KEY RECENT Publication ABSTRACTS
De novo design, synthesis, and mechanistic evaluation of short peptides that mimic heat shock protein 27 activity
Jessica Khoa, P. Chi Phama, Suhyeon Kwona, Alana Y. Huang a, Joel P. Riversa, Huixin Wanga, Heath Ecroydb, and W. Alexander Donalda, Shelli R. McAlpine*c ACS Med. Chem. Lett. V12 p713-719 2021 DOI: 10.1021/acsmedchemlett.0c0060
We report the first small molecule peptides based on the N-terminal sequence of Heat shock protein 27 (Hsp27, gene HSPB1) that demonstrates chaperone-like activity. The peptide, comprising of the SWDPF sequence located at Hsp27’s amino (N)-terminal domain, directly regulates protein aggregation events, maintaining the disaggregated state of the model protein, citrate synthase. While traditional inhibitors of protein aggregation act via regulation of a protein that facilitates aggregation or disaggregation, our molecules are the first small peptides between 5-8 amino acids in length, that are based on the N-terminus of Hsp27 and directly control protein aggregation. The presented strategy showcases a new approach for developing small peptides that control protein aggregation in proteins with high aggregate levels, making them a useful approach in developing new drugs.
Using NMR to identify binding regions for N and C-terminal Hsp90 inhibitors using Hsp90 domains
Jeanette R. McConnell, H. Jane Dyson*, Shelli R. McAlpine* RSC Med. Chem. V12, p410-415 2021
We present the first NMR study of the interaction between heat shock protein 90 (Hsp90) and amino (N)-terminal inhibitors 17-AAG, AUY922, and Carboxy (C)-terminal modulators SM253, and LB51. We show that the two ATP mimics, 17-AAG and AUY922, bind deeply within the ATP binding pocket of the N-terminal domain, consistent with the crystal structures. In contrast, SM253, a C-terminal Hsp90 modulator, binds to the linker region between the N and middle domains. We also show that C-terminal inhibitor LB51 binds to the C-terminus with a more significant spectroscopic change than previously reported using NMR binding studies of C-terminal inhibitors Novobiocin and Silybin. These data provide key insights into how the allosteric inhibitor SM253 controls the C-terminal co-chaperones and confirms the binding domain of LB51.
Cyclic peptides as drugs for intracellular targets: the next frontier in peptide therapeutic development Laura K. Buckton, Marwa N. Rahimi, and Shelli R. McAlpine* , Chem. Eur. J. V27, p1487-1513 2021 DOI:10.1002/chem.201905385
Developing macrocyclic peptides that can reach intracellular targets is a significant challenge. This review discusses the most recent strategies used to develop cell permeable cyclic peptides that maintain binding to their biological target inside the cell. Macrocyclic peptides are unique from small molecules because traditional calculated physical properties are unsuccessful for predicting cell membrane permeability. Peptide synthesis and experimental membrane permeability is the only strategy that effectively differentiates between cell permeable and cell impermeable molecules. Discussed are chemical strategies, including backbone N-methylation and stereochemical changes, which have produced molecular scaffolds with improved cell permeability. However, these improvements often come at the expense of biological activity as chemical modifications alter the peptide conformation, frequently impacting the compound’s ability to bind to the target. Highlighted is the most promising approach, which involves side chain alterations that improve cell permeability without impact binding events.
Real time monitoring of peptide delivery in vitro using high payload pH responsive nanogels Shegufta Farazi, Fan Chen, Henry Foster,Raelene Boquiren, Shelli R. McAlpine and Robert Chapman *Polym. Chem.V11, p425-432 2020, DOI: 10.1039/C9PY01120J
Nanogels are attractive delivery vehicles for small hydrophilic cargo, such as peptides, but there is a limited understanding of how the structure of both the nanogel and cargo affect the drug loading and release properties, particularly in biological environments. We have used Förster resonance energy transfer(FRET) to study the loading and release behaviour of a series of hydrophilic charged peptides (SNKAY, SNKKY and SNDDY) in a set of pH-responsive methacrylic acid (pMAA) core crosslinked nanogels that were prepared through miniemulsion polymerisation from a PEGMEMA–DMAEMA–tBuMA terblockcopoylmer. Our nanogels show an extremely high loading capacity of the positively charged peptides (400–800 wt% in the best cases), absorbing them from solution at pH 7.4 without any need for purification. At pH values below 6, the peptide is rapidly expelled from the nanogel due to the collapse of the core and protonation of the positively charged inner shell. By combining FRET with fluorescence lifetime imaging microscopy (FLIM), we were able to monitor this in vitro and found that most of the drug is released within the first 10 min after cell uptake.
