Katryna Cisek

Research Towards Tau Imaging

Tau-bearing neurofibrillary lesions present a promising biomarker for premortem diagnosis and staging of Alzheimers disease and certain forms of frontotemporal lobar degeneration by whole brain imaging methods. Although brain penetrating compounds capable of binding tau aggregates with high affinity have been disclosed for this purpose, the major barrier to progress remains the need for tau lesion binding selectivity relative to amyloid-beta plaques and other deposits of proteins in cross-beta-sheet conformation. Here we discuss challenges faced in the development of tau lesion-selective imaging agents, and recent preclinical advances in pursuit of this goal.

Structural Determinants of Tau Aggregation Inhibitor Potency

Background: Mechanistic insight into small-molecule Tau aggregation inhibitors is needed for their advancement as therapeutic agents. Results: Structure-activity relationship analysis identified polarizability as a common descriptor of inhibitor potency. Conclusion: Flat, highly polarizable ligands stabilize soluble oligomeric complexes at the expense of filamentous aggregates. Significance: The findings suggest a basis for rational improvement of ligand potency, whereas maintaining bioavailability. Small-molecule Tau aggregation inhibitors are under investigation as potential therapeutic agents against Alzheimer disease. Many such inhibitors have been identified in vitro, but their potency-driving features, and their molecular targets in the Tau aggregation pathway, have resisted identification. Previously we proposed ligand polarizability, a measure of electron delocalization, as a candidate descriptor of inhibitor potency. Here we tested this hypothesis by correlating the ground state polarizabilities of cyanine, phenothiazine, and arylmethine derivatives calculated using ab initio quantum methods with inhibitory potency values determined in the presence of octadecyl sulfate inducer under reducing conditions. A series of rhodanine analogs was analyzed as well using potency values disclosed in the literature. Results showed that polarizability and inhibitory potency directly correlated within all four series. To identify putative binding targets, representative members of the four chemotypes were added to aggregation reactions, where they were found to stabilize soluble, but SDS-resistant Tau species at the expense of filamentous aggregates. Using SDS resistance as a secondary assay, and a library of Tau deletion and missense mutants as targets, interaction with cyanine was localized to the microtubule binding repeat region. Moreover, the SDS-resistant phenotype was completely dependent on the presence of octadecyl sulfate inducer, but not intact PHF6/PH6* hexapeptide motifs, indicating that cyanine interacted with a species in the aggregation pathway prior to nucleus formation. Together the data suggest that flat, highly polarizable ligands inhibit Tau aggregation by interacting with folded species in the aggregation pathway and driving their assembly into soluble but highly stable Tau oligomers.

Structure and Mechanism of Action of Tau Aggregation Inhibitors

Since the discovery of phenothiazines as tau protein aggregation inhibitors, many additional small molecule inhibitors of diverse chemotype have been discovered and characterized in biological model systems. Although direct inhibition of tau aggregation has shown promise as a potential treatment strategy for depressing neurofibrillary lesion formation in Alzheimers disease, the mechanism of action of these compounds has been unclear. However, recent studies have found that tau aggregation antagonists exert their effects through both covalent and non-covalent means, and have identified associated potency and selectivity driving features. Here we review small-molecule tau aggregation inhibitors with a focus on compound structure and inhibitory mechanism. The elucidation of inhibitory mechanism has implications for maximizing on-target efficacy while minimizing off-target side effects.

Ligand polarizability contributes to tau fibril binding affinity

Whole brain imaging of tau-bearing neurofibrillary lesions has the potential to improve the premortem diagnosis and staging of Alzheimers disease. Diverse compounds with high affinity for tau aggregates have been reported from high-throughput screens, but the affinity driving features common among them have not been determined. To identify these features, analogs of compounds discovered by high-throughput screening, including phenothiazine, triarylmethine, benzothiazole, and oxindole derivatives, were tested for their ability to displace fluorescent thioflavin dyes from filaments made from recombinant tau protein or authentic paired helical filaments purified from Alzheimers disease tissue. When representative members of all scaffolds were assayed, the rank order of binding affinity determined for synthetic and authentic filaments correlated strongly, indicating that synthetic filaments have predictive utility for ligand development. Within individual scaffold families, binding affinity was found to correlate with compound polarizability, consistent with a role for dispersion forces in mediating ligand binding. Overall, the data indicate that polarizability is an important commonality among structurally diverse tau binding ligands, and that affinity for tau aggregates can be maximized by integrating formal assessment of this parameter into ligand discovery efforts.

