Koteswara Rao Valasani

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.

Structure-Based Design and Synthesis of Benzothiazole Phosphonate Analogues with Inhibitors of Human ABAD-Aβ for Treatment of Alzheimer’s Disease

Amyloid binding alcohol dehydrogenase, a mitochondrial protein, is a cofactor facilitating amyloid‐β peptide (Aß) induced cell stress. Antagonizing Aß‐ABAD interaction protects against aberrant mitochondrial and neuronal function and improves learning memory in the Alzheimer’s disease mouse model. Therefore, it offers a potential target for Alzheimer’s drug design, by identifying potential inhibitors of Aβ‐ABAD interaction. 2D QSAR methods were applied to novel compounds with known IC50 values, which formed a training set. A correlation analysis was carried out comparing the statistics of the measured IC50 with predicted values. These selectivity‐determining descriptors were interpreted graphically in terms of principle component analyses, which are highly informative for the lead optimization process with respect to activity enhancement. A 3D pharmacophore model also was created. The 2D QSAR and 3D pharmacophore models will assist in high‐throughput screening. In addition, ADME descriptors were also determined to study their pharmacokinetic properties. Finally, amyloid binding alcohol dehydrogenase molecular docking study of these novel molecules was undertaken to determine whether these compounds exhibit significant binding affinity with the binding site. We have synthesized only the compounds that have shown the best drug‐like properties as candidates for further studies.

Compartmentalized Accumulation of cAMP near Complexes of Multidrug Resistance Protein 4 (MRP4) and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Contributes to Drug-induced Diarrhea

Background: Diarrhea is an adverse side effect associated with many therapeutics. Results: Irinotecan induced hyperactive cystic fibrosis transmembrane conductance regulator (CFTR) function by inhibiting multidrug resistance protein 4 (MRP4) and formation of MRP4-CFTR macromolecular complexes. Conclusion: MRP4-CFTR-containing macromolecular complexes play an important role in drug-induced diarrhea. Significance: These studies help define molecular mechanisms of drug-induced diarrhea. Diarrhea is one of the most common adverse side effects observed in ∼7% of individuals consuming Food and Drug Administration (FDA)-approved drugs. The mechanism of how these drugs alter fluid secretion in the gut and induce diarrhea is not clearly understood. Several drugs are either substrates or inhibitors of multidrug resistance protein 4 (MRP4), such as the anti-colon cancer drug irinotecan and an anti-retroviral used to treat HIV infection, 3′-azido-3′-deoxythymidine (AZT). These drugs activate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluid secretion by inhibiting MRP4-mediated cAMP efflux. Binding of drugs to MRP4 augments the formation of MRP4-CFTR-containing macromolecular complexes that is mediated via scaffolding protein PDZK1. Importantly, HIV patients on AZT treatment demonstrate augmented MRP4-CFTR complex formation in the colon, which defines a novel paradigm of drug-induced diarrhea.

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 Alzheimers 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 Alzheimers 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.

Tetramethylguanidiniumchlorosulfonate ionic liquid (TMG IL): an efficient reusable catalyst for the synthesis of tetrahydro-1H-benzo[a]chromeno[2,3-c]phenazin-1-ones under solvent-free conditions and evaluation for their in vitro bioassay activity

Medicinally important tetrahydro-1H-benzo[a]chromeno[2,3-c]phenazin-1-ones were synthesized by the straightforward, efficient and convenient approach of a three-component reaction between aldehydes, benzo[a]phenazin-5-ol and active methylene compounds under neat conditions in the presence of an ionic liquid, tetramethylguanidiniumchlorosulfonate (TMG IL). The TMG IL was used as a solvent and as a catalyst under reusable conditions. The observed higher product yields in a shorter reaction time and the use of environmentally benign reaction conditions are the merits of this reaction. The title compounds were screened for their antioxidant and anticancer activities, with the majority of them showing good bioassay activity.

