Crystal structure and molecular modeling of 17-DMAG in complex with human Hsp90

Hsp90 is an attractive chemotherapeutic target because it chaperones the folding of proteins found in multiple signal transduction pathways. We describe the 1.75 A resolution crystal structure of human Hsp90 alpha (residues 9-236) complexed with 17-desmethoxy-17-N,N-dimethylaminoethylamino-geldanamycin (17-DMAG). The structure revealed an altered set of interactions between the 17-substituent and the protein compared to geldanamycin and the 17-dimethylaminoethyl moiety pointing into solvent, but otherwise was similar to that reported for the complex with geldanamycin. Targeted molecular dynamics simulations and energetic analysis indicate that geldanamycin undergoes two major conformational changes when it binds Hsp90, with the key step of the conversion being the trans to cis conformational change of the macrocycle amide bond. We speculate that 17-DMAG analogs constrained to a cis-amide in the ground state could provide a significant increase in affinity for Hsp90.     

Coupling of alkenes and alkynes: synthesis of the C1-C11 And C18-C28 fragments of miyakolide

A transition metal-mediated, atom-economical approach toward the crucial A and D rings of miyakolide is described. A Pd-catalyzed alkyne-alkyne coupling/6- endo- dig cyclization is employed to assemble the A ring fragment. The key D ring pyran is constructed utilizing an Ru-catalyzed alkene-alkyne coupling followed by a Pd-catalyzed allylic alkylation to establish the all-cis stereochemistry.     

Engineered biosynthesis of geldanamycin analogs for Hsp90 inhibition

Geldanamycin, a polyketide natural product, is of significant interest for development of new anticancer drugs that target the protein chaperone Hsp90. While the chemically reactive groups of geldanamycin have been exploited to make a number of synthetic analogs, including 17-allylamino-17-demethoxy geldanamycin (17-AAG), currently in clinical evaluation, the "inert" groups of the molecule remain unexplored for structure-activity relationships. We have used genetic engineering of the geldanamycin polyketide synthase (GdmPKS) gene cluster in Streptomyces hygroscopicus to modify geldanamycin at such positions. Substitutions of acyltransferase domains were made in six of the seven GdmPKS modules. Four of these led to production of 2-desmethyl, 6-desmethoxy, 8-desmethyl, and 14-desmethyl derivatives, including one analog with a four-fold enhanced affinity for Hsp90. The genetic tools developed for geldanamycin gene manipulation will be useful for engineering additional analogs that aid the development of this chemotherapeutic agent.     

Reinventing Hsp90 Inhibitors: Blocking C‐Terminal Binding Events to Hsp90 by Using Dimerized Inhibitors

Heat shock protein 90 (Hsp90) is a molecular chaperone (90 kDa) that functions as a dimer. This protein facilitates the folding, assembly, and stabilization of more than 400 proteins that are responsible for cancer development and progression. Inhibiting Hsp90’s function will shut down multiple cancer‐driven pathways simultaneously because oncogenic clients rely heavily on Hsp90, which makes this chaperone a promising anticancer target. Classical inhibitors that block the binding of adenine triphosphate (ATP) to the N‐terminus of Hsp90 are highly toxic to cells and trigger a resistance mechanism within cells. This resistance mechanism comprises a large increase in prosurvival proteins, namely, heat shock protein 70 (Hsp70), heat shock protein 27 (Hsp27), and heat shock factor 1 (HSF‐1). Molecules that modulate the C‐terminus of Hsp90 are effective at inducing cancer‐cell death without activating the resistance mechanism. Herein, we describe 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 relative to monomers.     

Design,Synthesis and Pharmacological Evaluation of Novel Hsp90N-terminal Inhibitors Without Induction of Heat Shock Response

Heat shock protein 90 (Hsp90) is a potential oncogenic target. However, Hsp90 inhibitors in clinical trial induce heat shock response, resulting in drug resistance and inefficiency. In this study, we designed and synthesized a series of novel triazine derivatives ( A1 - 26 , B1 - 13 , C1 - 23 ) as Hsp90 inhibitors. Compound A14 directly bound to Hsp90 in a different manner from traditional Hsp90 inhibitors, and degraded client proteins, but did not induce the concomitant activation of Hsp72. Importantly, A14 exhibited the most potent anti-proliferation ability by inducing autophagy, with the IC₅₀ values of 0.1 μM and 0.4 μM in A549 and SK-BR-3 cell lines, respectively. The in vivo study demonstrated that A14 could induce autophagy and degrade Hsp90 client proteins in tumor tissues, and exhibit anti-tumor activity in A549 lung cancer xenografts. Therefore, the compound A14 with potent antitumor activity and unique pharmacological characteristics is a novel Hsp90 inhibitor for developing anticancer agent without heat shock response.     

