Saktimayee M. Roy

A novel p38α MAPK inhibitor suppresses brain proinflammatory cytokine up-regulation and attenuates synaptic dysfunction and behavioral deficits in an Alzheimer's disease mouse model

BackgroundAn accumulating body of evidence is consistent with the hypothesis that excessive or prolonged increases in proinflammatory cytokine production by activated glia is a contributor to the progression of pathophysiology that is causally linked to synaptic dysfunction and hippocampal behavior deficits in neurodegenerative diseases such as Alzheimers disease (AD). This raises the opportunity for the development of new classes of potentially disease-modifying therapeutics. A logical candidate CNS target is p38α MAPK, a well-established drug discovery molecular target for altering proinflammatory cytokine cascades in peripheral tissue disorders. Activated p38 MAPK is seen in human AD brain tissue and in AD-relevant animal models, and cell culture studies strongly implicate p38 MAPK in the increased production of proinflammatory cytokines by glia activated with human amyloid-beta (Aβ) and other disease-relevant stressors. However, the vast majority of small molecule drugs do not have sufficient penetrance of the blood-brain barrier to allow their use as in vivo research tools or as therapeutics for neurodegenerative disorders. The goal of this study was to test the hypothesis that brain p38α MAPK is a potential in vivo target for orally bioavailable, small molecules capable of suppressing excessive cytokine production by activated glia back towards homeostasis, allowing an improvement in neurologic outcomes.MethodsA novel synthetic small molecule based on a molecular scaffold used previously was designed, synthesized, and subjected to analyses to demonstrate its potential in vivo bioavailability, metabolic stability, safety and brain uptake. Testing for in vivo efficacy used an AD-relevant mouse model.ResultsA novel, CNS-penetrant, non-toxic, orally bioavailable, small molecule inhibitor of p38α MAPK (MW01-2-069A-SRM) was developed. Oral administration of the compound at a low dose (2.5 mg/kg) resulted in attenuation of excessive proinflammatory cytokine production in the hippocampus back towards normal in the animal model. Animals with attenuated cytokine production had reductions in synaptic dysfunction and hippocampus-dependent behavioral deficits.ConclusionThe p38α MAPK pathway is quantitatively important in the Aβ-induced production of proinflammatory cytokines in hippocampus, and brain p38α MAPK is a viable molecular target for future development of potential disease-modifying therapeutics in AD and related neurodegenerative disorders.

Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction.

Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction.

Targeting Human Central Nervous System Protein Kinases: An Isoform Selective p38αMAPK Inhibitor That Attenuates Disease Progression in Alzheimer’s Disease Mouse Models

The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150’s exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior.

Molecular Properties and CYP2D6 Substrates: Central Nervous System Therapeutics Case Study and Pattern Analysis of a Substrate Database

CYP2D6 substrate status is a critical Go/No Go decision criteria in central nervous system (CNS) drug discovery efforts because the polymorphic nature of CYP2D6 can lead to variable patient safety and drug efficacy. In addition, CYP2D6 is disproportionately involved in the metabolism of CNS drugs compared with other drug classes. Therefore, identifying trends in small molecule properties of CNS-penetrant compounds that can help discriminate potential CYP2D6 substrates from nonsubstrates would allow additional prioritization in the synthesis and biological evaluation of new therapeutic candidates. We report here the conversion of the CNS drug minaprine from substrate to nonsubstrate, as well as the conversion of the related CNS drug minozac from nonsubstrate to substrate, through the use of analog synthesis and CYP2D6 enzyme kinetic analyses. No single molecular property strongly correlated with substrate status for this 3-amino-4-methyl-6-phenylpyridazine scaffold, although molecular volume and charge appeared to be indirectly related. A parsed database of CYP2D6 substrates across diverse chemical structures was assembled and analyzed for physical property trends correlating with substrate status. We found that a complex interplay of properties influenced CYP2D6 substrate status and that the particular chemical scaffold affects which properties are most prominent. The results also identified an unexpected issue in CNS drug discovery, in that some property trends correlative with CYP2D6 substrates overlap previously reported properties that correlate with CNS penetrance. These results suggest the need for a careful balance in the design and synthesis of new CNS therapeutic candidates to avoid CYP2D6 substrate status while maintaining CNS penetrance.

