Per-6-substituted-per-6-deoxy beta-cyclodextrins inhibit the formation of beta-amyloid peptide derived soluble oligomers.

Recent studies have indicated that the most important role of beta-amyloid peptide (Abeta) in the etiology of Alzheimer's disease may not be in plaque formation but in the formation of soluble, metastable Abeta(1-42) neurotoxic intermediates (called ADDLs). In the present work we describe the preparation of per-6-amino-6-deoxy-beta-cyclodextrins, which inhibit ADDLs formation in vitro.

Femtomole immunodetection of synthetic and endogenous amyloid-beta oligomers and its application to Alzheimer's disease drug candidate screening.

Alzheimer's disease (AD) is a fatal, progressive dementia for which there is no cure and for which a molecular basis has yet to be established. However, considerable evidence suggests that AD is linked to neurotoxic assemblies of the 42-amino-acid peptide amyloid beta (Abeta). There is now a clear body of evidence that shows this neurotoxicity resides not only in insoluble fibrils of Abeta but also in soluble Abeta ADDLs (Abeta-derived diffusible ligands) and larger protofibrils. Further, anti-Abeta antibodies have been reported to reverse memory failure in human amyloid precursor protein (hAPP)-expressed transgenic mice in a manner that suggests symptom reversal is attributable to targeting of ADDLs. Clearly, a search for drugs targeting the assembly of these soluble Abeta species represents a new and potentially important approach to the treatment of AD. In this work we describe the development of a dot-blot immunoassay to measure ADDL at the femtomole level, its use in defining the time course of ADDL formation, and its use in determining the presence of ADDLs in the hAPP transgenic mouse brain. Discussion of a protocol to screen agents for inhibition of neurotoxic ADDLformation both in vivo and in vitro is also presented. The methods are suitable for screening combinatorial libraries and, importantly, provide the potential for simultaneous information on candidate transport across the blood-brain barrier.

Regioselective covalent modification of hemoglobin in search of antisickling agents.

Although the molecular defect in sickle hemoglobin that produces sickle cell disease has been known for decades, there is still no effective drug treatment that acts on hemoglobin itself. In this work, a series of diversely substituted isothiocyanates (R-NCS) were examined for their regioselective reaction with hemoglobin in an attempt to alter the solubility properties of sickle hemoglobin. Electrospray mass spectrometry, molecular modeling, X-ray crystallography, and conventional protein chemistry were used to study this regioselectivity and the resulting increase in solubility of the modified hemoglobin. Depending on the attached R-group, the isothiocyanates were found to react either with the Cysbeta93 or the N-terminal amine of the alpha-chain. One of the most effective compounds in the series, 2-(N,N-dimethylamino)ethyl isothiocyanate, selectively reacts with the thiol of Cysbeta93 which, in conjunction with the cationic group, was seen to perturb the local hemoglobin structure. This modified HbS shows an approximately 30% increase in solubility for the fully deoxygenated state, along with a significant increase in oxygen affinity. This compound and a related analogue appear to readily traverse the erythrocyte membrane. A discussion of the relation of these structural changes to inhibition of gelation is presented. The dual activities of increasing HbS oxygen affinity and directly inhibiting deoxy HbS polymerization, in conjunction with facile membrane traversal, suggest that these cationic isothiocyanates show substantial promise as lead compounds for development of therapeutic agents for sickle cell disease.

Per-6-substituted beta-cyclodextrin libraries inhibit formation of beta-amyloid-peptide (A beta)-derived, soluble oligomers.

Alzheimer's disease is the most common cause of dementia in older individuals with compelling evidence favoring neuron dysfunction and death triggered by assembled forms of A beta(1-42). While large neurotoxic amyloid fibrils have been known for years, recent studies show that soluble protofibril and A beta(1-42)-derived diffusible ligands (ADDLs) may also be involved in neurotoxicity. In the present work, dot-blot immunoassays discriminating ADDLs from monomers were used to screen libraries of per-substituted beta-cyclodextrin (beta-CD) derivatives for inhibition of ADDLs formation. Libraries were prepared from per-6-iodo-beta-CD by treatment with various amine nucleophiles. The most active library tested (containing >2000 derivatives) was derived from imidazole, N, N-dimethylethylenediamine and furfurylamine, which at 10 microM total library, inhibited ADDLs formation (10 nM A beta(1-42)) over a period of 4 hours. The latter was confirmed by a western blot assay showing decreased amounts of the initially formed A beta(1-42) tetramer. These preliminary experiments suggest that derivatized forms of beta-CD can interfere with the oligomerization process of A beta(1-42).

Cocaine detoxification by combinatorially substituted beta-cyclodextrin libraries.

Per-6-substituted- and A,C,E(F)-tri-6-substituted-6-deoxy-beta-cyclodextrin (beta-CD) libraries were generated using solution-phase combinatorial chemistry techniques starting from the corresponding iodo precursors and different combinations of individual amine nucleophiles. Using a high throughput electrospray mass spectrometry (ESMS) screen to monitor the hydrolysis of cocaine, certain libraries showed the ability to specifically hydrolyze the methyl ester of cocaine, with the most active per-6-substituted beta-CD library I producing complete hydrolysis in 24h. The cocaine hydrolytic activity in this series showed structure-activity relationships which appeared to involve specific interaction between the amine side chains and the cocaine molecule. Comparison of the composition of the most active per-6-substiuted beta-CD libraries and A,C,E(F)-tri-6-substituted-6-deoxy-beta-CD libraries (I and XV) showed three common side chains (3, 4, and 5), suggesting that active side chains in the tri-substituted beta-CD library might be predicted from evaluation of the more easily prepared per-6-substituted-6-deoxy-beta-CD series.