Rajeshwar Narlawar

Substrate-targeting γ-secretase modulators

Selective lowering of Aβ42 levels (the 42-residue isoform of the amyloid-β peptide) with small-molecule γ-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer’s disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the γ-secretase complex, but instead labelled the β-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-β peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP γ-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28–36 of amyloid-β, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-β act as GSMs, and some GSMs alter the production of cell-derived amyloid-β oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Aβ42 production and inhibition of amyloid-β aggregation, which may synergistically reduce amyloid-β deposition in Alzheimer’s disease. These data also demonstrate the existence and feasibility of ‘substrate targeting’ by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of ‘druggable’ targets.

Curcumin-Derived Pyrazoles and Isoxazoles: Swiss Army Knives or Blunt Tools for Alzheimer's Disease?

Curcumin binds to the amyloidu2005β peptide (Aβ) and inhibits or modulates amyloid precursor protein (APP) metabolism. Therefore, curcumin‐derived isoxazoles and pyrazoles were synthesized to minimize the metal chelation properties of curcumin. The decreased rotational freedom and absence of stereoisomers was predicted to enhance affinity toward Aβ42 aggregates. Accordingly, replacement of the 1,3‐dicarbonyl moiety with isosteric heterocycles turned curcumin analogue isoxazoles and pyrazoles into potent ligands of fibrillar Aβ42 aggregates. Additionally, several compounds are potent inhibitors of tau protein aggregation and depolymerized tau protein aggregates at low micromolar concentrations.

Curcumin Derivatives Inhibit or Modulate Beta-Amyloid Precursor Protein Metabolism

Curcumin-derived oxazoles and pyrazoles were synthesized in order to minimize the metal chelation properties of curcumin. The reduced rotational freedom and the absence of stereoisomers was anticipated to enhance the inhibition of γ-secretase. Accordingly, the replacement of the 1,3-dicarbonyl moiety by isosteric heterocycles turned curcumin analogue oxazoles and pyrazoles into potent γ-secretase inhibitors. Compounds 4a-i were found to be potent inhibitors of γ-secretase and displayed activity in the low micromolar range.

Curcumin Cross-links Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Polypeptides and Potentiates CFTR Channel Activity by Distinct Mechanisms

Cystic fibrosis (CF) is caused by loss-of-function mutations in the CFTR chloride channel. Wild type and mutant CFTR channels can be activated by curcumin, a well tolerated dietary compound with some appeal as a prospective CF therapeutic. However, we show here that curcumin has the unexpected effect of cross-linking CFTR polypeptides into SDS-resistant oligomers. This effect occurred for CFTR channels in microsomes as well as in intact cells and at the same concentrations that are effective for promoting CFTR channel activity (5–50 μm). Both mature CFTR polypeptides at the cell surface and immature CFTR protein in the endoplasmic reticulum were cross-linked by curcumin, although the latter pool was more susceptible to this modification. Curcumin cross-linked two CF mutant channels (ΔF508 and G551D) as well as a variety of deletion constructs that lack the major cytoplasmic domains. In vitro cross-linking could be prevented by high concentrations of oxidant scavengers (i.e. reduced glutathione and sodium azide) indicating a possible oxidation reaction with the CFTR polypeptide. Importantly, cyclic derivatives of curcumin that lack the reactive β diketone moiety had no cross-linking activity. One of these cyclic derivatives stimulated the activities of wild type CFTR channels, Δ1198-CFTR channels, and G551D-CFTR channels in excised membrane patches. Like the parent compound, the cyclic derivative irreversibly activated CFTR channels in excised patches during prolonged exposure (>5 min). Our results raise a note of caution about secondary biochemical effects of reactive compounds like curcumin in the treatment of CF. Cyclic curcumin derivatives may have better therapeutic potential in this regard.

Drug development and PET-diagnostics for Alzheimer's disease.

The exact cause of Alzheimers disease is still unknown; despite the dramatic progress in understanding. Most gene mutations associated with Alzheimers disease point to the amyloid precursor protein and amyloid beta. The alpha-, beta- and gamma-secretases are the three executioners of amyloid precursor protein processing. Significant progress has been made in the selective inhibition of these proteases, regardless of the availability of structural information. Several peptidic and non-peptidic leads were identified and first drug candidates are in clinical trials. Cholesterol lowering drugs and metal chelators are also in advanced clinical stages as disease modifiers. Successful trials demand either large cohorts or reliable markers for Alzheimers disease. Therefore, several radiomarkers are under investigation to support such clinical trials.

Modulators and Inhibitors of γ- and β-Secretases

Most gene mutations associated with Alzheimer’s disease point to the metabolism of amyloid precursor protein as a potential cause. The β- and γ-secretases are two executioners of amyloid precursor protein processing resulting in amyloid-β. Significant progress has been made in the selective inhibition of both proteases, regardless of structural information for γ-secretase. Several peptidic and nonpeptidic leads were identified for both targets.

IC-P3-212: Mapping the binding site of gamma-secretase modulators by small (and not so small) organic molecules

Background: Non-steroidal anti-inflammatory drugs (NSAIDs) that modulate gamma-secretase cleavage of amyloid precursor protein (APP) affect the distance between APP and presenilin, the catalytic subunit of gammasecretase. They seem to interfere with substrate recognition/cleavage and shift the precision of gamma-secretase cleavage from the beta-amyloid 42 to the beta-amyloid 38 site to generate more Abeta 38 and less Abeta 42.New data indicate binding of selected gamma-secretase modulators to the substrate to induce a stabilisation of a non-pathological conformation. Methods: The optimisation of NSAID derived lead structure and introduction of photocrosslinkable fragment furnished suitable tools to address the binding of gamma-secretase modulators. Fluorescent derivatives and tethered dimers are utilized to investigate presence and distance of potential multiple binding sites. Results: The binding site of flurbiprofen derived gamma-secretase modulators was mapped to the vicinity of the GXXXG motive of amyloid precursor protein. Conclusions: The established binding site of flurbiprofen derived gamma-secretase modulators resides close to the membrane surface and thus mandates a high lipophilicity in combination with a strongly acidic functional group. This combination is unusual for therapeutic drug, but common to amphilic tensides, thus creates an obstacle for drug development.