C-terminal Hsp90 inhibitors block the HIF-1 hypoxic response by degrading HIF-1a through the oxygen-dependent degradation pathway Nalin Kataria, Chloe-Anne Martinez, Bernadette Kerr, Samantha S. Zaiter, Monica Morgan, Shelli R. McAlpine and Kristina M Cook* Cell. Physiol. Biochem V53, p480-495 2019 DOI: 10.33594/000000152.
Hypoxia Inducible Factor-1α (HIF-1α) is involved in cancer progression and is stabilized by the chaperone HSP90 (Heat Shock Protein 90), preventing degradation. Previously identified HSP90 inhibitors bind to the N-terminal pocket of HSP90, which blocks binding to HIF-1α and induces HIF-1α degradation. N-terminal inhibitors have failed in the clinic as single therapy treatments partially because they induce a heat shock response. SM molecules are HSP90 inhibitors that bind to the C-terminus of HSP90 and do not induce a heat shock response. The effects of these C-terminal inhibitors on HIF-1α are unreported. Results: We show that SM compounds decrease HIF-1α target expression at the mRNA and protein level under hypoxia in colorectal, breast and prostate cancer cells, leading to cell death, without inducing a heat shock response. Surprisingly, we found that when the C-terminal of HSP90 is inhibited, HIF-1α degradation occurs through the proteasome and prolyl hydroxylases in an oxygen-dependent manner even in very low levels of oxygen (tumor hypoxia levels). RACK1 was not required for proteasomal degradation of HIF-1α. Conclusion: Our results suggest that by targeting the C-terminus of HSP90 we can exploit the prolyl hydroxylase and proteasome pathway to induce HIF-1α degradation in hypoxic tumors.
Polymer mediate transport of the Hsp90 inhibitor LB76, a polar resulting cyclic peptide, produces an Hsp90 cellular phenotype Marwa N. Rahimi, Henry G. Foster, Shegufta N. Farazi, Robert Chapman*, and Shelli R. McAlpine* Chem Commun, V55, p4515-4518 2019 DOI:10.1039/C9CC00890J
LB76 is a cyclic peptide that shows great promise as a selective heat shock protein 90 (Hsp90) inhibitor. However despite strong binding to and inhibition of Hsp90 in cell lysate its polar structure prevents it from crossing the cell membrane. We have developed a pH responsive polymer nanoparticle that effectively encapsulates LB76 from solution without need for purification. The nanoparticle releases the molecule upon crossing the cell membrane. Treatment of human colon cancer HCT116 cells with nanoparticle laden with LB76 produce the typical phenotype associated with Hsp90 inhibition, providing evidence of a therapeutically active payload.
Delivering bioactive cyclic peptides that target Hsp90 as prodrugs Yuantao Huo, Laura K. Buckton, Jack L. Bennett, Eloise C. Smith, Frances, L. Byrne, Kyle L. Hoehn, Marwa N. Rahimi, and Shelli R. McAlpine* J. Enzyme Inhib. Med. Chem.V34, p728-739, 2019 DOI: 10.1080/14756366.2019.1580276
The most challenging issue facing peptide drug development is producing a molecule with optimal physical properties while maintaining target binding affinity. Masking peptides with protecting groups that can be removed inside the cell, produces a cell permeable peptide, which, theoretically can maintain its biological activity. Described are series of prodrugs masked using: (a) O-alkyl, (b) N-alkyl, and (c) acetyl groups, and their binding affinity for Hsp90. Alkyl moieties increased compound permeability, Papp, from 3.3 to 5.6, however alkyls could not be removed by liver microsomes or in-vivo and their presence decreased target binding affinity (IC50 of ≥ 10 µM). Thus, unlike small molecules, peptide masking groups cannot be predictably removed; their removal is related to the 3-D conformation. O-Acetyl groups were cleaved but are labile, increasing challenges during synthesis. Utilizing acetyl groups coupled with mono-methylated amines may decrease the polarity of a peptide, while maintaining binding affinity.
