Bruno Bulic

Tau protein and tau aggregation inhibitors

Alzheimer disease is characterized by pathological aggregation of two proteins, tau and Abeta-amyloid, both of which are considered to be toxic to neurons. In this review we summarize recent advances on small molecule inhibitors of protein aggregation with emphasis on tau, with activities mediated by the direct interference of self-assembly. The inhibitors can be clustered in several compound classes according to their chemical structure, with subsequent description of the structure-activity relationships, showing that hydrophobic interactions are prevailing. The description is extended to the pharmacological profile of the compounds in order to evaluate their drug-likeness, with special attention to toxicity and bioavailability. The collected data indicate that following the improvements of the in vitro inhibitory potencies, the consideration of the in vivo pharmacokinetics is an absolute prerequisite for the development of compounds suitable for a transfer from bench to bedside.

Amyloid beta 42 peptide (Aβ42)-lowering compounds directly bind to Aβ and interfere with amyloid precursor protein (APP) transmembrane dimerization

Following ectodomain shedding by β-secretase, successive proteolytic cleavages within the transmembrane sequence (TMS) of the amyloid precursor protein (APP) catalyzed by γ-secretase result in the release of amyloid-β (Aβ) peptides of variable length. Aβ peptides with 42 amino acids appear to be the key pathogenic species in Alzheimer’s disease, as they are believed to initiate neuronal degeneration. Sulindac sulfide, which is known as a potent γ-secretase modulator (GSM), selectively reduces Aβ42 production in favor of shorter Aβ species, such as Aβ38. By studying APP–TMS dimerization we previously showed that an attenuated interaction similarly decreased Aβ42 levels and concomitantly increased Aβ38 levels. However, the precise molecular mechanism by which GSMs modulate Aβ production is still unclear. In this study, using a reporter gene-based dimerization assay, we found that APP–TMS dimers are destabilized by sulindac sulfide and related Aβ42-lowering compounds in a concentration-dependent manner. By surface plasmon resonance analysis and NMR spectroscopy, we show that sulindac sulfide and novel sulindac-derived compounds directly bind to the Aβ sequence. Strikingly, the attenuated APP–TMS interaction by GSMs correlated strongly with Aβ42-lowering activity and binding strength to the Aβ sequence. Molecular docking analyses suggest that certain GSMs bind to the GxxxG dimerization motif in the APP–TMS. We conclude that these GSMs decrease Aβ42 levels by modulating APP–TMS interactions. This effect specifically emphasizes the importance of the dimeric APP–TMS as a promising drug target in Alzheimer’s disease.

Small‐Molecule Inhibitors of Islet Amyloid Polypeptide Fibril Formation

Newly synthesized proteins in the cell adopt a functional folded state resulting from a highly regulated process. Failure to form this functional state leads to the degradation of proteins inside the cell. However, under certain conditions, some proteins can also adopt an alternative state by the assembly of unfolded or partially folded monomers or protein fragments into a b-sheet structure called amyloid fibril. In spite of arising from diverse amino acid sequences, they have a similar fibrillar structure that binds the dye Congo red. These amyloids are involved in a number of devastating diseases including Alzheimer&s disease, prion diseases, and type-II diabetes mellitus. In the type-II diabetes, deposition of extracellular amyloid plaques in pancreatic beta cells has been observed in humans. Biochemical analysis of the plaques revealed the presence of a 37-residue peptide called islet amyloid polypeptide (IAPP) or amylin, which is co-secreted with insulin. It has a disulfide bond between residues 2–7 and the C-terminus is amidated. IAPP is also known to interact with lipid membranes which are able to induce and foster the fibril formation. Recently, a possible mechanism of IAPP fibril formation at anionic lipid interfaces has been proposed in which it has been shown that IAPP forms b-sheet-rich amyloid fibrils via an intermediate a-helical state. The presence of IAPP amyloid finally leads to the apoptosis of pancreatic beta cells. However, it is still not clear whether the fibrils themselves or their intermediate states are responsible for the cell death. In nature, IAPP amyloid fibril formation can be prevented by altering the primary amino acid sequence, such as in rat IAPP where three proline residues, which are absent in the human IAPP, are thought to prevent the amyloid fibril formation. Recently, Kapurniotu&s group succeeded in the synthesis of conformationally constrained analogues of IAPP, which are methylated at amide bonds and do not fibrillize. Inhibition of amyloid fibril formation is considered to be a potentially key therapeutic approach towards diabetes and other amyloid-related diseases. Surprisingly, very little attempt has been made to inhibit IAPP fibril formation by small-molecule inhibitors. Small-molecule inhibitors have advantages over peptide inhibitors because they could more easily cross the blood brain barrier, avoid immunological response, and are more stable in biological fluids and tissues. In addition, the high flexibility of peptide inhibitors may, for entropic reasons, prevent efficient binding. This problem may be overcome by synthesis of conformationally restricted peptides. The bottleneck in the discovery of small-molecule inhibitors of amyloid fibril formation is the lack of structural information about amyloids. However, this did not prevent the discovery of small-molecule inhibitors for other amyloid fibrils such as, Ab and tau, which are involved in Alzheimer&s disease. In a recent study on a cellular model of tau aggregate inhibition, two rhodanine-scaffold (2-thioxothiazolidin-4one) based inhibitors have been identified which have very low cell toxicity. These compounds were chosen because of the presence of a rhodanine heterocyclic core, which is biocompatible, non-mutagenic, and has a drug-like profile. Inspired by these results, we wanted to explore whether these compounds will also inhibit amyloid fibril formation of IAPP which has a completely different amino acid sequence but shares a similar fibrillar morphology with the tau aggregate. To our knowledge, this is the first study on such smallmolecule inhibitors of IAPP amyloid formation. The compounds 1 and 2 (Figure 1) were synthesized as described earlier. Amyloid fibril formation was carried out in 10 mm sodium phosphate buffer at pH 7.5 for 96 h. To reveal the effect of the two potential inhibitors, different concentrations of the compounds were added to the buffer solution. Fibril formation was quantified by measuring the fluorescence intensity of the amyloid-specific dye thioflavin T (ThT) at a wavelength of 480 nm. The fluorescence intensity of amyloid fibrils increases upon binding to ThT. The efficiency of inhibition was monitored by measuring the ThT fluorescence intensity with respect to that of pure IAPP aggregate without inhibitor (100%). It is evident from Figure 1b that both compounds have a marked inhibitory effect. The concentration at which half of the fibril formation is inhibited (IC50) is 1.23 mm for compound 1, and 0.45 mm for compound 2. A similar trend has been observed for the aggregation of tau, with IC50 values of 0.67 and 0.26 mm for compounds 1 and 2, respectively. From the results on tau and IAPP aggregation it is clear that compound 2 is a more [*] Dr. R. Mishra, D. Sellin, S. Jha, Prof. Dr. R. Winter Faculty of Chemistry Physical Chemistry I—Biophysical Chemistry Technical University Dortmund Otto-Hahn-Strasse 6, 44227 Dortmund (Germany) Fax. (+49)231-755-3901 E-mail: roland.winter@uni-dortmund.de

