The potassium (K+) ion channel KCNK13 is specifically expressed in human microglia with elevated expression observed in post-mortem human brain tissue from patients with Alzheimer's disease. Modulation of KCNK13 activity by a small-molecule inhibitor is proposed as a potential treatment for neurodegenerative diseases. Herein, we describe the evolution of a series of KCNK13 inhibitors derived from a high-throughput screening campaign, resulting in CVN293, a potent, selective, and brain permeable clinical candidate molecule. CVN293 demonstrated a concentration-dependent inhibition of the NLRP3-inflammasome mediated production of IL-1β from LPS-primed murine microglia. Cross-species pharmacokinetic data of CVN293 are also disclosed. These findings support the advancement of CVN293 in clinical trials.

CVN424 is a drug candidate, which is being investigated in clinical trials for the treatment of motor fluctuations associated with Parkinson's disease. We herein describe the process development of an efficient synthetic route that delivered several kilograms of the drug substance. The synthesis included diacylation of commercially available 3,4-diaminopyridine 1 with diethyl oxalate to give 2 and chlorination with POCl3 to give pyrido[3,4-b]pyrazine 3, followed by two sequential nucleophilic aromatic substitutions. A final hydrogenation and acetylation of intermediate 7 provided CVN424. Overall, a safe and robust synthesis was developed, which occurred in five linear steps with an overall yield of 15%.

From our NETSseq-derived human brain transcriptomics data, we identified GPR55 as a potential molecular target for the treatment of motor symptoms in patients with Parkinson's disease. From a high-throughput screen, we identified and optimized agonists with nanomolar potency against both human and rat GPR55. We discovered compounds with either strong or limited β-arrestin signaling and receptor desensitization, indicating biased signaling. A compound that showed minimal GPR55 desensitization demonstrated a reduction in firing frequency of medium spiny neurons cultured from rat striatum but did not reverse motor deficits in a rat hypolocomotion model. Further profiling of several desensitizing and non-desensitizing lead compounds showed that they are selective over related cannabinoid receptors CB1 and CB2 and that unbound brain concentrations well above the respective GPR55 EC50 can be readily achieved following oral administration. The novel brain-penetrant GPR55 agonists disclosed can be used to probe the role of this receptor in the brain.

Nirmatrelvir (1), a novel and specific inhibitor of the SARS-CoV-2 3C-like protease, was developed by Pfizer scientists in mid 2020. Efforts to develop a scalable process to manufacture nirmatrelvir were undertaken with a great sense of urgency, as there were no effective treatments available for the worldwide patient population at that time. We used a convergent approach to generate this molecule. The first two steps used to generate the western fragment of nirmatrelvir from l-tert-leucine, ethyl trifluoroacetate, and a [3.1.0] bicyclic proline derivative are described here. This is the first of a series of four papers describing the commercial process of the development of nirmatrelvir.

Brain concentrations of nicotinamide adenine dinucleotide (NAD(+)), an important cellular co-factor, tend to decrease with age and in neurodegeneration. As the NADase cluster of differentiation 38 (CD38) significantly contributes to NAD(+) consumption, we reasoned that CD38 inhibition may be of therapeutic value for CNS disorders. The new pyrazole compound was designed based on a known CD38 inhibitor and showed good inhibitory potency. Several attempts to co-crystallise this pyrazole with CD38 and cyclic adenosine diphosphate ribose (cADPR) culminated in a high-resolution X-ray structure, in which the pyrazolyl group in the new compound formed a covalent bond with one of the ribosyl units of cADPR. This reaction proceeded under retention of configuration and resulted in a neutral ribosyl-pyrazole conjugate that is embedded within the active site of the enzyme. An analysis of this structural complex gave rise to design principles that enabled the preparation of more potent CD38 inhibitors with drug-like properties.

Nicotinic acetylcholine receptor (nAChR) α6 subunit RNA expression is relatively restricted to midbrain regions and is located presynaptically on dopaminergic neurons projecting to the striatum. This subunit modulates dopamine neurotransmission and may have therapeutic potential in movement disorders. We aimed to develop potent and selective α6-containing nAChR antagonists to explore modulation of dopamine release and regulation of motor function in vivo. High-throughput screening (HTS) identified novel α6-containing nAChR antagonists and led to the development of CVN417. This molecule blocks α6-containing nAChR activity in recombinant cells and reduces firing frequency of noradrenergic neurons in the rodent locus coeruleus. CVN417 modulated phasic dopaminergic neurotransmission in an impulse-dependent manner. In a rodent model of resting tremor, CVN417 attenuated this behavioral phenotype. These data suggest that selective antagonism of α6-containing nAChR, with molecules such as CVN417, may have therapeutic utility in treating the movement dysfunctions observed in conditions such as Parkinson's disease.