Protein-protein inhibitors designed de-novo to target the C-terminus of Hsp90 block co-chaperone activity Marwa N. Rahimi and Shelli R. McAlpine* Chem. Commun. V55, p846-849, 2019 DOI: 10.1039/C8CC07576J
Protein-protein interactions control all cellular functions. Presented is the first de-novo designed protein-protein inhibitor that targets the C-terminus of Heat shock protein 90 (Hsp90) and blocks co-chaperones from binding. Compound LB76, which was created from an Hsp90 co-chaperone, selectively pulls down Hsp90 from cell lysates, binds to Hsp90’s C-terminal domain, and blocks the interactions between Hsp90 and TPR-containing co-chaperones. Through these interactions, LB76 inhibits the protein-folding function of Hsp90. Blocking these protein-protein interactions betweeen Hsp90 and C-terminal co-chaperones regulate the cell’s entire protein-folding machinery.
Designing de novo small molecules that control Heat shock protein 70 within the chaperone machinery Samantha S. Zaiter, Yuantao Huo, Fong Ying Tiew, Jason E. Gestwicki, and Shelli R. McAlpine* J. Med. Chem. V62, p742-761, 2019 DOI: 10.1021/acs.jmedchem.8b01436
Protein-protein interactions (PPIs) regulate all signalling pathways for cellular function. Developing molecules that modulate PPIs through the interface of their protein surfaces has been a significant challenge and there has been little success controlling PPI’s through standard molecular library screening approaches. PPIs control the cell’s protein-folding machinery, and this machinery relies on a multiprotein complex formed with heat shock protein 70 (Hsp70). Described is the design, synthesis and biological evaluation of molecules aimed to regulate the interaction between two proteins that are critical to the protein-folding machinery: heat shock protein 70 (Hsp70) and co-chaperone heat shock organizing protein (HOP). We report the first class of compounds that directly regulate these two protein-protein interactions and inhibit protein folding events.
Nanoparticles for bioapplications: Study of the cytotoxicity of water dispersible CdSe(S) and CdSe(S)/ZnO Quantum Dots F Mirnajafizadeh, D. Ramsey, S. R. McAlpine, F. Wang, P. Reece, J. Stride* Nanomaterials, V9 p465 2019 DOI: 10.3390/nano9030465
Semiconductor nanocrystals or quantum dots (QDs) have unique optical and physical properties that make them potential imaging tools in biological and medical applications. However, concerns over the aqueous dispersivity, toxicity to cells, and stability in biological environments may limit the use of QDs in such applications. Here, we report an investigation into the cytotoxicity of aqueously dispersed CdSe(S) and CdSe(S)/ZnO core/shell QDs in the presence of human colorectal carcinoma cells (HCT-116) and a human skin fibroblast cell line (WS1). The cytotoxicity of the precursor solutions used in the synthesis of the CdSe(S) QDs was also determined in the presence of HCT-116 cells. CdSe(S) QDs were found to have a low toxicity at concentrations up to 100 µg/mL, with a decreased cell viability at higher concentrations, indicating a highly dose-dependent response. Meanwhile, CdSe(S)/ZnO core/shell QDs exhibited lower toxicity than uncoated QDs at higher concentrations. Confocal microscopy images of HCT-116 cells after incubation with CdSe(S) and CdSe(S)/ZnO QDs showed that the cells were stable in aqueous concentrations of 100 µg of QDs per mL, with no sign of cell necrosis, confirming the cytotoxicity data
