discovery of VU6005649, a CNS Penetrant mGlu7/8 Receptor PAM Derived from a Series of Pyrazolo[1,5-a]pyrimidines.

Herein, we report the structure-activity relationships within a series of mGlu7 PAMs based on a pyrazolo[1,5-a]pyrimidine core with excellent CNS penetration (Kps > 1 and Kp,uus > 1). Analogues in this series proved to display a range of Group III mGlu receptor selectivity, but VU6005649 emerged as the first dual mGlu7/8 PAM, filling a void in the Group III mGlu receptor PAM toolbox and demonstrating in vivo efficacy in a mouse contextual fear conditioning model.

Diverse Effects on M1 Signaling and Adverse Effect Liability within a Series of M1 Ago-PAMs.

Both historical clinical and recent preclinical data suggest that the M1 muscarinic acetylcholine receptor is an exciting target for the treatment of Alzheimer's disease and the cognitive and negative symptom clusters in schizophrenia; however, early drug discovery efforts targeting the orthosteric binding site have failed to afford selective M1 activation. Efforts then shifted to focus on selective activation of M1 via either allosteric agonists or positive allosteric modulators (PAMs). While M1 PAMs have robust efficacy in rodent models, some chemotypes can induce cholinergic adverse effects (AEs) that could limit their clinical utility. Here, we report studies aimed at understanding the subtle structural and pharmacological nuances that differentiate efficacy from adverse effect liability within an indole-based series of M1 ago-PAMs. Our data demonstrate that closely related M1 PAMs can display striking differences in their in vivo activities, especially their propensities to induce adverse effects. We report the discovery of a novel PAM in this series that is devoid of observable adverse effect liability. Interestingly, the molecular pharmacology profile of this novel PAM is similar to that of a representative M1 PAM that induces severe AEs. For instance, both compounds are potent ago-PAMs that demonstrate significant interaction with the orthosteric site (either bitopic or negative cooperativity). However, there are subtle differences in efficacies of the compounds at potentiating M1 responses, agonist potencies, and abilities to induce receptor internalization. While these differences may contribute to the differential in vivo profiles of these compounds, the in vitro differences are relatively subtle and highlight the complexities of allosteric modulators and the need to focus on in vivo phenotypic screening to identify safe and effective M1 PAMs.

Prefrontal Cortex-Mediated Impairments in a Genetic Model of NMDA Receptor Hypofunction Are Reversed by the Novel M1 PAM VU6004256.

Abnormalities in the signaling of the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) within cortical and limbic brain regions are thought to underlie many of the complex cognitive and affective symptoms observed in individuals with schizophrenia. The M1 muscarinic acetylcholine receptor (mAChR) subtype is a closely coupled signaling partner of the NMDAR. Accumulating evidence suggests that development of selective positive allosteric modulators (PAMs) of the M1 receptor represent an important treatment strategy for the potential normalization of disruptions in NMDAR signaling in patients with schizophrenia. In the present studies, we evaluated the effects of the novel and highly potent M1 PAM, VU6004256, in ameliorating selective prefrontal cortical (PFC)-mediated physiologic and cognitive abnormalities in a genetic mouse model of global reduction in the NR1 subunit of the NMDAR (NR1 knockdown [KD]). Using slice-based extracellular field potential recordings, deficits in muscarinic agonist-induced long-term depression (LTD) in layer V of the PFC in the NR1 KD mice were normalized with bath application of VU6004256. Systemic administration of VU6004256 also reduced excessive pyramidal neuron firing in layer V PFC neurons in awake, freely moving NR1 KD mice. Moreover, selective potentiation of M1 by VU6004256 reversed the performance impairments of NR1 KD mice observed in two preclinical models of PFC-mediated learning, specifically the novel object recognition and cue-mediated fear conditioning tasks. VU6004256 also produced a robust, dose-dependent reduction in the hyperlocomotor activity of NR1 KD mice. Taken together, the current findings provide further support for M1 PAMs as a novel therapeutic approach for the PFC-mediated impairments in schizophrenia.

Hypocretin-1 (orexin-A) concentrations in cerebrospinal fluid are low in patients with Guillain-Barré syndrome.

It is reported that cerebrospinal fluid (CSF) hypocretin-1 (orexin-A) concentrations in patients with narcolepsy are significantly low. Human narcolepsy is also known to be closely associated with a specific human histocompatibility leukocyte antigen (HLA), suggesting that autoimmunity is involved in the pathophysiology of the disease. Thus, it is important to know whether hypocretin changes are found in definite neuroimmunological diseases such as multiple sclerosis and Guillain-Barré syndrome (GBS). The results of the present study indicate that some patients with GBS have lower levels of CSF hypocretin-1.

Effects of Yoku-kan-san-ka-chimpi-hange on the sleep of normal healthy adult subjects.

Yoku-kan-san-ka-chimpi-hange (YKCH) is a drug used for insomnia in Japanese traditional herbal medicine. The present study evaluated the effects of YKCH on sleep by all-night polysomnography using the double-blind method. Yoku-kan-san-ka-chimpi-hange increased the total sleep time significantly, and tended to cause an increase in sleep efficiency and of stage 2 sleep, as well as a decrease of sleep latency and of stage 3 + 4 sleep. There was no apparent influence on rapid eye movement (REM) sleep. In terms of non-REM sleep, the effects of YKCH exhibit a profile similar to those of benzodiazepines.

Effects of donepezil (Aricept) on the rapid eye movement sleep of normal subjects.

Donepezil (Aricept) is a therapeutic drug for the treatment of Alzheimer's disease (AD). However, there have been only two reports describing the effects of donepezil on sleep as assessed by nocturnal polysomnography (PSG). With this in mind, the effects of donepezil on the sleep of healthy subjects was evaluated using PSG. The results indicated that the percentage of rapid eye movement sleep to total sleep time was increased significantly by a single dose of 5 mg of donepezil when given to healthy subjects immediately before retiring to bed.