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Based on the structural analysis of tricyclic scaffolds as butyrylcholinesterase (BuChE) inhibitors, a series of pyrazolo[1,5-c][1,3]benzoxazin-5(5H)-one derivatives were designed, synthesized and evaluated for their acetylcholinesterase (AChE) and BuChE inhibitory activity. Compounds with 5-carbonyl and 7- or/and 9-halogen substitutions showed potential BuChE inhibitory activity, among which compounds 6a, 6c and 6g showed the best BuChE inhibition (IC50 = 1.06, 1.63 and 1.63 µM, respectively). The structure-activity relationship showed that the 5-carbonyl and halogen substituents significantly influenced BuChE activity. Compounds 6a and 6g were found nontoxic, lipophilic and exhibited remarkable neuroprotective activity and mixed-type inhibition against BuChE (Ki = 7.46 and 3.09 µM, respectively). Docking studies revealed that compound 6a can be accommodated into BuChE via five hydrogen bonds, one Pi-Sigma interaction and three Pi-Alkyl interactions. [ABSTRACT FROM AUTHOR]

The present review article presents the available synthetic routes for the preparation of different types of imidazothiadiazines. The classification of imidazothiadiazines into seven types is based on the position of the linkage between the imidazole and the thiadiazine rings. This review is meant to be a guide for both synthetic and medicinal chemists to discover and design new imidazothiadiazine derivatives for medical purposes. [ABSTRACT FROM PUBLISHER]

Abstract: What changes occur when a natural protein that had been under low mutation rates is subjected to a neutral drift at high mutational loads, thus generating genetically diverse (polymorphic) gene ensembles that all maintain the protein''s original function and structure? To address this question we subjected large populations of TEM-1 β-lactamase to a prolonged neutral drift, applying high mutation rates and purifying selection to maintain TEM-1''s existing penicillinase activity. Purging of deleterious mutations and enrichment of beneficial ones maintained the sequence of these ensembles closer to TEM-1''s family consensus and inferred ancestor. In particular, back-to-consensus/ancestor mutations that increase TEM-1''s kinetic and thermodynamic stability were enriched. These acted as global suppressors and enabled the tolerance of a broad range of deleterious mutations, thus further increasing the genetic diversity of the drifting populations. The probability of a new function emerging (cefotaxime degradation) was also substantially increased in these ensembles owing to the presence of many gene variants carrying the global suppressors. Our findings indicate the unique features of large, polymorphic neutral ensembles generated under high mutational loads and prompt the speculation that the progenitors of today''s proteins may have evolved under high mutational loads. The results also suggest that predictable back-to-consensus/ancestor changes can be used in the laboratory to generate highly diverse and evolvable gene libraries. [Copyright &y& Elsevier]

Abstract: Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrPSc). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90–105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrPSc is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95–104) peptide by modeling the corresponding mutations. [Copyright &y& Elsevier]