The low affinity metabotropic glutamate receptor mGluR7 has been implicated in numerous CNS disorders; however, a paucity of potent and selective activators has hampered full delineation of the functional role and therapeutic potential of this receptor. In this work, we present the identification, optimization, and characterization of highly potent, novel mGluR7 agonists. Of particular interest is the chromane CVN636, a potent (EC50 7 nM) allosteric agonist which demonstrates exquisite selectivity for mGluR7 compared to not only other mGluRs, but also a broad range of targets. CVN636 demonstrated CNS penetrance and efficacy in an in vivo rodent model of alcohol use disorder. CVN636 thus has potential to progress as a drug candidate in CNS disorders involving mGluR7 and glutamatergic dysfunction.

Neuroinflammation, specifically the NLRP3 inflammasome cascade, is a common underlying pathological feature of many neurodegenerative diseases. Evidence suggests that NLRP3 activation involves changes in intracellular K+. Nuclear Enriched Transcript Sort Sequencing (NETSseq), which allows for deep sequencing of purified cell types from human post-mortem brain tissue, demonstrated a highly specific expression of the tandem pore domain halothane-inhibited K+ channel 1 (THIK-1) in microglia compared to other glial and neuronal cell types in the human brain. NETSseq also showed a significant increase of THIK-1 in microglia isolated from cortical regions of brains with Alzheimer's disease (AD) relative to control donors. Herein, we report the discovery and pharmacological characterisation of C101248, the first selective small-molecule inhibitor of THIK-1. C101248 showed a concentration-dependent inhibition of both mouse and human THIK-1 (IC50: ∼50 nM) and was inactive against K2P family members TREK-1 and TWIK-2, and Kv2.1. Whole-cell patch-clamp recordings of microglia from mouse hippocampal slices showed that C101248 potently blocked both tonic and ATP-evoked THIK-1 K+ currents. Notably, C101248 had no effect on other constitutively active resting conductance in slices from THIK-1-depleted mice. In isolated microglia, C101248 prevented NLRP3-dependent release of IL-1β, an effect not seen in THIK-1-depleted microglia. In conclusion, we demonstrated that inhibiting THIK-1 (a microglia specific gene that is upregulated in brains from donors with AD) using a novel selective modulator attenuates the NLRP3-dependent release of IL-1β from microglia, which suggests that this channel may be a potential therapeutic target for the modulation of neuroinflammation in AD.

Food intake and body weight are tightly regulated by neurons within specific brain regions, including the brainstem, where acute activation of dorsal raphe nucleus (DRN) glutamatergic neurons expressing the glutamate transporter Vglut3 (DRNVglut3) drive a robust suppression of food intake and enhance locomotion. Activating Vglut3 neurons in DRN suppresses food intake and increases locomotion, suggesting that modulating the activity of these neurons might alter body weight. Here, we show that DRNVglut3 neurons project to the lateral hypothalamus (LHA), a canonical feeding center that also reduces food intake. Moreover, chronic DRNVglut3 activation reduces weight in both leptin-deficient (ob/ob) and leptin-resistant diet-induced obese (DIO) male mice. Molecular profiling revealed that the orexin 1 receptor (Hcrtr1) is highly enriched in DRN Vglut3 neurons, with limited expression elsewhere in the brain. Finally, an orally bioavailable, highly selective Hcrtr1 antagonist (CVN45502) significantly reduces feeding and body weight in DIO. Hcrtr1 is also co-expressed with Vglut3 in the human DRN, suggesting that there might be a similar effect in human. These results identify a potential therapy for obesity by targeting DRNVglut3 neurons while also establishing a general strategy for developing drugs for central nervous system disorders. Schneeberger et al. show that glutamatergic neurons within the dorsal raphe nucleus of the brainstem are enriched with the orexin 1 receptor Hcrtr1, which can be pharmacologically targeted to treat obesity in mice.

A series of pyrrolidine amino nitrile DPP1 inhibitors have been developed and characterized. The S2 pocket structure-activity relationship for these compounds shows significant gains in potency for DPP1 from interacting further with target residues and a network of water molecules in the binding pocket. Herein we describe the X-ray crystal structures of several of these compounds alongside an analysis of factors influencing the inhibitory potency toward DPP1 of which stabilization of the water network, demonstrated using Grand Canonical Monte Carlo simulations and free energy calculations, is attributed as a main factor.

A novel series of second generation DPP1 inhibitors free from aorta binding liabilities found for earlier compound series was discovered. This work culminated in the identification of compound 30 (AZD7986) as a highly potent, reversible, and selective clinical candidate for COPD, with predicted human PK properties suitable for once daily human dosing.