QSAR studies for prediction of cross-β sheet aggregate binding affinity and selectivity

Protein aggregates that accumulate in neurodegenerative diseases are important targets of radiotracer discovery efforts. Although multiple scaffold classes have been reported to bind cross-β sheet structure, their mechanism of binding and their ability to interact selectively with aggregates of varying protein composition are not well understood. Here we take a ligand-based quantitative structure-activity relationship approach to identify descriptors of binding affinity and selectivity for a series of 50 closely related benzothiazole derivatives reported to displace Thioflavin T fluorescent probe from synthetic aggregates composed of β-amyloid peptide and insulin. Using a two-step workflow involving both partial least squares and multiple linear regression methods, compound polarizability and hydrophobicity were identified as tunable mediators of binding selectivity. The correlations also revealed how polarizability could be modulated in neutral compounds having push-pull character. These data suggest that the relative affinity of small molecules for binding sites exposed on aggregate surfaces can be modulated by simple chemical design considerations that are compatible with multiple scaffolds.

Imaging as a Strategy for Premortem Diagnosis and Staging of Tauopathies

Alzheimers disease is diagnosed by postmortem detection of pathological lesions that accumulate in specific brain regions. Although the presence of both beta-amyloid plaques and tau-bearing neurofibrillary lesions defines Alzheimers disease, the distribution of neurofibrillary lesions alone correlates strongly with neurodegeneration and cognitive decline. A whole-brain imaging test capable of detecting these lesions in premortem cases could have great potential for staging and differentially diagnosing Alzheimers disease. Here we discuss the challenges in developing a whole-brain imaging approach for detection of this intracellular target.

Ligand electronic properties modulate tau filament binding site density

Small molecules that bind tau-bearing neurofibrillary lesions are being sought for premortem diagnosis, staging, and treatment of Alzheimers disease and other tauopathic neurodegenerative diseases. The utility of these agents will depend on both their binding affinity and binding site density (B(max)). Previously we identified polarizability as a descriptor of protein aggregate binding affinity. To examine its contribution to binding site density, we investigated the ability of two closely related benzothiazole derivatives ((E)-2-[[4-(dimethylamino)phenyl]azo]-6-methoxybenzothiazole) and ((E)-2-[2-[4-(dimethylamino)phenyl]ethenyl]-6-methoxybenzothiazole) that differed in polarizability to displace probes of high (Thioflavin S) and low (radiolabeled (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene; IMSB) density sites. Consistent with their site densities, Thioflavin S completely displaced radiolabeled IMSB, but IMSB was incapable of displacing Thioflavin S. Although both benzothiazoles displaced the low B(max) IMSB probe, only the highly polarizable analog displaced near saturating concentrations of the Thioflavin S probe. Quantum calculations showed that high polarizability reflected extensive pi-electron delocalization fostered by the presence of electron donating and accepting groups. These data suggest that electron delocalization promotes ligand binding at a subset of sites on tau aggregates that are present at high density, and that optimizing this aspect of ligand structure can yield tau-directed agents with superior diagnostic and therapeutic performance.

Rational Design of Early Premortem Alzheimer's Disease Diagnostic Agents

Alzheimers disease (AD) is a global burden; it affects over five million people in the US, and nearly 30 million worldwide. It is a progressive neurodegenerative disease that develops over many years before cognitive and behavioral symptoms appear. During that time, characteristic lesions called neurofibrillary tangles (NFTs) and senile plaques accumulate in the brain. Although the presence of both plaques and tangles defines AD, NFTs have special utility for diagnosis because their appearance correlates strongly with neurodegeneration and decline in memory. In fact, the spatial distribution of NFTs is the gold standard of postmortem assessment and AD staging. A challenge for porting NFT detection to premortem diagnosis is the identification of a potent and selective NFT probe for a whole brain imaging technique, such as Positron Emission Tomography (PET). Thus, the factors essential for generating differential binding affinity for various proteinaceous deposits must be elucidated.To identify the major sources of binding affinity for protein filaments, a family of 50 benzothiazole derivatives disclosed in patent literature was investigated using a computational approach. The published values for compound potency on synthetic aggregates composed of Abeta peptide and insulin using Thioflavin T displacement fluorescence assays were rationalized via calculated molecular properties using both partial and multiple least squares (PLS, MLS) regression methods. Correlation of over 280 molecular properties through PLS analysis identified molecular polarizability, hydrophobicity, valence connectivity, and the rotatable bond fraction as top ranked determinants of binding affinity. MLS revealed that the correlation coefficient for calculated vs. measured potency exceeded 0.7 when just these four descriptors were used to build the structure-activity relationship. Most importantly, the analysis revealed that polarizability and hydrophobicity were most important for generating differential binding affinity for Abeta relative to insulin filaments (p < 0.01, and 0.001, respectively).