Complementary protocols to evaluate inhibitors against the DnaK chaperone network.

Bacterial DnaK belongs to the Hsp70 chaperone family, which plays a critical role in maintaining proteostasis by catalyzing protein folding, and is a proposed antibacterial target in the pathogen Mycobacterium tuberculosis. Here, we describe an experimental toolbox for evaluating inhibitors against the mycobacterial DnaK chaperone network: a coupled-enzymatic assay to monitor ATPase activity, a proteolytic cleavage assay to study DnaK conformational changes upon ligand addition, as well as a protein renaturation assay to assess chaperone function. For complete details on the use and execution of this protocol, please refer to Hosfelt et al. (2021).

A Dual-Pronged Approach: A Ruthenium(III) Complex That Modulates Amyloid-ß Aggregation and Disrupts Its Formed Aggregates.

Alzheimer's disease (AD) is a devastating neurological disorder for which soluble oligomers of the peptide amyloid-ß (Aß) are now recognized as the neurotoxic species. Metal-based therapeutics are uniquely suited to target Aß, with ruthenium-based (Ru) complexes emerging as propitious candidates. Recently, azole-based Ru(III) complexes were observed to modulate the aggregation of Aß in solution, where the inclusion of a primary amine proximal to the ligand coordination site improved the activity of the complexes. To advance these structure-activity relationships, a series of oxazole-based Ru complexes were prepared and evaluated for their ability to modulate Aß aggregation. From these studies, a lead candidate, Oc, emerged that had superior activity relative to its azole predecessors in modulating the aggregation of soluble Aß and diminishing its cytotoxicity. Further evaluation of Oc demonstrated its ability to disrupt formed Aß aggregates, resulting in smaller amorphous species. Because altering both sides of the aggregation equilibrium for Aß has not been previously suggested for metal-based complexes for AD, this work represents an exciting new avenue for improved therapeutic success.

Importance of Hydrogen Bonding: Structure-Activity Relationships of Ruthenium(III) Complexes with Pyridine-Based Ligands for Alzheimer's Disease Therapy.

Alzheimer's disease (AD) is the most common form of dementia, where one of the pathological hallmarks of AD is extracellular protein deposits, the primary component of which is the peptide amyloid-ß (Aß). Recently, the soluble form of Aß has been recognized as the primary neurotoxic species, making it an important target for therapeutic development. Metal-based drugs are promising candidates to target Aß, as the interactions with the peptide can be tuned by ligand design. In the current study, 11 ruthenium complexes containing pyridine-based ligands were prepared, where the functional groups at the para position on the coordinated pyridine ligand were varied to determine structure-activity relationships. Overall, the complexes with terminal primary amines had the greatest impact on modulating the aggregation of Aß and diminishing its cytotoxicity. These results identify the importance of specific intermolecular interactions and are critical in the advancement of metal-based drugs for AD therapy.

Ruthenium(III) complexes with imidazole ligands that modulate the aggregation of the amyloid-ß peptide via hydrophobic interactions.

Alzheimer's disease (AD) is the most common form of dementia, characterized by extracellular protein deposits, comprised primarily of the peptide amyloid-beta (Aß), are a pathological indicator of the disease. Commonly known as Aß plaques, these deposits contain a relatively high concentration of metals, making metallotherapeutics uniquely suited to target soluble Aß, thereby limiting its aggregation and cytotoxicity. Ruthenium-based complexes are promising candidates for advancement, as the complex PMRU20 (2-aminothiazolium [trans-RuCl4(2-aminothiazole)2]) and several thiazole-based derivatives were found to prevent the aggregation of Aß, with hydrogen-bonding functional groups improving their performance. Further investigation into the impact of the heteroatom in the azole ring on the activity of Ru complexes was achieved through the synthesis and evaluation of a small set of imidazole-based compounds. The ability of the complexes to prevent the aggregation of Aß was determined where the same sample was subjected to analysis by three complementary methods: ThT fluorescence, dynamic light scattering (DLS), and transmission electron microscopy (TEM). It was found that hydrophobic interactions, along with hydrogen-bonding via the imidazole nitrogen heteroatom, promoted interactions with the Aß peptide, thereby limiting its aggregation. Furthermore, it was found that having rapid and sequential exchange proved detrimental as it resulted in a decreased association with Aß. These results highlight important considerations between a balance of intermolecular interactions and ligand exchange kinetics in the design of further therapeutic candidates.

Ruthenium(iii) complexes containing thiazole-based ligands that modulate amyloid-ß aggregation.

Alzheimer's Disease (AD) is a devastating neurodegenerative disorder where one of the commonly observed pathological hallmarks is extracellular deposits of the peptide amyloid-ß (Aß). These deposits contain a high concentration of metals and initially presented a promising target for therapy; however it has become increasingly evident that the soluble form of the peptide is neurotoxic, not the amyloidogenic species. Metal-based therapeutics are uniquely suited to target soluble Aß and have shown considerable promise to prevent the aggregation and induced cytotoxicity of the peptide in vitro. Herein, we have prepared a small series of derivatives of two promising Ru(iii) complexes NAMI-A (imidazolium [trans-RuCl4(1H-imidazole)(dimethyl sulfoxide-S)]) and PMRU20 (2-aminothiazolium [trans-RuCl4(2-aminothiazole)2]), to determine structure-activity relationships (SAR) for Ru(iii) therapeutics for AD. Using the three complementary methods of Thioflavin T fluorescence, dynamic light scattering (DLS), and transmission electron microscopy (TEM), it was determined that the symmetry around the metal center did not significantly impact the activity of the complexes, but rather the attached thiazole ligand(s) mitigated Aß aggregation. Across both families of Ru(iii) complexes the determined SAR for the functional groups on the thiazole ligands to modulate Aß aggregation were NH2 > CH3 > H. These results highlight the importance of secondary interactions between the metallotherapeutic and the Aß peptide where hydrogen-bonding has the greatest impact on modulating Aß aggregation.