A Conditionally Fluorescent Peptide Reporter of Secondary Structure Modulation

Proteins containing intrinsic disorder often form secondary structure upon interaction with a binding partner. Modulating such structures presents an approach for manipulating the resultant functional outcomes. Translational repressor protein 4E‐BP1 is an example of an intrinsically disordered protein that forms an α‐helix upon binding to its protein ligand, eIF4E. Current biophysical methods for analyzing binding‐induced structural changes are low‐throughput, require large amounts of sample, or are extremely sensitive to signal interference by the ligand itself. Herein, we describe the discovery and development of a conditionally fluorescent 4E‐BP1 peptide that reports structural changes of its helix in high‐throughput format. This reporter peptide is based on conditional quenching of fluorescein by thioamides. In this case, fluorescence signal increases as the peptide becomes more ordered. Conversely, destabilization of the α‐helix results in decreased fluorescence signal. The low concentration and low volume of peptide required make this approach amenable for high‐throughput screening to discover ligands that alter peptide secondary structure.PET lights up peptide dynamics: Photoinduced electron transfer (PET) quenching of fluorescence by thioamides presents an elegant method for monitoring changes in macromolecular conformation. Here we apply this approach to monitor peptide dynamics in a 384‐well plate format. Using a fluorescein‐conjugated, 4E‐BP1‐based peptide containing an embedded thioamide, we probe its transition from disorder to a short α‐helix.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146852/1/cbic201800377.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146852/2/cbic201800377-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146852/3/cbic201800377_am.pd

Proteostasis perturbation of N-Myc leveraging HSP70 mediated protein turnover improves treatment of neuroendocrine prostate cancer

N-Myc is a key driver of neuroblastoma and neuroendocrine prostate cancer (NEPC). One potential way to circumvent the challenge of undruggable N-Myc is to target the protein homeostasis (proteostasis) system that maintains N-Myc levels. Here, we identify heat shock protein 70 (HSP70) as a top partner of N-Myc, which binds a conserved "SELILKR" motif and prevents the access of E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), possibly through steric hindrance. When HSP70's dwell time on N-Myc is increased by treatment with the HSP70 allosteric inhibitor, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites and forms polyubiquitination chains linked by the K11 and K63 sites. Notably, HSP70 inhibition significantly suppressed NEPC tumor growth, increased the efficacy of aurora kinase A (AURKA) inhibitors, and limited the expression of neuroendocrine-related pathways

Multivalent protein-protein interactions are pivotal regulators of eukaryotic Hsp70 complexes.

Heat shock protein 70 (Hsp70) is a molecular chaperone and central regulator of protein homeostasis (proteostasis). Paramount to this role is Hsp70's binding to client proteins and co-chaperones to produce distinct complexes, such that understanding the protein-protein interactions (PPIs) of Hsp70 is foundational to describing its function and dysfunction in disease. Mounting evidence suggests that these PPIs include both "canonical" interactions, which are universally conserved, and "non-canonical" (or "secondary") contacts that seem to have emerged in eukaryotes. These two categories of interactions involve discrete binding surfaces, such that some clients and co-chaperones engage Hsp70 with at least two points of contact. While the contributions of canonical interactions to chaperone function are becoming increasingly clear, it can be challenging to deconvolute the roles of secondary interactions. Here, we review what is known about non-canonical contacts and highlight examples where their contributions have been parsed, giving rise to a model in which Hsp70's secondary contacts are not simply sites of additional avidity but are necessary and sufficient to impart unique functions. From this perspective, we propose that further exploration of non-canonical contacts will generate important insights into the evolution of Hsp70 systems and inspire new approaches for developing small molecules that tune Hsp70-mediated proteostasis

Two distinct classes of cochaperones compete for the EEVD motif in heat shock protein 70 to tune its chaperone activities.

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions with many cochaperones. One "hotspot" for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70's EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70-CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70-DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein-protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded

Analogs of the Heat Shock Protein 70 Inhibitor MKT-077 Suppress Medullary Thyroid Carcinoma Cells

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the RET proto-oncogene. We previously demonstrated that depletion of the mitochondrial molecular chaperone, mortalin, can effectively suppress human MTC cells in culture and in mouse xenografts, by disrupting mitochondrial bioenergetics and subsequently inducing apoptosis and RET downregulation. Similar effects were induced by MKT-077, a water-soluble rhodocyanine dye analog known to inhibit mortalin, but with notable toxicity in animals. These observations led us to evaluate recently developed MKT-077 analogs that exhibited higher selectivity to HSP70 proteins and improved bioavailability. We validated the MTC cell-suppressive effects of mortalin depletion in three-dimensional cultures of the human MTC lines, TT, and MZ-CRC-1, and then evaluated different MKT-077 analogs in two- and three-dimensional cell cultures, to show that the MKT-077 analogs, JG-98 and JG-194, effectively and consistently inhibited propagation of TT and MZ-CRC-1 cells in these cultures. Of note, these compounds also effectively suppressed the viability of TT and MZ-CRC-1 progenies resistant to vandetanib and cabozantinib. Moreover, JG-231, an analog with improved microsomal stability, consistently suppressed TT and MZ-CRC-1 xenografts in mice. These data suggest that mortalin inhibition may have therapeutic potential for MTC

Analogs of the Heat Shock Protein 70 Inhibitor MKT-077 Suppress Medullary Thyroid Carcinoma Cells.