Inhibition of Hsp90 with synthetic macrolactones: synthesis and structural and biological evaluation of ring and conformational analogs of radicicol

A series of benzo-macrolactones of varying ring size and conformation has been prepared by chemical synthesis and evaluated by structural and biological techniques. Thus, 12- to 16-membered lactones were obtained by concise routes, involving ring-closing metathesis as a key step. In enzyme assays, the 13-, 15-, and 16-membered analogs are good inhibitors, suggesting that they can adopt the required conformation to fit in the ATP-binding site. This was confirmed by cocrystallization of 13-, 14-, and 15-membered lactones with the N-terminal domain of yeast Hsp90, showing that they bind similarly to the "natural" 14-membered radicicol. The most active compounds in the ATPase assays also showed the greatest growth-inhibitory potency in HCT116 human colon cancer cells and the established molecular signature of Hsp90 inhibition, i.e., depletion of client proteins with upregulation of Hsp70.     

Iridium complex catalyzed carbonylative alkyne-alkyne coupling for the synthesis of cyclopentadienones

[reaction: see text]. Catalytic carbonylative alkyne-alkyne coupling proceeds using iridium-phosphine complexes under carbon monoxide at atmospheric pressure or a partial pressure of 0.2 atm. This reaction provides various cyclopentadienones in high isolated yields.     

Rational design,synthesis, and biological evaluation of novel C6-modified geldanamycin derivatives as potent Hsp90 inhibitors and anti-tumor agents

Heat shock protein 90 (Hsp90) is an appealing anticancer drug target that provoked a tremendous wave of investigations. Geldanamycin (GA) is the first identified Hsp90 inhibitor that exhibited potent anti-cancer activity, but the off-target toxicity associated with the benzoquinone moiety hampered its clinical application. Until now, structure optimization of GA is still in need to fully exploit the therapeutic value of Hsp90. Due to the structural complexity and synthetic challenge of this compound family, conventional optimization is bound to be costly but high efficiency is expected to be reachable by combining the art of rational design and total synthesis. Described in this paper is our first attempt at this approach aiming at rational modification of the C6-position of GA. The binding affinities towards Hsp90 of compound 1 (C6-ethyl) and 2 (C6-methyl) were designed and predicted by using Discovery Studio. These compounds were synthesized and further subjected to a thorough in vitro biological evaluation. We found that compounds 1 and 2 bind to Hsp90 protein with the IC₅₀ of 34.26 nmol/L and 163.7 nmol/L, respectively. Both compounds showed broad-spectrum antitumor effects. Replacing by ethyl, compound 1 exhibited more potent bioactivity than positive control GA, such as in G2/M cell cycle arrest, cell apoptosis and client proteins degradations. The results firstly indicated that the docking study is able to provide a precise prediction of Hsp90 affinities of GA analogues, and the C6 substituent of GA is not erasable without affecting its biological activity.     

Chemical Perturbation of Oncogenic Protein Folding: from the Prediction of Locally Unstable Structures to the Design of Disruptors of Hsp90–Client Interactions

Protein folding quality control in cells requires the activity of a class of proteins known as molecular chaperones. Heat shock protein-90 (Hsp90), a multidomain ATP driven molecular machine, is a prime representative of this family of proteins. Interactions between Hsp90, its co-chaperones, and client proteins have been shown to be important in facilitating the correct folding and activation of clients. Hsp90 levels and functions are elevated in tumor cells. Here, we computationally predict the regions on the native structures of clients c-Abl, c-Src, Cdk4, B-Raf and Glucocorticoid Receptor, that have the highest probability of undergoing local unfolding, despite being ordered in their native structures. Such regions represent potential ideal interaction points with the Hsp90-system. We synthesize mimics spanning these regions and confirm their interaction with partners of the Hsp90 complex (Hsp90, Cdc37 and Aha1) by Nuclear Magnetic Resonance (NMR). Designed mimics selectively disrupt the association of their respective clients with the Hsp90 machinery, leaving unrelated clients unperturbed and causing apoptosis in cancer cells. Overall, selective targeting of Hsp90 protein–protein interactions is achieved without causing indiscriminate degradation of all clients, setting the stage for the development of therapeutics based on specific chaperone:client perturbation.     

