Protein homeostasis is one of the key mechanisms that determine cellular life, and the Hsp90 family of molecular chaperones plays a key role in it. While Hsp90 dysregulation is a hallmark of numerous diseases, ranging from cancer to neurodegeneration, traditional inhibitors targeting its highly conserved ATPase site have largely failed in the clinic due to off-target toxicity and compensatory stress responses. One of the challenges in drug discovery, as well as in the development of chemical tools to investigate the specific roles of single family members, lies in achieving isoform specificity across the cytoplasm, endoplasmic reticulum, and mitochondria.Here, we exploit the intrinsic asymmetry of mitochondrial isoform TRAP1 and combine it with a fragment-design inspired approach to develop new possible TRAP1 targeting leads. We start from the consideration that the TRAP1 catalytic cycle relies on a strained, asymmetric dimer conformation that enforces sequential ATP hydrolysis. By integrating advanced computational dynamics with biochemical profiling, we demonstrate that small molecules can be rationally designed to target these transient asymmetric states. Our findings reveal that targeting allosteric, symmetry-breaking interfaces allows for the modulation of TRAP1, offering a novel platform and starting point for next-generation, isoform-specific anticancer therapeutics.

The molecular chaperone HSP90 drives the folding and activation of a broad spectrum of client proteins through dynamic, transient multiprotein assemblies. Although HSP90 has been widely pursued as an anticancer target, ATP-competitive inhibitors can trigger indiscriminate client depletion and protective heat shock responses, limiting clinical utility. Here, we report a client-directed strategy to perturb HSP90-dependent maturation by targeting unstable client regions that act as transient recognition sites for chaperone networks. Using energy-decomposition analysis of the ligand-binding domain of the stringent HSP90 client glucocorticoid receptor (GR), we identified surface-exposed, weakly coupled substructures predicted to sample locally unfolded conformations. We translated these motifs into a set of short GR-derived peptide mimics designed to compete with chaperone engagement events required for productive GR folding. The peptides selectively bind purified HSP90, are cell permeable, and induce GR degradation in triple-negative breast cancer cells. Functionally, the most active mimic potentiates paclitaxel response under hormone-supplemented conditions and suppresses glucocorticoid-induced tumor cell quiescence. These results establish a framework that integrates computation-guided identification of client unfolding regions to the rational design of chemical probes that modulate chaperone-dependent signaling pathways, with relevance for cancer progression. More broadly, our study illustrates how targeting weak, client-specific interactions within proteostasis assemblies can yield new entry points for therapeutic development.

Mitochondrial chaperonin Heat Shock Protein 60 kDa (Hsp60) oversees the correct folding of client proteins in cooperation with Hsp10. Hsp60 monomers M first form 7-meric Single rings (S), which then pair into 14-meric Double rings (D) that accommodate clients in their lumen. Recruitment of 7 Hsp10 molecules per pole yields a sealed 28-meric Football-shaped complex (F). ATP hydrolysis in each Hsp60 unit drives client folding and F disassembly. The V72I mutation in hereditary spastic paraplegia form SPG13 impairs Hsp60 function despite being distant from the active site. We here investigate this impairment with atomistic molecular dynamics (MD) simulations of M, S, D, and F for both WT and mutant Hsp60, considering catalytic aspartates in D and F in different protonation states (even simulating one such state of D post-hydrolysis). Our findings show that-as observed experimentally-V72I rigidifies Hsp60 assemblies, significantly impacting internal dynamics. In monomers, V72I introduces a new allosteric route that bypasses the ATP binding site and affects mechanisms driving reactivity. These insights highlight a multiscale complexity of Hsp60 that could inspire the design of experiments to better understand both its WT and V72I variants.

Essential chaperones heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) collaborate in oncoprotein folding. Dual inhibition of these chaperones has shown synergy in preclinical studies but remains challenging to achieve. Using a computational approach, we designed peptides mimicking the predicted unfolding regions of Kinase CDK4, a client protein of both Hsp70 and Hsp90. Peptide Cdk4-2 is shown to simultaneously bind Hsp70, Hsp90, and co-chaperone Cdc37. Cdk4-2 is membrane permeable, inhibits CDK4-mediated retinoblastoma phosphorylation, and induces apoptosis in renal carcinoma cells. Structure-function studies identified a minimal pharmacophore for Hsp70 binding and critical interactions for peptide affinity. These findings demonstrate the feasibility of rationally designing multi-target modulators of chaperone networks. Cdk4-2 is a promising lead for therapeutic development, expanding the molecular space of modulators of cancer-associated multiprotein machineries. While focused on chaperones, the idea behind our strategy is general and immediately transferable to other multiprotein targets and networks.

Adenosine 5'-triphosphate (ATP) hydrolysis is one of the most significant reactions in biochemistry. In chaperone proteins, energy released by hydrolysis enables them to carry out their function and help other proteins (called "clients") to fold into their functional form. Here, we run Density Functional Theory calculations on three cluster models of the Hsp60 active site extracted from our previous molecular dynamics simulations of the 14-meric Hsp60 double-ring complex: our aim is to qualitatively investigate the mechanisms of ATP hydrolysis in different scenarios where the chaperone closes a dyad composed of catalytic aspartates Asp50 and Asp397. Since dyad closure raises Asp pKa values and increases likelihood of protonation, we modeled the active site both in the presence and absence of a proton. Comparison of reaction barriers suggests that hydrolysis is favored when aspartates become deprotonated, explaining increased ATPase activity observed in V72I mutant Hsp60 (known to favor dyad closure).

