Inhibition of class IIa histone deacetylase (HDAC) enzymes have been suggested as a therapeutic strategy for a number of diseases, including Huntington’s disease. Catalytic-site small molecule ...inhibitors of the class IIa HDAC4, -5, -7, and -9 were developed. These trisubstituted diarylcyclopropanehydroxamic acids were designed to exploit a lower pocket that is characteristic for the class IIa HDACs, not present in other HDAC classes. Selected inhibitors were cocrystallized with the catalytic domain of human HDAC4. We describe the first HDAC4 catalytic domain crystal structure in a “closed-loop” form, which in our view represents the biologically relevant conformation. We have demonstrated that these molecules can differentiate class IIa HDACs from class I and class IIb subtypes. They exhibited pharmacokinetic properties that should enable the assessment of their therapeutic benefit in both peripheral and CNS disorders. These selective inhibitors provide a means for evaluating potential efficacy in preclinical models in vivo.
Autophagy is an essential cellular process that removes harmful protein species, and autophagy upregulation may be able to protect against neurodegeneration and various pathogens. Here, we have ...identified the essential protein VCP/p97 (VCP, valosin-containing protein) as a novel regulator of autophagosome biogenesis, where VCP regulates autophagy induction in two ways, both dependent on Beclin-1. Utilizing small-molecule inhibitors of VCP ATPase activity, we show that VCP stabilizes Beclin-1 levels by promoting the deubiquitinase activity of ataxin-3 towards Beclin-1. VCP also regulates the assembly and activity of the Beclin-1-containing phosphatidylinositol-3-kinase (PI3K) complex I, thus regulating the production of PI(3)P, a key signaling lipid responsible for the recruitment of downstream autophagy factors. A decreased level of VCP, or inhibition of its ATPase activity, impairs starvation-induced production of PI(3)P and limits downstream recruitment of WIPI2, ATG16L and LC3, thereby decreasing autophagosome formation, illustrating an important role for VCP in early autophagy initiation.
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.
Enhancing the removal of aggregate-prone toxic proteins is a rational therapeutic strategy for a number of neurodegenerative diseases, especially Huntington's disease and various spinocerebellar ...ataxias. Ideally, such approaches should preferentially clear the mutant/misfolded species, while having minimal impact on the stability of wild-type/normally-folded proteins. Furthermore, activation of both ubiquitin-proteasome and autophagy-lysosome routes may be advantageous, as this would allow effective clearance of both monomeric and oligomeric species, the latter which are inaccessible to the proteasome. Here we find that compounds that activate the D1 ATPase activity of VCP/p97 fulfill these requirements. Such effects are seen with small molecule VCP activators like SMER28, which activate autophagosome biogenesis by enhancing interactions of PI3K complex components to increase PI(3)P production, and also accelerate VCP-dependent proteasomal clearance of such substrates. Thus, this mode of VCP activation may be a very attractive target for many neurodegenerative diseases.
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.
Histone deacetylase (HDAC) inhibitors have received considerable attention as potential therapeutics for a variety of cancers and neurological disorders. Recent publications on a class of pimelic ...diphenylamide HDAC inhibitors have highlighted their promise in the treatment of the neurodegenerative diseases Friedreich's ataxia and Huntington's disease, based on efficacy in cell and mouse models. These studies' authors have proposed that the unique action of these compounds compared to hydroxamic acid-based HDAC inhibitors results from their unusual slow-on/slow-off kinetics of binding, preferentially to HDAC3, resulting in a distinctive pharmacological profile and reduced toxicity. Here, we evaluate the HDAC subtype selectivity, cellular activity, absorption, distribution, metabolism and excretion (ADME) properties, as well as the central pharmacodynamic profile of one such compound, HDACi 4b, previously described to show efficacy in vivo in the R6/2 mouse model of Huntington's disease. Based on our data reported here, we conclude that while the in vitro selectivity and binding mode are largely in agreement with previous reports, the physicochemical properties, metabolic and p-glycoprotein (Pgp) substrate liability of HDACi 4b render this compound suboptimal to investigate central Class I HDAC inhibition in vivo in mouse per oral administration. A drug administration regimen using HDACi 4b dissolved in drinking water was used in the previous proof of concept study, casting doubt on the validation of CNS HDAC3 inhibition as a target for the treatment of Huntington's disease. We highlight physicochemical stability and metabolic issues with 4b that are likely intrinsic liabilities of the benzamide chemotype in general.
BACE1 is responsible for the first step in APP proteolysis, leading to toxic Aβ production, and has been indicated to play a key role in the pathogenesis of Alzheimer’s disease. The related isoform ...BACE2 is thought to be involved in processing of the pigment cell-specific melanocyte protein. To avoid potential effects on pigmentation, we investigated the feasibility for developing isoform-selective BACE1 inhibitors. Cocrystal structures of 47 compounds were analyzed and clustered according to their selectivity profiles. Selective BACE1 inhibitors were found to exhibit two distinct conformational features proximal to the flap and the S3 subpocket. Several new molecules were designed and tested to make use of this observation. The combination of a pyrimidinyl C-ring and a methylcyclohexyl element resulted in lead molecule 28, which exhibited ∼50-fold selectivity. Compared to a nonselective BACE1/2 inhibitor, 28 showed significantly less inhibition of PMEL processing in human melanocytes, indicating good functional selectivity of this inhibitor class.
Neuroinflammation is highly influenced by microglia, particularly through activation of the NLRP3 inflammasome and subsequent release of IL-1β. Extracellular ATP is a strong activator of NLRP3 by ...inducing K
efflux as a key signaling event, suggesting that K
-permeable ion channels could have high therapeutic potential. In microglia, these include ATP-gated THIK-1 K
channels and P2X7 receptors, but their interactions and potential therapeutic role in the human brain are unknown. Using a novel specific inhibitor of THIK-1 in combination with patch-clamp electrophysiology in slices of human neocortex, we found that THIK-1 generated the main tonic K
conductance in microglia that sets the resting membrane potential. Extracellular ATP stimulated K
efflux in a concentration-dependent manner only via P2X7 and metabotropic potentiation of THIK-1. We further demonstrated that activation of P2X7 was mandatory for ATP-evoked IL-1β release, which was strongly suppressed by blocking THIK-1. Surprisingly, THIK-1 contributed only marginally to the total K
conductance in the presence of ATP, which was dominated by P2X7. This suggests a previously unknown, K
-independent mechanism of THIK-1 for NLRP3 activation. Nuclear sequencing revealed almost selective expression of THIK-1 in human brain microglia, while P2X7 had a much broader expression. Thus, inhibition of THIK-1 could be an effective and, in contrast to P2X7, microglia-specific therapeutic strategy to contain neuroinflammation.
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 pyrido3,4-bpyrazine 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%.
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.
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