Mansuo L. Hayashi

Identification of direct target engagement biomarkers for kinase-targeted therapeutics.

Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is often unknown. By definition, proximal PD biomarkers aim to measure the interaction of a drug with its biological target. For kinase drug discovery, protein substrate phosphorylation sites represent candidate PD biomarkers. However, substrate phosphorylation is often controlled by input from multiple converging pathways complicating assessment of how potently a small molecule drug hits its target based on substrate phoshorylation measurements alone. Here, we report the use of quantitative, differential mass-spectrometry to identify and monitor novel drug-regulated phosphorylation sites on target kinases. Autophosphorylation sites constitute clinically validated biomarkers for select protein tyrosine kinase inhibitors. The present study extends this principle to phosphorylation sites in serine/threonine kinases looking beyond the T-loop autophosphorylation site. Specifically, for the 3′-phosphoinositide-dependent protein kinase 1 (PDK1), two phospho-residues p-PDK1Ser410 and p-PDK1Thr513 are modulated by small-molecule PDK1 inhibitors, and their degree of dephosphorylation correlates with inhibitor potency. We note that classical, ATP-competitive PDK1 inhibitors do not modulate PDK1 T-loop phosphorylation (p-PDK1Ser241), highlighting the value of an unbiased approach to identify drug target-regulated phosphorylation sites as these are complementary to pathway PD biomarkers. Finally, we extend our analysis to another protein Ser/Thr kinase, highlighting a broader utility of our approach for identification of kinase drug-target engagement biomarkers.

Structure guided design of a series of selective pyrrolopyrimidinone MARK inhibitors

The initial structure activity relationships around an isoindoline uHTS hit will be described. Information gleaned from ligand co-crystal structures allowed for rapid refinements in both MARK potency and kinase selectivity. These efforts allowed for the identification of a compound with properties suitable for use as an in vitro tool compound for validation studies on MARK as a viable target for Alzheimers disease.

MARK inhibitors: Declaring a No-Go decision on a chemical series based on extensive DMPK experimentation

Attempts to optimize pharmacokinetic properties in a promising series of pyrrolopyrimidinone MARK inhibitors for the treatment of Alzheimers disease are described. A focus on physical properties and ligand efficiency while prosecuting this series afforded key tool compounds that revealed a large discrepancy in the rat in vitro-in vivo DMPK (Drug Metabolism/Pharmacokinetics) correlation. These differences prompted an in vivo rat disposition study employing a radiolabeled representative of the series, and the results from this experiment justified the termination of any further optimization efforts.

Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties.

Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimers disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.

MEASUREMENT OF ENDOGENOUS MOUSE TAU IN CEREBROSPINAL FLUID FROM AGED PDAPP MICE FOLLOWING TREATMENT WITH AB-LOWERING COMPOUNDS

Background:Dementia is usually attributable to one or a combination of neuropathologic abnormalities, including Alzheimer amyloid plaques and neurofibrillary tangles. Most demented individuals have at least two types of dementia-related abnormality. Older decedents without abnormalities may be viewed as showing resistance to the relevant pathogenic processes. Others with brain or cognitive reserves may maintain normal cognitive performance even when 1 or 2 types of histopathologic abnormality have developed. We hypothesize essential resilience as a third defense against established neuropathology. Methods: Analyses involved 572 autopsied HAAS participants (Japanese-American men) dying at age 80 or older whose baseline Cognitive Assessment and Screening Instrument (CASI) score had been normal (>1⁄474). End-of-life severe impairment was based on a final CASI score <60. Four overlapping reserve elements were each dichotomized as 0 or 1: (i) negligible brain atrophy, (ii) higher education, (iii) higher occupational complexity, (iv) higher adult life cognitive test scores. A neuropathologic burden index (Neurology, March 2016) was the sum of four histopathologic abnormalities, each scored 0.4 (moderate) or 1.0 (severe) for Alzheimer lesions, microinfarcts, Lewy bodies, and hippocampal sclerosis. Results:Among 159 participants with a neuropathologic burden index of zero, 145 (91%) maintained normal cognition. Among 387 with burden indices 0.4 1.8 (most with two moderate or severe lesion types), the proportions developing severe impairment fell from 49%, to 35%, to 29%, to 25%, to 13% respectively in decedents with zero, 1, 2, 3, or all 4 reserve elements. Of 26 decedents with neuropathologic burden indices >1⁄42, 22 (85%) developed severe cognitive impairment. We continue to search for factors determining essential resilience. Conclusions: In this autopsied cohort, brain and/or cognitive reserves provided a powerful attenuation of cognitive impairment attributable to a panel of neuropathological abnormalities. A hypothesized additional contribution by essential resilience remains to be demonstrated.

CHARACTERIZATION OF BIOFLUID STREM2 USING A NOVEL STREM2 ASSAY

Background:Apolipoprotein E (apoE) is a key lipid transport protein in brain and plasma. Humans have three apoE isoforms; namely, apoE2, apoE3 and apoE4. Of the three isoforms, apoE4 is strongly associated with late-onset and sporadic forms of Alzheimer’s disease (AD). In addition to its role in neuronal repair, synaptogenesis, and clearance of toxic Aß fragments from brain, apoE has been suggested to play a critical role in inflammation in the brain. Using pan LXR agonists as a tool compound for increasing brain and astrocytic apoE levels, we sought to investigate the effect of increasing astrocytic apoE on brain inflammation induced by lipopolysaccharide (LPS) both in vitro and in vivo. Methods:Using primary mouse microglia and astrocytes and C57/bl6 wild type mice as our model system, we evaluated the effect of increasing apoE protein levels using T0901317 (pan LXR agonist) on LPS induced inflammation. We used RT-qPCR to evaluate the mRNA levels of apoE and apoE lipidating genes and MSD assays to evaluate cytokine protein levels in cell media and brain homogenates. Results: TO901317 treatment led to an increase in brain apoE levels in vivo in wild type mice and in vitro, in both primary murine astrocytes and microglia. This increase in apoE was accompanied by an increase in apoE lipidating genes (Abca1 and Abcg1). Treatment with LPS led to a significant inflammatory response as seen by an increase in pro-inflammatory cytokines, TNFa and IL6. However, TO901317 treatment led to a significant reduction in the levels of the pro-inflammatory cytokines both in vitro (astrocytes and microglia) and in vivo in the brain. Conclusions:Our results indicate that increasing brain and gial apoE using a pan LXR agonist attenuates LPS induced inflammation. Our data suggests that apoE could play an important role in promoting antiinflammatory effects in the brain. Given the strong association of apoE with AD, increasing lipidated apoE in the brain could have multi-prong benefits including anti-inflammatory effects.