Functionalization of Quinazolin-4-Ones Part 3: Synthesis, structures,elucidation DNA-PK, PI3K and cytotoxicity of novel 8-aryl-2-morpholino-quinazolin-4-ones Jacob T. Heppella, MD. Amirul Islamc Shelli R McAlpine and Jasim M. A. Al-Rawi* J. Heterocycl. Chem., V56, p124-142 2019 DOI: 10.1002/jhet.3385 2019
A series of novel 8‐aryl‐2‐morpholino quinazolines (11a–n, 12a–d, 14a–f, and 15) were synthesized from the precursor 2‐thioxo quinazolin‐4‐ones 8. The 8‐aryl‐2‐morpholino quinazolines compounds were assayed for DNA‐PK and PI3K. All compounds showed low DNA‐PK % inhibition activity at 10 μM compound concertation, and the most active was 8‐(dibenzo[b,d]thiophen‐4‐yl) 12d with 38% inhibition. Similar pattern of PI3K α, β, γ, and δ isoforms inhibition activity at 10 μM were observed. The most active isoform was PI3K δ of 41% inhibition for 8‐(dibenzo[b,d]furan‐4‐yl) compound 11. Most compounds were less active than expected in spite of the strong structural resemblance to known inhibitors (NU7441, 3, 4, and 6). Loss of activity could be attributed to the tautomerization to the aromatic enol (4‐OH), which could specify that the important functional group for the activity is the 4‐carbonyl (C=O) group. Alternatively, the aromatization of the pyrimidine heterocyclic ring could alter the conformation, and thus binding site, of the 2‐morpholine ring, which could reduce the compound‐receptor hydrogen bonding to the morpholine 4‐oxygen. Selected compounds displayed appreciable cytotoxicity with 6‐chloro‐8‐(dibenzo[b,d]thiophen‐4‐yl)‐2‐morpholinoquinazolin‐4(1H)‐one 11j exhibiting the greatest activity with an IC50 of 9.95 μM. Therefore, the mechanism of the cytotoxicity of compound 11j were not through DNA‐PK or PI3K inhibition activity.
Hsp90 mediates membrane deformation and exosome release Elsa Lauwers,* Yu-Chung Wang, Rodrigo Gallardo, Rob Van der Kant, Emiel Michiels, Jef Swerts, Pieter Baatsen, Samantha S. Zaiter, Shelli R. McAlpine, Natalia V. Gounko, Frederic Rousseau, Joost Schymkowitz, and Patrik Verstreken* Molecular Cell V71, p689-702, e9 2018 DOI: 10.1016/j.molcel.2018.07.016
Hsp90 is an essential chaperone that guards proteome integrity and amounts to 2% of cellular protein. We now find that Hsp90 also has the ability to directly interact with and deform membranes via an evolutionarily conserved amphipathic helix. Using a new cell-free system and in vivo measurements, we show this amphipathic helix allows exosome release by promoting the fusion of multivesicular bodies (MVBs) with the plasma membrane. We dissect the relationship between Hsp90 conformation and mem-brane-deforming function and show that mutations and drugs that stabilize the open Hsp90 dimer expose the helix and allow MVB fusion, while these effects are blocked by the closed state. Hence, we structurally separated the Hsp90 membrane-deform-ing function from its well-characterized chaperone activity, and we show that this previously unrecognized function is required for exosome release.
Converting Polar cyclic peptides into cell permeable molecules using N-methylation Leo L. H. Lee, Laura K. Buckton* and Shelli R. McAlpine* Peptide Science, V110, e24063. 2018 DOI: 10.1002/pep2.24063
Described are the design, synthesis, and biological evaluation of 5 N-methylated analogs that are based on a lead drug structure LB51. LB51 is a cyclic pentapeptide that inhibits heat shock protein 90 and although a potent inhibitor of the protein function, it has poor cell permeability. Introduction of an N-methyl moiety at each amino acid produces 5 analogs of LB51, where all 5 show significantly improved membrane permeability over the lead molecule despite the presence of 4 highly polar side chains.
