Thioflavin T (ThT) dye fluorescence is used regularly to quantify the formation and inhibition of amyloid fibrils in the presence of anti-amyloidogenic compounds such as polyphenols. However, in this study, it was shown, using three polyphenolics (curcumin, quercetin and resveratrol), that ThT fluorescence should be used with caution in the presence of such exogenous compounds. The strong absorptive and fluorescent properties of quercetin and curcumin were found to significantly bias the ThT fluorescence readings in both in situ real-time ThT assays and single time-point dilution ThT-type assays. The presence of curcumin at concentrations as low as 0.01 and 1 lm was sufficient to interfere with the ThT fluorescence associated with fibrillar amyloid-b(1-42) (0.5 lm) and fibrillar reduced and carboxymethylated j-casein (50 lm), respectively. The ThT fluorescence associated with fibrillar amyloid-b(1-42) was also biased using higher concentrations of resveratrol, a polyphenol that is not spectroscopically active at the wavelengths of ThT fluorescence, implying that there can be direct interactions between ThT and the exogenous compound and ⁄ or competitive binding with ThT for the fibrils. Thus, in all cases where ThT is used in the presence of an exogenous compound, biases for amyloid-associated ThT fluorescence should be tested, regardless of whether the additive is spectroscopically active. Simple methods to conduct these tests were described. The Congo red spectral shift assay is demonstrated as a more viable spectrophotometric alternative to ThT, but allied methods, such as transmission electron microscopy, should also be used to assess fibril formation independently of dye-based assays. Abbreviations CR, congo red; RCMj-CN, reduced and carboxymethylated j-casein; TEM, transmission electron microscopy; ThT, thioflavin T.

Fragment-based approaches to finding novel small molecules that bind to proteins are now firmly established in drug discovery and chemical biology. Initially developed primarily in a few centers in the biotech and pharma industry, this methodology has now been adopted widely in both the pharmaceutical industry and academia. After the initial success with kinase targets, the versatility of this approach has now expanded to a broad range of different protein classes. Herein we describe recent fragment-based approaches to a wide range of target types, including Hsp90, β-secretase, and allosteric sites in human immunodeficiency virus protease and fanesyl pyrophosphate synthase. The role of fragment-based approaches in an academic research environment is also examined with an emphasis on neglected diseases such as tuberculosis. The development of a fragment library, the fragment screening process, and the subsequent fragment hit elaboration will be discussed using examples from the literature.

The polyphenol (-)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-beta, alphasynuclein and huntingtin, the amyloid-fibril-forming proteins involved in Alzheimer's, Parkinson's and Huntington's diseases, respectively. EGCG redirects the aggregation of these polypeptides to a disordered offfolding pathway that results in the formation of non-toxic amorphous aggregates. whether this anti-fibril activity is specific to these disease-related target proteins or ismore generic remains to be established. In addition, the mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic polyphenols, remains unclear. To address these aspects, we have investigated the ability of EGCG to inhibit amyloidogenesis of the generic model fibril-forming protein RCMkappa-CN (reduced and carboxymethylated kappa-casein) and thereby protect pheochromocytoma-12 cells from RCMkappa-CN amyloid-induced toxicity. We found that EGCG potently inhibits in vitro fibril formation byRCMkappa-CN [the IC50 for 50 uM RCMkappa-CN is 1 uM]. Biophysical studies reveal that EGCG prevents RCMkappa-CN fibril formation by stabilising RCMkappa-CN in its nativelike state rather than by redirecting its aggregation to the disordered, amorphous aggregation pathway. Thus, while it appears that EGCG is a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves this inhibition is specific to the target fibril-forming polypeptide. It is proposed that EGCG is directed to the amyloidogenic sheet-turn-sheet motif of monomeric RCMkappa-CN with high affinity by strong non-specific hydrophobic associations. Additional non-covalent pi-pi stacking interactions between the polyphenolic and aromatic residues common to the amyloidogenic sequence are also implicated. The polyphenol (−)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-β, α-synuclein and huntingtin, the amyloid-fibril-forming proteins involved in Alzheimer's, Parkinson's and Huntington's diseases, respectively. EGCG redirects the aggregation of these polypeptides to a disordered off-folding pathway that results in the formation of non-toxic amorphous aggregates. Whether this anti-fibril activity is specific to these disease-related target proteins or is more generic remains to be established. In addition, the mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic polyphenols, remains unclear. To address these aspects, we have investigated the ability of EGCG to inhibit amyloidogenesis of the generic model fibril-forming protein RCMκ-CN (reduced and carboxymethylated κ-casein) and thereby protect pheochromocytoma-12 cells from RCMκ-CN amyloid-induced toxicity. We found that EGCG potently inhibits in vitro fibril formation by RCMκ-CN [the IC 50 for 50 μM RCMκ-CN is 13 ± 1 μM]. Biophys...

No abstract

The essential enzyme CYP121 is a target for drug development against antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to bind to CYP121 using a cascade of biophysical assays. Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 μM). Deconstruction of 2 into its component retrofragments allowed the group efficiency of structural motifs to be assessed, the identification of more LE scaffolds for optimization and highlighted binding affinity hotspots. Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands. Elaboration of these compounds to target binding hotspots in the distal active site afforded compounds with excellent selectivity against human drug-metabolizing P450s. Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV–vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.