Characterization of Novel CSF Tau and ptau Biomarkers for Alzheimer’s Disease

<div><p>Cerebral spinal fluid (CSF) Aβ42, tau and p181tau are widely accepted biomarkers of Alzheimer’s disease (AD). Numerous studies show that CSF tau and p181tau levels are elevated in mild-to-moderate AD compared to age-matched controls. In addition, these increases might predict preclinical AD in cognitively normal elderly. Despite their importance as biomarkers, the molecular nature of CSF tau and ptau is not known. In the current study, reverse-phase high performance liquid chromatography was used to enrich and concentrate tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of tau were detected in pooled CSF with apparent sizes ranging from <20 kDa to ~40 kDa. The pattern of tau fragments in AD and control samples were similar. In contrast, full-length tau and C-terminal-containing fragments were not detected. To quantify levels, five tau ELISAs and three ptau ELISAs were developed to detect different overlapping regions of the protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples. Of the tau ELISAs, the two assays specific for tau containing N-terminal sequences, amino acids 9-198 (numbering based on tau 441) and 9-163, exhibited the most significant differences between AD and control samples. In contrast, CSF tau was not detected with an ELISA specific for a more C-terminal region (amino acids 159-335). Significant discrimination was also observed with ptau assays measuring amino acids 159-p181 and 159-p231. Interestingly, the discriminatory potential of p181 was reduced when measured in the context of tau species containing amino acids 9-p181. Taken together, these results demonstrate that tau in CSF occurs as a series of fragments and that discrimination of AD from control is dependent on the subset of tau species measured. These assays provide novel tools to investigate CSF tau and ptau as biomarkers for other neurodegenerative diseases.</p> </div

Detection of tau fragments in human CSF.

<p>Human control and AD CSF subjected to RP-HPLC, fractions collected and run on SDS-PAGE gels followed by western-blotting with different tau antibodies. A) HT7 (mid domain antibody). B) Tau 12 (N-terminal antibody). C) K9JA (C-terminal microtubule repeat domain antibody). D) IgG1 isotype control. On each blot, human recombinant tau441 (tau) is included in lane 1 and molecular weight markers (mw) in lane 2 followed by the HPLC fractions from 1 to 6 or HPLC fractions 7 to 11. Fractions 1 and 2 were pooled and run as a single sample, while fractions 3-10 were run as individual samples. Control CSF (C) and AD CSF (D) samples for each fraction were run side by side for comparison.</p

Tau and ptau levels in 20 AD and 20 control CSF samples.

<p>A set of 20 AD and 20 age-matched normal control CSF samples were analyzed using the tau ELISAs (HT7-BT2, HT7-Tau5, Tau12-BT2, Tau12-HT7 and HT7-77G7) and pTau ELISAs (HT7-AT270, HT7-PHF6 and Tau12-AT270). Dashed lines indicate the assay LLQ corrected for CSF dilution. Statistics based on 2-tailed Student’s t test comparison of log-transformed data, * p < 0.05; ** p< 0.01; *** p < 0.001. </p

Discovery of S3-Truncated, C‑6 Heteroaryl Substituted Aminothiazine β‑Site APP Cleaving Enzyme‑1 (BACE1) Inhibitors

Truncation of the S3 substituent of the biaryl aminothiazine <b>2</b>, a potent BACE1 inhibitor, led to a low molecular weight aminothiazine <b>5</b> with moderate activity. Despite its moderate activity, compound <b>5</b> demonstrated significant brain Aβ reduction in rodents. The metabolic instability of <b>5</b> was overcome by the replacement of the 6-dimethylisoxazole, a metabolic soft spot, with a pyrimidine ring. Thus, truncation of the S3 substituent represents a viable approach to the discovery of orally bioavailable, brain-penetrant BACE1 inhibitors

Targeting the BACE1 Active Site Flap Leads to a Potent Inhibitor That Elicits Robust Brain Aβ Reduction in Rodents