Improving the cell permeability of polar cyclic peptides by replacing residues with alkylated amino acids, asparagines, and D-amino acids Laura K. Buckton and Shelli R. McAlpine*Org. Lett. V20, p506-509, 2018
The design, synthesis, and cell permeability of 19 hydrophilic macrocyclic peptides is presented. By systematically analyzing the impact of three different approaches (alkylated amino acids, asparagines, and d-amino acids) on the permeability of polar peptides, a well-defined strategy for optimizing cell permeability is provided. These three new methods can be used individually or in combination to effectively convert polar peptides into cell permeable molecules, and the results can be applied to the rapidly expanding peptide therapeutic industry.
Synthesis and structure-activity relationships of inhibitors that target the C-terminal MEEVD on Heat shock protein 90 (Hsp90) Marwa N. Rahimi, Laura K. Buckton, Samantha S. Zaiter, Jessica Kho, Vickie Chan, Aldwin Guo, Jenane Konesan, SuHyeon Kwon, Lok K. O. Lam, Michael F. Lawler, Michael Leong, Gabriel D. Moldovan, David A. Neale, Gillian Thornton, and Shelli R. McAlpine* ACS Med. Chem. Lett. V9 p73-77 2018
Herein we describe the synthesis and structure-activity relationships of cyclic peptides designed to target heat shock protein 90 (Hsp90). Generating 19 compounds and evaluating their binding affinity reveals that increasing electrostatic interactions allows the compounds to bind more effectively with Hsp90 compared to the lead structure. Exchanging specific residues for lysine improves binding affinity for Hsp90, indicating some residues are not critical for interacting with the target, whereas others are essential. Replacing l- for d- amino acids produced compounds with decreased binding affinity compared to the parent structure, confirming the importance of conformation and identifying key residues most important for binding. Thus, a specific conformation and electrostatic interactions are required in order for these inhibitors to bind to Hsp90.
RITA mimics: Synthesis and mechanistic evaluation of asymmetric linked Trithiazoles Adrian L. Pietkiewicz,† Yuqi Zhang,† Marwa N. Rahimi, Michael Stramandinoli, Matthew Teusner, and Shelli R. McAlpine* ACS Med. Chem. Lett. V8 p401-406 2017
The established cytotoxic agent RITA contains a thiophene-furan-thiophene backbone and two terminal alcohol groups. Herein we investigate the effect of using thiazoles as the backbone in RITA-like molecules, and modifying the terminal groups of these trithiazoles, thereby generating 41 unique structures. Incorporating side chains with varied steric bulk allowed us to investigate how size and a stereocenter impacted biological activity. Subjecting compounds to growth inhibition assays on HCT-116 cells showed that the most potent compounds 7d, 7e, and 7h had GI50 values of 4.4 µM, 4.4 µM and 3.4 µM respectively, versus RITA (GI50 of 800 nM). Analysis of these compounds in apoptosis assays proved that 7d, 7e, and 7h were as effective as RITA at inducing apoptosis. Evaluating the impact of 7h on proteins targeted by RITA (p53, c-Myc, and Mcl-1) indicated that it acts via a different mechanism of action to that of RITA. RITA suppressed Mcl-1 protein via p53, whereas compound 7h suppressed Mcl-1 expression via an alternative mechanism independent of p53.
Redefining the phenotype of Heat shock protein 90 (Hsp90) inhibitors Yao Wang, Yen Chin Koay, and Shelli R. McAlpine*Chem. Eur. J. V23 2010-2013 2017
The phenotypes produced when cells are treated with the Hsp90 inhibitors AUY922 or 17-AAG (classical inhibitors) are different to those produced when cells are knocked down with Hsp90a. Pull-down assays using classical inhibitors suggest that these molecules bind to multiple targets other than Hsp90. Classical inhibitors also induce similar protein markers as other anti-cancer therapies Cisplatin and bortezomib that do not target Hsp90. Together these data suggest that AUY922 and 17-AAG acts on multiple targets and likely kills cells through multiple mechanisms. Comparing these classical inhibitors to the effects seen when treating cells with C-terminal Hsp90 modulators reveals that C-terminal modulators effectively bind to Hsp90, and induce phenotypic markers consistent with the Hsp90a CRISPR knockdown data. Our findings challenge the current interpretation of Hsp90 inhibitors and suggest that a large body of literature that describes the Hsp90 phenotype and inhibitors is re-examined in this context.