A small molecule designed to bind to the adenine nucleotide pocket of Hsp90 causes Her2 degradation and the growth arrest and differentiation of breast cancer cells

BACKGROUND: The Hsp90s contain a conserved pocket that binds ATP/ADP and plays an important role in the regulation of chaperone function. Occupancy of this pocket by several natural products (geldanamycin (GM) and radicicol) alters Hsp90 function and results in the degradation of a subset of proteins (i.e. steroid receptors, Her2, Raf). We have used the structural features of this pocket to design a small molecule inhibitor of Hsp90. RESULTS: The designed small molecule PU3 competes with GM for Hsp90 binding with a relative affinity of 15-20 microM. PU3 induces degradation of proteins, including Her2, in a manner similar to GM. Furthermore, PU3 inhibits the growth of breast cancer cells causing retinoblastoma protein hypophosphorylation, G1 arrest and differentiation. CONCLUSIONS: PU3 is representative of a novel class of synthetic compounds that binds to Hsp90 and inhibits the proliferation of cancer cells. These reagents could provide a new strategy for the treatment of cancers.     

Hsp90 inhibitors disrupt mitochondrial homeostasis in cancer cells

Hsp90 is a cytosolic molecular chaperone whose paralog in mitochondria, TRAP1, protects cells from oxidative stress. The recent study in Cell by Kang et al. now identifies the molecular components of the proapoptotic network regulated by TRAP1, that includes Hsp90. Targeting Hsp90/TRAP1 inhibitors to mitochondria induces rapid tumor cell-specific apoptosis.     

Hsp90 inhibitors identified from a library of novobiocin analogues

Novobiocin is a C-terminal inhibitor of the Hsp90 protein folding machinery, which is responsible for the conformational maturation of numerous proteins involved in cancer growth and survival. Due to novobiocin's poor inhibitory activity ( approximately 700 muM), very little attention has been paid toward the development of novobiocin analogues for Hsp90 inhibition. In this study, a parallel library of 20 novobiocin derivatives was prepared and the biological activity of each evaluated by Western blot analysis of Hsp90 client proteins. A4 was found to be a potent inhibitor of Hsp90 as determined by its ability to cause the degradation of several Hsp90 client proteins in both breast and prostate cancer cell lines. In the presence of 1 muM A4, several Hsp90 client proteins were degraded, including AKT, Her2, Hif-1alpha, and the androgen receptor.     

Novobiocin: redesigning a DNA gyrase inhibitor for selective inhibition of hsp90

Novobiocin is a member of the coumermycin family of antibiotics and is a well-established inhibitor of DNA gyrase. Recent studies have shown that novobiocin binds to a previously unrecognized ATP-binding site at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins at approximately 700 microM. In an effort to develop more efficacious inhibitors of the C-terminal binding site, a library of novobiocin analogues was prepared and initial structure-activity relationships revealed. These data suggested that the 4-hydroxy moiety of the coumarin ring and the 3'-carbamate of the noviose appendage were detrimental to Hsp90 inhibitory activity. In an effort to confirm these findings, 4-deshydroxy novobiocin (DHN1) and 3'-descarbamoyl-4-deshydroxynovobiocin (DHN2) were prepared and evaluated against Hsp90. Both compounds were significantly more potent than the natural product, and DHN2 proved to be more active than DHN1. In an effort to determine whether these moieties are important for DNA gyrase inhibition, these compounds were tested for their ability to inhibit DNA gyrase and found to exhibit significant reduction in gyrase activity. Thus, we have established the first set of compounds that clearly differentiate between the C-terminus of Hsp90 and DNA gyrase, converted a well-established gyrase inhibitor into a selective Hsp90 inhibitor, and confirmed essential structure-activity relationships for the coumermycin family of antibiotics.     

Post-Synthetic Modification of a Porous Hydrocarbon Cage to Give a Discrete Co24 Organometallic Complex**

A new alkyne-based hydrocarbon cage was synthesized in high overall yield using alkyne-alkyne coupling in the cage forming step. The cage is porous and displays a moderately high BET surface area (546 m² g⁻¹). The cage loses crystallinity on activation and thus is porous in its amorphous form, while very similar cages have been either non-porous, or retained crystallinity on activation. Reaction of the cage with Co₂(CO)₈ results in exhaustive metalation of its 12 alkyne groups to give the Co₂₄(CO)₇₂ adduct of the cage in good yield.     

Biotinylated geldanamycin

Inhibition of the 90 kDa heat shock proteins (Hsp90) represents a promising new chemotherapeutic approach for the treatment of several cancers. Hsp90 is essential to the survival of cancer cells and is inhibited by members of the ansamycin family of antibiotics. In particular, the quinone-containing antibiotics geldanamycin (GDA) and herbimycin A inhibit Hsp90 function in vitro at low micromolar concentrations via interaction with an ATP binding domain. Many proteins bind ATP, and the discovery of selective Hsp90 inhibitors requires the identification of other proteins that bind GDA and may cause undesired effects. Biotinylated analogues of GDA with varying tether lengths have been synthesized to elucidate other proteins that competitively bind GDA. Analogues containing a photolabile tether have also been prepared as a complementary method for the removal of GDA-bound proteins from neutravidin-containing resin. Preliminary studies indicate several proteins other than Hsp90 are isolated with biotinylated GDA.     