Heat Shock Protein 60 kDa (Hsp60) is a mitochondrial chaperonin that cooperates with Hsp10 to drive the correct folding of client proteins. Monomers M of Hsp60 (featuring equatorial, intermediate, and apical domains) first assemble into 7-meric Single rings ( S ), then pairs of S interface equatorially to form 14-meric Double rings ( D ) that accommodate clients into their lumen. Recruitment of 7 Hsp10 molecules per pole turns D into a 28-meric Football-shaped complex ( F ). Sequential hydrolysis of ATP present in each Hsp60 unit of F finally drives client folding and F disassembly. Equatorial domain mutation V72I occurs in SPG13, a form of hereditary spastic paraplegia: while distal to the active site, this severely impairs the chaperone cycle and stability. To understand the molecular bases of this impairment we have run atomistic molecular dynamics (MD) simulations of M , S , D , and F for both WT and mutant Hsp60, with two catalytically relevant Hsp60 aspartates in D and F modelled in three different protonation states. Additionally, D in one protonation state was modelled post-hydrolysis (total production time: 36 µs). By combining complementary experimental and computational approaches for the analysis of functional dynamics and allosteric mechanisms, we consistently find that mutation V72I significantly rewires allosteric routes present in WT Hsp60 across its complexes, from isolated M units right up to F , rigidifying them—as observed experimentally—by introducing a direct allosteric link between equatorial and apical Hsp60 domains that bypasses the ATP binding site (wherein we observe the alteration of mechanisms driving reactivity). Our results reveal a multiscale complexity of functional mechanisms for Hsp60 and its pathogenic mutant, and may lay the foundation for the design of experiments to fully understand both variants. ### Competing Interest Statement The authors have declared no competing interest.

Protein-protein interactions (PPIs) have emerged in the past years as significant pharmacological targets in the development of new therapeutics due to their key roles in determining pathological pathways. Herein, we present fragments on energy surfaces, a simple and general design strategy that integrates the analysis of the dynamic and energetic signatures of proteins to unveil the substructures involved in PPIs, with docking, selection, and combination of drug-like fragments to generate new PPI inhibitor candidates. Specifically, structural representatives of the target protein are used as inputs for the blind physics-based prediction of potential protein interaction surfaces using the matrix of low coupling energy decomposition method. The predicted interaction surfaces are subdivided into overlapping windows that are used as templates to direct the docking and combination of fragments representative of moieties typically found in active drugs. This protocol is then applied and validated using structurally diverse, important PPI targets as test systems. We demonstrate that our approach facilitates the exploration of the molecular diversity space of potential ligands, with no requirement of prior information on the location and properties of interaction surfaces or on the structures of potential lead compounds. Importantly, the hit molecules that emerge from our ab initio design share high chemical similarity with experimentally tested active PPI inhibitors. We propose that the protocol we describe here represents a valuable means of generating initial leads against difficult targets for further development and refinement.

c-Src tyrosine kinase is a renowned key intracellular signaling molecule and a potential target for cancer therapy. Secreted c-Src is a recent observation, but how it contributes to extracellular phosphorylation remains elusive. Using a series of domain deletion mutants, we show that the N-proximal region of c-Src is essential for its secretion. The tissue inhibitor of metalloproteinases 2 (TIMP2) is an extracellular substrate of c-Src. Limited proteolysis-coupled mass spectrometry and mutagenesis studies verify that the Src homology 3 (SH3) domain of c-Src and the P31VHP34 motif of TIMP2 are critical for their interaction. Comparative phosphoproteomic analyses identify an enrichment of PxxP motifs in phosY-containing secretomes from c-Src-expressing cells with cancer-promoting roles. Inhibition of extracellular c-Src using custom SH3-targeting antibodies disrupt kinase-substrate complexes and inhibit cancer cell proliferation. These findings point toward an intricate role for c-Src in generating phosphosecretomes, which will likely influence cell-cell communication, particularly in c-Src-overexpressing cancers.

Herein we examine the determinants of the allosteric inhibition of the mitochondrial chaperone TRAP1 by a small molecule ligand. The knowledge generated is harnessed into the design of novel derivatives with interesting biological properties. TRAP1 is a member of the Hsp90 family of proteins, which work through sequential steps of ATP processing coupled to client-protein remodeling. Isoform selective inhibition of TRAP1 can provide novel information on the biomolecular mechanisms of molecular chaperones, as well as new insights into the development of small molecules with therapeutic potential. Our analysis of the interactions between an active first-generation allosteric ligand and TRAP1 shows how the small molecule induces long-range perturbations that influence the attainment of reactive poses in the active site. At the same time, the dynamic adaptation of the allosteric binding pocket to the presence of the first-generation compound sets the stage for the design of a set of second-generation ligands: the characterization of the formation/disappearance of pockets around the allosteric site that is used to guide optimize the ligands' fit for the allosteric site and improve inhibitory activities. The effects of the newly designed molecules are validated experimentally in vitro and in vivo. We discuss the implications of our approach as a promising strategy towards understanding the molecular determinants of allosteric regulation in chemical and molecular biology, and towards speeding up the design of allosteric small molecule modulators.

Computational chemistry has come of age in drug discovery. Indeed, most pharmaceutical development programs rely on computer-based data and results at some point. Herein, we discuss recent applications of advanced simulation techniques to difficult challenges in drug discovery. These entail the characterization of allosteric mechanisms and the identification of allosteric sites or cryptic pockets determined by protein motions, which are not immediately evident in the experimental structure of the target; the study of ligand binding mechanisms and their kinetic profiles; and the evaluation of drug-target affinities. We analyze different approaches to tackle challenging and emerging biological targets. Finally, we discuss the possible perspectives of future application of computation in drug discovery.