How selective are Hsp90 inhibitors for cancer cells over normal cells? Yao Wang, Yen Chin Koay, and Shelli R. McAlpine*ChemMedChem V12 p353-357 2017
Selectively inhibiting target proteins in cancer cells over normal cells is one of the most critical features of a successful protein inhibitor for clinical applications. By evaluating and comparing the impact of a clinical N-terminal heat shock protein 90 (Hsp90) inhibitor, AUY922 (luminespib), on Hsp90 inhibition-associated cellular events in cancer cells versus normal cells, we found that it produces similar phenotype characteristics in both cell types, indicating that AUY922 is not selective for targeting Hsp90 in tumor cells. By comparison, the C-terminal Hsp90 modulator SM258 suppresses cell proliferation, triggers apoptosis, regulates the expression of Hsp90-associated heat shock proteins, and enhances the degradation of Hsp90's client proteins preferentially in cancer cells over normal cells. Our findings support a new paradigm that AUY922 is not tumor selective, whereas SM258 is more selective and likely acts through an Hsp90-dependent mechanism.
Allosteric Modulators of Heat Shock Protein 90 (HSP90) Yen Chin Koay and Shelli R. McAlpine * RSC Drug discovery series: “Allosterism in Drug Discovery” DOI:10.1039/9781782629276, p404-426 2016
Three classes of allosteric inhibitors have been described in this chapter. The first were molecules targeting the ATP binding site at the N-terminus of HSP90 (classical inhibitors), while influencing clients that bind to the middle domain. These molecules are the only ones that have reached clinical trials, but they had significant problems including induction of pro-survival responses and dose-limiting toxicities. Thus, these classical inhibitors are being used as part of dual inhibition regiments in current clinical trials. Data was presented in this chapter to suggest that the poor results are also due to the classical inhibitor’s poor selectivity for HSP90 in a cellular environment. Indeed, knocking down HSP90 in cells produces a phenotype that is distinct from that reported with these classical inhibitors. The second class of molecules described are those that bind to the C-terminus (e.g., Novobiocin and KU-174), which have anti-cancer cellular activity without inducing high levels of HSF-1, HSP70, or HSP27. Although these compounds affected several clients that bound to the middle domain, efforts focused on improving the potency and pharmacokinetics of these molecules for in vivo efficacy.
The third class of compounds described are the SM series; these molecules bind between the N- and middle domains of HSP90 but affect the C-terminus. The SM molecules bind to the flexible charged linker region and stop the N-terminus from rotating, thereby blocking access to the C-terminal MEEVD region. By inhibiting access to the MEEVD region, the SM series blocks all co-chaperones with a TRP domain from binding to HSP90. Downstream effects attributed to blocking these co-chaperones produce a reduction of immunophilins FKBP52, and FKBP51, and hormone receptor levels. These SM molecules are also the first to be proven to target HSP90 in the cell. Specifically, as determined using pulldown assays with tagged variants of the molecules in multiple cell lysates, the compounds selectively pull out HSP90. Evaluating their efficacy in cells treated with siRNA for HSP90 we see
that the compounds are more effective when HSP90 is knocked down and less effective when HSP90 is over-expressed. Finally, cells treated with the SM compounds match the phenotype produced when the HSP90 alpha gene is knocked down. Thus, it is possible that there has not yet been a clinical evaluation of HSP90 inhibitors. However, as with all allosteric modulators, there are significant challenges associated with predicting active structures, and as such new direct C-terminal modulators are currently under evaluation.