By targeting the flap backbone of the BACE1 active site, we discovered 6-dimethylisoxazole-substituted biaryl aminothiazine <b>18</b> with 34-fold improved BACE1 inhibitory activity over the lead compound <b>1</b>. The cocrystal structure of <b>18</b> bound to the active site indicated two hydrogen-bond interactions between the dimethylisoxazole and threonine 72 and glutamine 73 of the flap. Incorporation of the dimethylisoxazole substitution onto the related aminothiazine carboxamide series led to pyrazine-carboxamide <b>26</b> as a very potent BACE1 inhibitor (IC₅₀ < 1 nM). This compound demonstrated robust brain Aβ reduction in rat dose–response studies. Thus, compound <b>26</b> may be useful in testing the amyloid hypothesis of Alzheimer’s disease

Acyl Guanidine Inhibitors of β‑Secretase (BACE-1): Optimization of a Micromolar Hit to a Nanomolar Lead via Iterative Solid- and Solution-Phase Library Synthesis

This report describes the discovery and optimization of a BACE-1 inhibitor series containing an unusual acyl guanidine chemotype that was originally synthesized as part of a 6041-membered solid-phase library. The synthesis of multiple follow-up solid- and solution-phase libraries facilitated the optimization of the original micromolar hit into a single-digit nanomolar BACE-1 inhibitor in both radioligand binding and cell-based functional assay formats. The X-ray structure of representative inhibitors bound to BACE-1 revealed a number of key ligand:protein interactions, including a hydrogen bond between the side chain amide of flap residue Gln73 and the acyl guanidine carbonyl group, and a cation−π interaction between Arg235 and the isothiazole 4-methoxyphenyl substituent. Following subcutaneous administration in rats, an acyl guanidine inhibitor with single-digit nanomolar activity in cells afforded good plasma exposures and a dose-dependent reduction in plasma Aβ levels, but poor brain exposure was observed (likely due to Pgp-mediated efflux), and significant reductions in brain Aβ levels were not obtained

Model of antibody engagement of tau in-vivo.

<p>A compartmental model depicting tau and antibody (IgG) levels in brain, interstitial fluid (ISF), cerebrospinal fluid (CSF) and plasma. CSF tau is truncated with levels of ~1 nM, while tau in ISF exists as a full-length molecule with levels of 3–5 nM (4-5x greater than in CSF) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125614#pone.0125614.ref047" target="_blank">47</a>]. Concentrations of p-tau are estimated to be about 1–10% of total tau levels. Tau antibody concentrations are 1–3 nM in CSF and ISF [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125614#pone.0125614.ref044" target="_blank">44</a>]. Antibody engagement of p-tau in CSF and ISF would enable clearance of tau via a variety of antibody-mediated mechanisms. Full-length tau is indicated as a molecule containing N-terminal (orange line), mid-domain (blue line), microtubule-binding repeat region (black box), and C-terminal (grey line) regions, whereas truncated tau in the CSF compartment is indicated by the mid-domain and N-terminal fragments.</p

Immunodepletion with p-Tau antibodies reduces tau pathology in a primary neuronal model of tau aggregation.

<p><b>A.</b> Kinetics of association and dissociation of PHF6 antibody and PHF13 antibody. The table summarizes the calculated association rates, dissociation rates, and K_D values for PHF6 and PHF13. The vertical dotted lines demarcate, from left to right—peptide loading, wash/equilibration, antibody association and antibody dissociation. <b>B.</b> Representative images from Triton-insoluble MC1 staining of primary neurons that received no PFF treatment (- PFF), treatment with 100 nM K18PL PFF (+ PFF), rTg4510 brain extract (Tg4510) or tTA brain extract (tTA). <b>C.</b> Change in Triton-insoluble MC1 staining intensity in primary neurons treated as in B, relative to untreated cultures (n = 6 wells/condition; ANOVA with Dunnett’s test vs. no treatment). <b>D.</b> Immunodepletion of rTg4510 extracts with PHF6 or PHF13 led to significant declines in Triton-insoluble MC1staining (Red bar) in neurons relative to control antibody (ANOVA with Dunnett’s test vs. Control IgG). Also displayed are total tau (black bar) and p-tau levels (blue bar) measured by ELISA after immunodepletion (*p<0.05, *** p<0.001, n = 3 per group).</p