A PLATFORM FOR NOVEL IN VIVO MOLECULAR PROBES FOR ATTENUATION OF SYNAPTIC DYSFUNCTION AND PROINFLAMMATORY CYTOKINE OVERPRODUCTION

pathology with the severity of cognitive impairment, as well as the key role of CAA in the pathophysiology leading to development of amyloid-related imaging abnormalities, our studies were designed to determine whether stimulation of innate immunity with TLR9 agonist CpG oligodeoxynucleotides (ODNs) could have beneficial effects in a non-human primate model of sporadic CAA, squirrel monkey (Saimiri Boliviensis). Our prior work has shown that treatment with CpG ODN is effective at improving cognitive deficits in aged monkeys without inducing adverse events. Methods: Elderly female monkeys with expected CAA deposits were subcutaneously injected with either predetermined non-toxic dosages of the class B CpG ODN or saline for a 25 month period. Animals were continuously examined for signs of toxicity. Plasma taken at specific times in the course of treatment was analyzed to identify immune responses and AD biomarkers. Histological and biochemical brain analyses commenced upon completion of the behavioral protocols. Results: Long term treatment with CpG ODN significantly reduced total amyloid burden (6E10/4G8 immunohistochemistry), which in this model corresponds primarily to CAA. Histological evaluation of CpG ODN effect on pyroglutamate levels (AbpE3 immunohistochemistry) revealed region specific reduction in AbpE3 burden in monkeys treated with CpG ODN. These favorable histological data will be corroborated by measurements of Ab levels in the brain homogenates. Our initial biomarker analyses exhibited a noticeable increase in AbpE3 plasma levels in CpG ODNtreated animals. Subsequent evaluation showed no increase in the extent of Iba1 microgliosis and GFAP astrogliosis in CpG ODN group at the end of the treatment. Additionally, there was no evidence of increased microhemorrhages or T cell infiltration in association with CpG ODN treatment, further supporting the viability of our approach. Concluding assessment of microglia/macrophage activation is currently in progress. Conclusions:Overall, the present findings together with our earlier experimental evidence validate this novel concept of immunomodulation as a safe method to successfully ameliorate AD related pathology, suggesting CpG ODN would have a significant potential of achieving clinical efficacy.

An optimized and isoform-selective p38aMAPK inhibitor that attenuates disease progression in Alzheimer's disease mouse models

Background: Stress-induced activation of p38aMAPK in both neurons and glia raises a novel therapeutic hypothesis whereby enhanced efficacy might be generated via targeting a common target in two distinct cell types involved in a pathophysiological axis. However, pursuit of this neurotherapeutic hypothesis requires kinase isofor-selective inhibitors with appropriate neuropharmacology features. Methods: A scaffold repurposing approach employed high-resolution crystallography of complexes containing human p38aMAPK, driven by pharmacoinformatics-based design. Novel inhibitors were synthesized and filtered based on activity, pharmacological screening, and efficacy in Alzheimer’s disease (AD)-relevant mouse models in order to select best-in-class candidates. Results: We report an optimized, isoform-selective p38aMAPK inhibitor, MW01-18-150SRM (1⁄4 MW150) that has attractive pharmacological and functional features that render it a promising candidate for preclinical drug development. Highresolution crystallography documents MW150 active site binding and suggests a structural explanation for MW150’s exquisite kinome selectivity. Screens for target engagement, selectivity and property compliance with ICH guidelines indicate the high development potential of MW150. MW150 is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two independent AD synaptic dysfunction mouse models when administered either before full-fledged pathology is evident or after pathology is present. Conclusions: MW150 is a highly selective p38aMAPK inhibitor that attenuates synaptic and cognitive dysfunction in AD-relevant mouse models, and shows potential for use in either a prevention mode or disease treatment mode. The successful medicinal chemistry optimization campaign and pharmacological de-risking of MW150 render it appropriate for investigational new drug (IND) enabling development.