Reinventing Hsp90 inhibitors: Blocking C-terminal binding events to Hsp90 using dimerized inhibitors Yen Chin Koay, Hendra Wahyudi, and Shelli R. McAlpine* Chem. Eur. J. V22 p18572-18582 2016
Heat shock protein 90 (Hsp90) is a 90 kDa molecular chaperone that functions as a dimer. It facilitates the folding, assembly and stabilization of more than 400 proteins that are responsible for cancer development and progression. The reliance of the clients on Hsp90 has made it a promising anticancer target. Classical inhibitors that block the binding of ATP to the Hsp90’s N-terminus are highly toxic to cells and, trigger a resistance mechanism within cells. This resistance mechanism comprises of a large increase in the pro-survival heat shock proteins (HSF-1, Hsp70 and Hsp27). Molecules that modulate the C-terminus of Hsp90 are effective at inducing cancer cell death without activating the resistance mechanism. Herein we describe here the design, synthesis and biological binding affinity for a series of dimerized C-terminal Hsp90 modulators. We show that dimers of these C-terminal modulators synergistically inhibit Hsp90 compared to monomers.
A novel class of Hsp90 C-terminal modulators have preclinical efficacy in prostate tumor cells without induction of a heat shock response
Heather K. Armstrong, Yen Chin Koay, Swati Irani, Rajdeep Das, Zeyad D. Nassar, The Australian Prostate Cancer Bio resource, Luke A. Set, Margaret M. Centenera, Shelli R. McAlpine* and Lisa A. Butler* The Prostate V76, p1546-1559 2016
While there is compelling rationale to use heat shock protein 90 (Hsp90) inhibitors for treatment of advanced prostate cancer, agents that target the N-terminal ATP-binding site of Hsp90 have shown little clinical benefit. These N-terminal binding agents induce a heat shock response that activates compensatory HSP chaperones, which is believed to contribute in part to the agents’ lack of efficacy. Here, we describe the functional characterization of two novel agents, SM253 and SM258, that bind the N-middle linker region of Hsp90, resulting in reduced client protein activation and preventing C-terminal co-chaperones and client proteins from binding to Hsp90.SM253 and SM258 exhibit antiproliferative and pro-apoptotic activity in multiple prostate cancer cell lines (LNCaP, 22Rv1and PC-3) at low micromolar concentrations. Unlike the N-terminal inhibitors AUY922 and 17-AAG, these SM agents do not induce expression of Hsp27, Hsp40 or Hsp70, proteins that are characteristic of the heat shock response, in any of the prostate cell lines analyzed. Notably, SM258 significantly reduced proliferation within 2 days in human primary prostate tumors cultured ex vivo, without the significant induction of Hsp70 that was caused by AUY922 in the tissues. Our findings provide the first evidence of efficacy of this class of C-terminal modulators of Hsp90 in human prostate tumors, and indicate that further evaluation of these promising new agents is warranted.
The first report of direct inhibitors that target the C-terminus MEEVD region on heat shock protein 90
Laura K. Buckton, Hendra Wahyudi, and Shelli R. McAlpine* Chem. Commun. V52, p501-504 2016
Sixteen linear and cyclic peptides were designed de novo and synthesised. Protein binding data indicates that three compounds directly block acccess to heat shock protein 90 (hsp90)’s C-terminus. These molecules are valuable tools useful for investigating the impact of inhibiting hsp90 via a novel mechanism.
Hitting a moving target: How does an N-Methyl group impact biological activity? Yen Chin Koay, Nicole L. Richardson, Samantha S. Zaiter, Jessica Kho, Sheena Y. Nguyen, Daniel H. Tran, Ka Wai Lee, Laura K. Buckton, and Shelli R. McAlpine* ChemMedChem. V11, p881-892 2016
Macrocycles have several advantages over small-moleculedrugs when it comes to addressing specific protein–protein interactionsas therapeutic targets. Herein we report the synthesisof seven new cyclic peptide molecules and their biologicalactivity. These macrocycles were designed to understand howmoving an N-methyl moiety around the peptide backbone impactsbiological activity. Because the lead non-methylated structure inhibits the oncogenic regulator heat-shock protein90 (Hsp90), two of the most potent analogues were evaluated for their Hsp90 inhibitory activity. We show that incorporating an N-methyl moiety controls the conformation of the macrocycle, which dramatically impacts cytotoxicity and binding affinity for Hsp90. Thus, the placement of an N-methylated amino acid within a macrocycle generates an unpredictable change to the compound’s conformation and hence biological activity.