Although therapeutically targeting a single signaling pathway that drives tumor development and/or progression has been effective for a number of cancers, in many cases this approach has not been successful. Targeting networks of signaling pathways, instead of isolated pathways, may overcome this problem, which is probably due to the extreme heterogeneity of human tumors. However, the possibility that such networks may be spatially arranged in specialized subcellular compartments is not often considered in pathway-oriented drug discovery and may influence the design of new agents. Hsp90 is a chaperone protein that controls the folding of proteins in multiple signaling networks that drive tumor development and progression. Here, we report the synthesis and properties of Gamitrinibs, a class of small molecules designed to selectively target Hsp90 in human tumor mitochondria. Gamitrinibs were shown to accumulate in the mitochondria of human tumor cell lines and to inhibit Hsp90 activity by acting as ATPase antagonists. Unlike Hsp90 antagonists not targeted to mitochondria, Gamitrinibs exhibited a “mitochondriotoxic” mechanism of action, causing rapid tumor cell death and inhibiting the growth of xenografted human tumor cell lines in mice. Importantly, Gamitrinibs were not toxic to normal cells or tissues and did not affect Hsp90 homeostasis in cellular compartments other than mitochondria. Therefore, combinatorial drug design, whereby inhibitors of signaling networks are targeted to specific subcellular compartments, may generate effective anticancer drugs with novel mechanisms of action.

Pompe disease (PD) is a metabolic myopathy due to the deficiency of the lysosomal enzyme α-glucosidase (GAA). The only approved treatment for this disorder, enzyme replacement with recombinant human GAA (rhGAA), has shown limited therapeutic efficacy in some PD patients. Pharmacological chaperone therapy (PCT), either alone or in combination with enzyme replacement, has been proposed as an alternative therapeutic strategy. However, the chaperones identified so far also are active site-directed molecules and potential inhibitors of target enzymes. We demonstrated that N-acetylcysteine (NAC) is a novel allosteric chaperone for GAA. NAC improved the stability of rhGAA as a function of pH and temperature without disrupting its catalytic activity. A computational analysis of NAC–GAA interactions confirmed that NAC does not interact with GAA catalytic domain. NAC enhanced the residual activity of mutated GAA in cultured PD fibroblasts and in COS7 cells overexpressing mutated GAA. NAC also enhanced rhGAA efficacy in PD fibroblasts. In cells incubated with NAC and rhGAA, GAA activities were 3.7–8.7-fold higher than those obtained in cells treated with rhGAA alone. In a PD mouse model the combination of NAC and rhGAA resulted in better correction of enzyme activity in liver, heart, diaphragm and gastrocnemia, compared to rhGAA alone.

The self-organization of peptides into amyloidogenic oligomers is one of the key events for a wide range of molecular and degenerative diseases. Atomic-resolution characterization of the mechanisms responsible for the aggregation process and the resulting structures is thus a necessary step to improve our understanding of the determinants of these pathologies. To address this issue, we combine the accelerated sampling properties of replica exchange molecular dynamics simulations based on the OPEP coarse-grained potential with the atomic resolution description of interactions provided by all-atom MD simulations, and investigate the oligomerization process of the GNNQQNY for three system sizes: 3-mers, 12-mers and 20-mers. Results for our integrated simulations show a rich variety of structural arrangements for aggregates of all sizes. Elongated fibril-like structures can form transiently in the 20-mer case, but they are not stable and easily interconvert in more globular and disordered forms. Our extensive characterization of the intermediate structures and their physico-chemical determinants points to a high degree of polymorphism for the GNNQQNY sequence that can be reflected at the macroscopic scale. Detailed mechanisms and structures that underlie amyloid aggregation are also provided. The formation of amyloid fibrils is associated with many neurodegenerative diseases such as Alzheimer's, Creutzfeld-Jakob, Parkinson's, the Prion disease and diabetes mellitus. In all cases, proteins misfold to form highly ordered insoluble aggregates called amyloid fibrils that deposit intra- and extracellularly and are resistant to proteases. All proteins are believed to have the instrinsic capability of forming amyloid fibrils that share common specific structural properties that have been observed by X-ray crystallography and by NMR. However, little is known about the aggregation dynamics of amyloid assemblies, and their toxicity mechanism is therefore poorly understood. It is believed that small amyloid oligomers, formed on the aggregation pathway of full amyloid fibrils, are the toxic species. A detailed atomic characterization of the oligomerization process is thus necessary to further our understanding of the amyloid oligomer's toxicity. Our approach here is to study the aggregation dynamics of a 7-residue amyloid peptide GNNQQNY through a combination of numerical techniques. Our results suggest that this amyloid sequence can form fibril-like structures and is polymorphic, which agrees with recent experimental observations. The ability to fully characterize and describe the aggregation pathway of amyloid sequences numerically is key to the development of future drugs to target amyloid oligomers.

Molecular switching and ligand-based modulation of the 90-kDa heat-shock protein (Hsp90) chaperone activity may ultimately facilitate conformational coupling to the ATPase cycle along with activation and recruitment of the broad range of client proteins. We present an atomic resolution analysis of the Hsp90 N-terminal domain (NTD) binding energy landscape by simulating protein dynamics with a range of binding partners. We show that the activity of the molecular chaperone may be linked to ( i ) local folding-unfolding transitions and conformational switching of the “active site lid” upon binding and ( ii ) differences in the underlying protein dynamics as a function of the binding partner. This study suggests that structural plasticity of the Hsp90 NTD can be exploited by the molecular chaperone machinery to modulate enhanced structural rigidity during ATP binding and increased protein flexibility as a consequence of the inhibitor binding. The present study agrees with the experimental structural data and provides a plausible molecular model for understanding mechanisms of modulation of molecular chaperone activities by binding partners.