Identification and analysis of novel R308K mutation in glucokinase of type 2 diabetic patient and its kinetic correlation

Glucokinase (GK) plays a critical role in glucose homeostasis and the mutations in GK gene result in pathogenic complications known as Maturity Onset Diabetes of the Young 2, an autosomal dominant form of diabetic condition. In the present study, GK was purified from human liver tissue and the pure enzyme showed single band in SDS‐PAGE with a molecular weight of 50 kDa. The kinetics of pure GK showed enzyme activity of 0.423 ± 0.02 µM glucose‐6‐phosphate (G6P)/mL/Min and Km value of 6.66 ± 0.02 µM. These values were compared in the liver biopsy of a clinically proven type 2 diabetic patient, where GK kinetics showed decreased enzyme activity of 0.16 ± 0.025 µM G6P/mL/Min and increased Km of 23 ± 0.9 µM, indicating the hyperglycemic condition in the patient. The genetic analysis of 10th exon of GK gene from this patient showed a R308K mutation. To substantiate these results, comparative molecular dynamics and docking studies were carried out where a higher docking score (−10.218 kcal/mol) was observed in the mutated GK than wild‐type GK structure (−12.593 kcal/mol) indicating affinity variations for glucose. During the simulation process, glucose was expelled out from the mutant conformation but not from wild‐type GK, making glucose unavailable for phosphorylation. Therefore, these results conclusively explain hyperglycemic condition in this patient.

Mutations in exons 10 and 11 of human glucokinase result in conformational variations in the active site of the structure contributing to poor substrate binding – explains hyperglycemia in type 2 diabetic patients

Mutations in the glucokinase (GK) gene play a critical role in the establishment of type 2 diabetes. In our earlier study, R308K mutation in GK in a clinically proven type 2 diabetic patient showed, structural and functional variations that contributed immensely to the hyperglycemic condition. In the extension of this work, a cohort of 30 patients with established type 2 diabetic condition were chosen and the exons 10 and 11 of GK were PCR-amplified and sequenced. The sequence alignment showed A379S, D400Y, E300A, E395A, E395G, H380N, I348N, L301M, M298I, M381G, M402R, R308K, R394P, R397S, and S398R mutations in 12 different patients. The structural analysis of these mutated GKs, showed a variable number of β-α-β units, hairpins, β-bulges, strands, helices, helix–helix interactions, β-turns, and γ-turns along with the RMSD variations when compared to wild-type GK. Molecular modeling studies revealed that the substrate showed variable binding orientations and could not fit into the active site of these mutated structures; moreover, it was expelled out of the conformations. Therefore, these structural variations in GK due to mutations could be one of the strongest reasons for the hyperglycemic levels in these type 2 diabetic patients.

Structural Variations of Human Glucokinase Glu256Lys in MODY2 Condition Using Molecular Dynamics Study

Glucokinase (GK) is the predominant hexokinase that acts as glucose sensor and catalyses the formation of Glucose-6-phosphate. The mutations in GK gene influence the affinity for glucose and lead to altered glucose levels in blood causing maturity onset diabetes of the young type 2 (MODY2) condition, which is one of the prominent reasons of type 2 diabetic condition. In view of the importance of mutated GK resulting in hyperglycemic condition, in the present study, molecular dynamics simulations were carried out in intact and 256 E-K mutated GK structures and their energy values and conformational variations were correlated. Energy variations were observed in mutated GK (3500 Kcal/mol) structure with respect to intact GK (5000 Kcal/mol), and it showed increased γ-turns, decreased β-turns, and more helix-helix interactions that affected substrate binding region where its volume increased from 1089.152 Å2 to 1246.353 Å2. Molecular docking study revealed variation in docking scores (intact = −12.199 and mutated = −8.383) and binding mode of glucose in the active site of mutated GK where the involvement of A53, S54, K56, K256, D262 and Q286 has resulted in poor glucose binding which probably explains the loss of catalytic activity and the consequent prevailing of high glucose levels in MODY2 condition.

High-resolution crystal structures of two crystal forms of human cyclophilin D in complex with PEG 400 molecules

Cyclophilin D (CypD) is a key mitochondrial target for amyloid-β-induced mitochondrial and synaptic dysfunction and is considered a potential drug target for Alzheimers disease. The high-resolution crystal structures of primitive orthorhombic (CypD-o) and primitive tetragonal (CypD-t) forms have been determined to 1.45 and 0.85 Å resolution, respectively, and are nearly identical structurally. Although an isomorphous structure of CypD-t has previously been reported, the structure reported here was determined at atomic resolution, while CypD-o represents a new crystal form for this protein. In addition, each crystal form contains a PEG 400 molecule bound to the same region along with a second PEG 400 site in CypD-t which occupies the cyclosporine A inhibitor binding site of CypD. Highly precise structural information for CypD should be extremely useful for discerning the detailed interaction of small molecules, particularly drugs and/or inhibitors, bound to CypD. The 0.85 Å resolution structure of CypD-t is the highest to date for any CypD structure.