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the RET proto-oncogene. We previously demonstrated that depletion of the mitochondrial molecular chaperone, mortalin, can effectively suppress human MTC cells in culture and in mouse xenografts, by disrupting mitochondrial bioenergetics and subsequently inducing apoptosis and RET downregulation. Similar effects were induced by MKT-077, a water-soluble rhodocyanine dye analog known to inhibit mortalin, but with notable toxicity in animals. These observations led us to evaluate recently developed MKT-077 analogs that exhibited higher selectivity to HSP70 proteins and improved bioavailability. We validated the MTC cell-suppressive effects of mortalin depletion in three-dimensional cultures of the human MTC lines, TT, and MZ-CRC-1, and then evaluated different MKT-077 analogs in two- and three-dimensional cell cultures, to show that the MKT-077 analogs, JG-98 and JG-194, effectively and consistently inhibited propagation of TT and MZ-CRC-1 cells in these cultures. Of note, these compounds also effectively suppressed the viability of TT and MZ-CRC-1 progenies resistant to vandetanib and cabozantinib. Moreover, JG-231, an analog with improved microsomal stability, consistently suppressed TT and MZ-CRC-1 xenografts in mice. These data suggest that mortalin inhibition may have therapeutic potential for MTC

Chemical validation of a druggable site on Hsp27/HSPB1 using in silico solvent mapping and biophysical methods

Destabilizing mutations in small heat shock proteins (sHsps) are linked to multiple diseases; however, sHsps are conformationally dynamic, lack enzymatic function and have no endogenous chemical ligands. These factors render sHsps as classically "undruggable" targets and make it particularly challenging to identify molecules that might bind and stabilize them. To explore potential solutions, we designed a multi-pronged screening workflow involving a combination of computational and biophysical ligand-discovery platforms. Using the core domain of the sHsp family member Hsp27/HSPB1 (Hsp27c) as a target, we applied mixed solvent molecular dynamics (MixMD) to predict three possible binding sites, which we confirmed using NMR-based solvent mapping. Using this knowledge, we then used NMR spectroscopy to carry out a fragment-based drug discovery (FBDD) screen, ultimately identifying two fragments that bind to one of these sites. A medicinal chemistry effort improved the affinity of one fragment by ~50-fold (16 µM), while maintaining good ligand efficiency (~0.32 kcal/mol/non-hydrogen atom). Finally, we found that binding to this site partially restored the stability of disease-associated Hsp27 variants, in a redox-dependent manner. Together, these experiments suggest a new and unexpected binding site on Hsp27, which might be exploited to build chemical probes

Allosteric inhibition of HSP70 in collaboration with STUB1 augments enzalutamide efficacy in antiandrogen resistant prostate tumor and patient-derived models

Ubiquitin proteasome activity is suppressed in enzalutamide resistant prostate cancer cells, and the heat shock protein 70/STIP1 homology and U-box-containing protein 1 (HSP70/STUB1) machinery are involved in androgen receptor (AR) and AR variant protein stabilization. Targeting HSP70 could be a viable strategy to overcome resistance to androgen receptor signaling inhibitor (ARSI) in advanced prostate cancer. Here, we showed that a novel HSP70 allosteric inhibitor, JG98, significantly suppressed drug-resistant C4-2B MDVR and CWR22Rv1 cell growth, and enhanced enzalutamide treatment. JG98 also suppressed cell growth in conditional reprogramed cell cultures (CRCs) and organoids derived from advanced prostate cancer patient samples. Mechanistically, JG98 degraded AR/AR-V7 expression in resistant cells and promoted STUB1 nuclear translocation to bind AR-V7. Knockdown of the E3 ligase STUB1 significantly diminished the anticancer effects and partially restored AR-V7 inhibitory effects of JG98. JG231, a more potent analog developed from JG98, effectively suppressed the growth of the drug-resistant prostate cancer cells, CRCs, and organoids. Notably, the combination of JG231 and enzalutamide synergistically inhibited AR/AR-V7 expression and suppressed CWR22Rv1 xenograft tumor growth. Inhibition of HSP70 using novel small-molecule inhibitors coordinates with STUB1 to regulate AR/AR-V7 protein stabilization and ARSI resistance