Design and synthesis of 3′,5′-ansa-adenosines as potential Hsp90 inhibitors

3′,5′-Ansa-adenosine derivatives, rationally designed as an Hsp90 inhibitor by extracting and fusing a natural product, geldanamycin, and a natural substrate, ATP, were efficiently synthesized by the ring-closing metathesis assisted by the 2,4-dimethoxybenzyl group. This simpler scaffold design provides a practical synthesis of a set of analogs and demonstrates synthetic innovation.     

Structure-Based Design,Synthesis, and Biological Evaluation of Hsp90β-Selective Inhibitors

The 90 kDa heat shock proteins (Hsp90) are molecular chaperones that are responsible for the folding and/or trafficking of ∼400 client proteins, many of which are directly associated with cancer progression. Consequently, inhibition of Hsp90 can exhibit similar activity as combination therapy as multiple signaling nodes can be targeted simultaneously. In fact, seventeen small-molecule inhibitors that bind the Hsp90 N-terminus entered clinical trials for the treatment of cancer, all of which exhibited pan-inhibitory activity against all four Hsp90 isoforms. Unfortunately, most demonstrated undesired effects alongside induction of the pro-survival heat shock response. As a result, isoform-selective inhibitors have been sought to overcome these detriments. Described herein is a structure-based approach to design Hsp90β-selective inhibitors along with preliminary SAR. In the end, compound 5 was shown to manifest ∼370-fold selectivity for Hsp90β versus Hsp90α, and induced the degradation of select Hsp90β-dependent clients. These data support the development of Hsp90β-selective inhibitors as a new paradigm to overcome the detriments associated with pan-inhibition of Hsp90.     

Artificial Accelerators of the Molecular Chaperone Hsp90 Facilitate Rate‐Limiting Conformational Transitions

The molecular chaperone Hsp90 undergoes an ATP‐driven cycle of conformational changes in which large structural rearrangements precede ATP hydrolysis. Well‐established small‐molecule inhibitors of Hsp90 compete with ATP‐binding. We wondered whether compounds exist that can accelerate the conformational cycle. In a FRET‐based screen reporting on conformational rearrangements in Hsp90 we identified compounds. We elucidated their mode of action and showed that they can overcome the intrinsic inhibition in Hsp90 which prevents these rearrangements. The mode of action is similar to that of the co‐chaperone Aha1 which accelerates the Hsp90 ATPase. However, while the two identified compounds influence conformational changes, they target different aspects of the structural transitions. Also, the binding site determined by NMR spectroscopy is distinct. This study demonstrates that small molecules are capable of triggering specific rate‐limiting transitions in Hsp90 by mechanisms similar to those in protein cofactors.     

Asymmetric synthesis of dihydroindanes by convergent alkoxide-directed metallacycle-mediated bond formation

A convergent synthesis of highly substituted and stereodefined dihydroindanes is described from alkoxide-directed Ti-mediated cross-coupling of internal alkynes with substituted 4-hydroxy-1,6-enynes (substrates that derive from 2-directional functionalization of readily available epoxy alcohol derivatives). In addition to describing a new and highly stereoselective approach to bimolecular [2 + 2 + 2] annulation that delivers products not available with other methods in this area of chemical reactivity, evidence is provided to support annulation by way of regioselective alkyne-alkyne coupling, followed by metal-centered [4 + 2] rather than stepwise alkene insertion and reductive elimination. Overall, the reaction proceeds with exquisite stereochemical control and defines a convenient, convergent, and enantiospecific entry to fused carbocycles of great potential value in target-oriented synthesis and medicinal chemistry.     

基于抗肿瘤活性化合物MED烟酸类衍生物的设计及作用靶点

通过变性荧光素酶的再复性实验发现真菌环氧二烯(Mycoepoxydiene,MED)对热休克蛋白(Heat shock protein 90,Hsp90)具有抑制作用.Western Blot实验结果表明,MED影响人宫颈癌细胞株HeLa中Hsp70及Hsp90的客户蛋白质(Akt,Raf-1)的表达,表明MED是Hsp90的抑制剂.通过靶向对接技术预测了MED与人Hsp90 N端ATP结合位点的结合情况,并在此基础上发现,MED的烟酸类衍生物4-NDM与Hsp90的结合具有比MED与Hsp90更强的亲和作用.体外实验结果证明,MED的烟酸类衍生物4-NDM及3-NDM对HeLa细胞表现出比MED更强的细胞毒活性;可通过上调Hsp70,并下调Akt和Raf-1而影响HeLa细胞中Hsp90相关蛋白质的表达.由此推测,MED及其烟酸类衍生物可以通过抑制Hsp90,而使其客户蛋白Akt或Raf-1发生降解,发挥其抗肿瘤作用.