Progress and developments in tau aggregation inhibitors for Alzheimer disease

Pharmacological approaches directed toward Alzheimer disease are diversifying in parallel with a growing number of promising targets. Investigations on the microtubule-associated protein tau yielded innovative targets backed by recent findings about the central role of tau in numerous neurodegenerative diseases. In this review, we summarize the recent evolution in the development of nonpeptidic small molecules tau aggregation inhibitors (TAGIs) and their advancement toward clinical trials. The compounds are classified according to their chemical structures, providing correlative insights into their pharmacology. Overall, shared structure-activity traits are emerging, as well as specific binding modes related to their ability to engage in hydrogen bonding. Medicinal chemistry efforts on TAGIs together with encouraging in vivo data argue for successful translation to the clinic.

Presenilin‐1 but not amyloid precursor protein mutations present in mouse models of Alzheimer’s disease attenuate the response of cultured cells to γ‐secretase modulators regardless of their potency and structure

J. Neurochem. (2011) 116, 385–395.

Presenilin is the molecular target of acidic γ-secretase modulators in living cells.

The intramembrane-cleaving protease γ-secretase catalyzes the last step in the generation of toxic amyloid-β (Aβ) peptides and is a principal therapeutic target in Alzheimers disease. Both preclinical and clinical studies have demonstrated that inhibition of γ-secretase is associated with prohibitive side effects due to suppression of Notch processing and signaling. Potentially safer are γ-secretase modulators (GSMs), which are small molecules that selectively lower generation of the highly amyloidogenic Aβ42 peptides but spare Notch processing. GSMs with nanomolar potency and favorable pharmacological properties have been described, but the molecular mechanism of GSMs remains uncertain and both the substrate amyloid precursor protein (APP) and subunits of the γ-secretase complex have been proposed as the molecular target of GSMs. We have generated a potent photo-probe based on an acidic GSM that lowers Aβ42 generation with an IC50 of 290 nM in cellular assays. By combining in vivo photo-crosslinking with affinity purification, we demonstrated that this probe binds the N-terminal fragment of presenilin (PSEN), the catalytic subunit of the γ-secretase complex, in living cells. Labeling was not observed for APP or any of the other γ-secretase subunits. Binding was readily competed by structurally divergent acidic and non-acidic GSMs suggesting a shared mode of action. These findings indicate that potent acidic GSMs target presenilin to modulate the enzymatic activity of the γ-secretase complex.

