Identification of human presequence protease (hPreP) agonists for the treatment of Alzheimer’s disease

Amyloid-β (Aβ), a neurotoxic peptide, is linked to the onset of Alzheimer’s disease (AD). Increased Aβ content within neuronal cell mitochondria is a pathological feature in both human and mouse models with AD. This accumulation of Aβ within the mitochondrial landscape perpetuates increased free radical production and activation of the apoptotic pathway. Human Presequence Protease (hPreP) is responsible for the degradation of mitochondrial amyloid-β peptide in human neuronal cells, and is thus an attractive target to increase the proteolysis of Aβ. Therefore, it offers a potential target for Alzheimer’s drug design, by identifying potential activators of hPreP. We applied structure-based drug design, combined with experimental methodologies to investigate the ability of various compounds to enhance hPreP proteolytic activity. Compounds 3c & 4c enhanced hPreP-mediated proteolysis of Aβ (1–42), pF1β (2–54) and fluorogenic-substrate V. These results suggest that activation of hPreP by small benzimidazole derivatives provide a promising avenue for AD treatment

Inhibitory effect of gemcitabine and brucine on MDA MB-231human breast cancer cells

Combination of natural compounds and cytotoxic drugs represents a logical therapeutic approach for breast cancer. Brucine, a natural plant alkaloid is reported to possess cytotoxic, anti-inflammatory and anti-cancer activities. Gemcitabine is a nucleoside analog, commonly used in the treatment of several solid tumors, including breast cancer. In the present study we have examined the anticancer effect of brucine and gemcitabine on MDA MB-231 human breast cancer cells. Cell proliferation was assessed using MTT assay. Soft agar assay was used to evaluate the in-vitro clonogencity of MDA MB-231 cells. Cell migration was determined by in-vitro scratch assay and expression of p65 (NF-kB subunit) was evaluated by western blot analysis. Combination treatment with brucine and gemcitabine resulted in a significant inhibition of cell proliferation than either brucine or gemcitabine alone. Cells treated with combination of brucine and gemcitabine showed additive inhibition in colony formation and cell migration than treated with individual agents. The cells treated with brucine at 300 µM showed a significant decrease in p65-NF-kB expression but in combination treatment there was no additive inhibition of p65 expression compared to brucine treated cells. Overall, our results suggested that brucine in combination with gemcitabine showed supra-additive anticancer effects in MDA MB-231 cells and warrants further in-vivo studies in experimental animal models.

Determination of Small Molecule ABAD Inhibitors Crossing Blood Brain Barrier and Pharmacokinetics

A major obstacle to the development of effective treatment of Alzheimer’s disease (AD) is successfully delivery of drugs to the brain. We have previously identified a series of benzothiazole phosphonate compounds that block the interaction of amyloid beta peptide (Aβ) with amyloid-beta binding alcohol dehydrogenase (ABAD). A selective and sensitive method for the presence of three new benzothiazole ABAD inhibitors in mouse plasma, brain and artificial cerebrospinal fluid has been developed and validated based on high performance liquid chromatography tandem mass spectrometry. Mass spectra were generated using Micromass Quattro Ultima “triple” quadrupole mass spectrometer equipped with Electrospray ionization interface. Good linearity was obtained over a concentration range of 0.05–2.5 µg/ml. The lowest limit of quantification and detection was 0.05µg/ml. All inter-day accuracies and precisions were within ±15% of the nominal value and ±20%, respectively, at the lower limit of quantitation. The tested compounds were stable at various conditions with recoveries >90.0 % (RSD<10%). The method used for pharmacokinetic studies of compounds in mouse cerebrospinal fluid, plasma, and brain is accurate, precise, and specific with no matrix effect. Pharmacokinetic data showed these compounds penetrate the blood–brain barrier (BBB) yielding 4–50 ng/ml peak brain concentrations and 2 µg/ml peak plasma concentrations from a 10mg/kg dose. These results indicate that our newly synthesized small molecule ABAD inhibitor have good drug properties with the ability to cross the blood brain barrier, which holds a great potential for AD therapy

Structure Based Design, Synthesis, Pharmacophore Modeling, Virtual Screening, and Molecular Docking Studies for Identification of Novel Cyclophilin D Inhibitors

Cyclophilin D (CypD) is a peptidyl prolyl isomerase F that resides in the mitochondrial matrix and associates with the inner mitochondrial membrane during the mitochondrial membrane permeability transition. CypD plays a central role in opening the mitochondrial membrane permeability transition pore (mPTP) leading to cell death and has been linked to Alzheimer’s disease (AD). Because CypD interacts with amyloid beta (Aβ) to exacerbate mitochondrial and neuronal stress, it is a potential target for drugs to treat AD. Since appropriately designed small organic molecules might bind to CypD and block its interaction with Aβ, 20 trial compounds were designed using known procedures that started with fundamental pyrimidine and sulfonamide scaffolds know to have useful therapeutic effects. Two-dimensional (2D) quantitative structure–activity relationship (QSAR) methods were applied to 40 compounds with known IC₅₀ values. These formed a training set and were followed by a trial set of 20 designed compounds. A correlation analysis was carried out comparing the statistics of the measured IC₅₀ with predicted values for both sets. Selectivity-determining descriptors were interpreted graphically in terms of principle component analyses. These descriptors can be very useful for predicting activity enhancement for lead compounds. A 3D pharmacophore model was also created. Molecular dynamics simulations were carried out for the 20 trial compounds with known IC₅₀ values, and molecular descriptors were determined by 2D QSAR studies using the Lipinski rule-of-five. Fifteen of the 20 molecules satisfied all 5 Lipinski rules, and the remaining 5 satisfied 4 of the 5 Lipinski criteria and nearly satisfied the fifth. Our previous use of 2D QSAR, 3D pharmacophore models, and molecular docking experiments to successfully predict activity indicates that this can be a very powerful technique for screening large numbers of new compounds as active drug candidates. These studies will hopefully provide a basis for efficiently designing and screening large numbers of more potent and selective inhibitors for CypD treatment of AD