Chemical Biology, Molecular Mechanism and Clinical Perspective of γ-Secretase Modulators in Alzheimer’s Disease

Comprehensive evidence supports that oligomerization and accumulation of amyloidogenic Aβ42 peptides in brain is crucial in the pathogenesis of both familial and sporadic forms of Alzheimers disease. Imaging studies indicate that the buildup of Aβ begins many years before the onset of clinical symptoms, and that subsequent neurodegeneration and cognitive decline may proceed independently of Aβ. This implies the necessity for early intervention in cognitively normal individuals with therapeutic strategies that prioritize safety. The aspartyl protease γ-secretase catalyses the last step in the cellular generation of Aβ42 peptides, and is a principal target for anti-amyloidogenic intervention strategies. Due to the essential role of γ-secretase in the NOTCH signaling pathway, overt mechanism-based toxicity has been observed with the first generation of γ-secretase inhibitors, and safety of this approach has been questioned. However, two new classes of small molecules, γ-secretase modulators (GSMs) and NOTCH-sparing γ-secretase inhibitors, have revitalized γ-secretase as a drug target in AD. GSMs are small molecules that cause a product shift from Aβ42 towards shorter and less toxic Ab peptides. Importantly, GSMs spare other physiologically important substrates of the γ-secretase complex like NOTCH. Recently, GSMs with nanomolar potency and favorable in vivo properties have been described. In this review, we summarize the knowledge about the unusual proteolytic activity of γ-secretase, and the chemical biology, molecular mechanisms and clinical perspective of compounds that target the γ-secretase complex, with a particular focus on GSMs.

No improvement after chronic ibuprofen treatment in the 5XFAD mouse model of Alzheimer's disease

Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) that has been reported to reduce the risk of developing Alzheimers disease (AD). Its preventive effects in AD are likely pleiotropic as ibuprofen displays both anti-inflammatory activity by inhibition of cyclooxygenases and anti-amyloidogenic activity by modulation of γ-secretase. In order to study the anti-inflammatory properties of ibuprofen independent of its anti-amyloidogenic activity, we performed a long-term treatment study with ibuprofen in 5XFAD mice expressing a presenilin-1 mutation that renders this AD model resistant to γ-secretase modulation. As expected, ibuprofen treatment for 3 months resulted in a reduction of the inflammatory reaction in the 5XFAD mouse model. Importantly, an unchanged amyloid beta (Aβ) plaque load, an increase in soluble Aβ42 levels, and an aggravation of some behavioral parameters were noted, raising the question whether suppression of inflammation by nonsteroidal anti-inflammatory drug is beneficial in AD.

Synthesis of a potent photoreactive acidic γ-secretase modulator for target identification in cells

Supramolecular self-assembly of amyloidogenic peptides is closely associated with numerous pathological conditions. For instance, Alzheimer´s disease (AD) is characterized by abundant amyloid plaques originating from the proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. Compounds named γ-secretase modulators (GSMs) can shift the substrate cleavage specificity of γ-secretase toward the production of non-amyloidogenic, shorter Aβ fragments. Herein, we describe the synthesis of highly potent acidic GSMs, equipped with a photoreactive diazirine moiety for photoaffinity labeling. The probes labeled the N-terminal fragment of presenilin (the catalytic subunit of γ-secretase), supporting a mode of action involving binding to γ-secretase. This fundamental step toward the elucidation of the molecular mechanism governing the GSM-induced shift in γ-secretase proteolytic specificity should pave the way for the development of improved drugs against AD.

Presenilin-1 (PS1) and amyloid precursor protein (APP) mutations present in mouse models of Alzheimer's disease in their response to γ-secretase inhibitors and modulators

peptides mainly generate two isoform, Aß40 and Aß42 by enzymatic proteolysis of amyloid precursor protein (APP). In particular, the Aß42 is believed to be the major etiologic agent in pathogenesis of AD due to its higher fibrillation or oligomerization properties than that of Aß40. Recently we have established conformation dependent human antibody, B6, which binds to Aß42 fibril, but not to soluble form of Aß42, inhibiting Aß42 fibril formation. Concurrently, we have identified a mimotope of B6, B6-C15, using the PhD.C7C phage library. We chemically synthesized TAT-conjugated B6-C15 peptide, TATB6-C15. This synthetic peptide has inhibitory activity on Aß42 fibrillation. Furthermore, TAT-B6-C15 specifically binds to the oligomer form of Aß42, but not to freshly prepared monomer Aß42 nor its fibril form. In this study, we investigated the effect of this TAT-B6-C15 peptide on Aß40 assembly. Methods: Aß42 or Aß40 was incubated at 37 C in the presence or absence of TAT-B6-C15 peptide. Aß fibrillation was monitored by amyloid specific fluorescence dye, Thioflavin T. To identify the Aß conformers which specifically bound to TAT-B6-C15 peptide, we performed dot blot analysis. Aß conformers were periodically sampled after the onset of Aß40 or 42 fibrillation assay and hand-spotted onto nitrocellulose membrane, followed by incubation with detection probes such as TAT-B6-C15 or anti-Aß antibody. Results: The TAT-B6-C15 peptide exhibited inhibitory effect on Aß42, but not Aß40 fibrillation. Furthermore, the TAT-B6-C15 showed binding activity to the Aß42 prefibrillar oligomer, but not any Aß40 conformers. Conclusions: The mimotope peptide which identified as conformation dependent antibody epitope, specifically binds to prefibrillar oligomers of Aß42, inhibiting Aß42 but not Aß40 fibril formation.