An Hsp90 modulator that exhibits a unique mechanistic profile.

Described is the synthesis of two biotinylated derivatives of a cytotoxic macrocycle. Pull-down assays indicate that this macrocycle targets the N-middle domain of Hsp90. Untagged compound can effectively compete away tagged compound-Hsp90 protein complexes, confirming the binding specificity of the macrocycle for Hsp90. The macrocycle is similar in potency to other structurally-related analogs of Sansalvamide A (San A) and induces apoptosis via a caspase 3 mechanism. Unlike other San A derivatives, we show that the macrocycle does not inhibit binding between C-terminal client proteins and co-chaperones and Hsp90, suggesting that it has a unique mechanism of action.

Synthesis of sansalvamide A peptidomimetics: triazole, oxazole, thiazole, and pseudoproline containing compounds.

Peptidomimetic-based macrocycles typically have improved pharmacokinetic properties over those observed with peptide analogs. Described are the syntheses of 13 peptidomimetic derivatives that are based on active Sansalvamide A structures, where these analogs incorporate heterocycles (triazoles, oxazoles, thiazoles, or pseudoprolines) along the macrocyclic backbone. The syntheses of these derivatives employ several approaches that can be applied to convert a macrocyclic peptide into its peptidomimetic counterpart. These approaches include peptide modifications to generate the alkyne and azide for click chemistry, a serine conversion into an oxazole, a Hantzsch reaction to generate the thiazole, and protected threonine to generate the pseudoproline derivatives. Furthermore, we show that two different peptidomimetic moieties, triazoles and thiazoles, can be incorporated into the macrocyclic backbone without reducing cytotoxicity: triazole and thiazole.

A small molecule that preferentially binds the closed conformation of Hsp90.

Described is the synthesis of three different fluorescein-tagged derivatives of a macrocycle, and their binding affinity to heat shock protein 90 (Hsp90). Using fluorescence polarization anisotropy, we report the binding affinity of these fluorescein-labeled compounds to Hsp90 in its open state and ATP-dependent closed state. We show that the compounds demonstrate a conformation-dependent preference for binding to the closed state.

Macrocycles that inhibit the binding between heat shock protein 90 and TPR-containing proteins.

Heat shock protein 90 (Hsp90) accounts for 1-2% of the total proteins in normal cells and functions as a molecular chaperone that folds, assembles, and stabilizes client proteins. Hsp90 is overexpressed (3- to 6-fold increase) in stressed cells, including cancer cells, and regulates over 200 client and co-chaperone proteins. Hsp90 client proteins are involved in a plethora of cellular signaling events including numerous growth and apoptotic pathways. Since pathway-specific inhibitors can be problematic in drug-resistant cancers, shutting down multiple pathways at once is a promising approach when developing new therapeutics. Hsp90's ability to modulate many growth and signaling pathways simultaneously makes this protein an attractive target in the field of cancer therapeutics. Herein we present evidence that a small molecule modulates Hsp90 via binding between the N and middle domain and allosterically inhibiting the binding interaction between Hsp90 and four C-terminal binding client proteins: IP6K2, FKBP38, FKBP52, and HOP. These last three clients contain a tetratricopeptide-repeat (TPR) region, which is known to interact with the MEEVD sequence on the C-terminus of Hsp90. Thus, this small molecule modulates the activity between co-chaperones that contain TPR motifs and Hsp90's MEEVD region. This mechanism of action is unique from that of all Hsp90 inhibitors currently in clinical trials where these molecules have no effect on proteins that bind to the C-terminus of Hsp90. Further, our small molecule induces a Caspase-3 dependent apoptotic event. Thus, we describe the mechanism of a novel scaffold that is a useful tool for studying cell-signaling events that result when blocking the MEEVD-TPR interaction between Hsp90 and co-chaperone proteins.

Synthesis and evaluation of biotinylated sansalvamide A analogs and their modulation of Hsp90.

Described are the syntheses of three sansalvamide A derivatives that contain biotinylated tags at individual positions around the macrocycle. The tagged derivatives indicated in protein pull-down assays that they bind to Hsp90 at the same binding site (N-Middle domain) as the San A-amide peptide. Further, these compounds inhibit binding between Hsp90 and multiple C-terminal client proteins. This interaction is unique to the San A analogs indicating they can be tuned for selectivity against Hsp90 client/co-chaperone proteins.

Histone Deacetylase Inhibitors: Synthesis of Cyclic Tetrapeptides and their Triazole Analogues.

Synthesis of nine macrocyclic peptide HDAC inhibitors and three triazole derivatives are described. HDAC inhibitory activity of these compounds against HeLa cell lysate is evaluated. The biological data demonstrates that incorporation of a triazole unit improves the HDAC inhibitory activity.

Design and synthesis of Hsp90 inhibitors: exploring the SAR of Sansalvamide A derivatives.

Utilizing the structure-activity relationship we have developed during the synthesis of the first two generations and mechanism of action studies that point to the interaction of these molecules with the key oncogenic protein Hsp90, we report here the design of 32 new Sansalvamide A derivatives and their synthesis. Our new structures, designed from previously reported potent compounds, were tested for cytotoxicity on the HCT116 colon cancer cell line, and their binding to the biological target was analyzed using computational studies involving blind docking of derivatives using Autodock. Further, we show new evidence that our molecules bind directly to Hsp90 and modulate Hsp90's binding with client proteins. Finally, we demonstrate that we have integrated good ADME properties into a new derivative.

Macrocyclic inhibitors of hsp90.

Heat shock proteins (HSP) are a family of highly conserved proteins, whose expression increases in response to stresses that may threaten cell survival. Over the past decade, heat shock protein 90 (Hsp90) has emerged as a potential therapeutic target for cancer as it plays a vital role in normal cell maturation and acts as a molecular chaperone for proper folding, assembly, and stabilization of many oncogenic proteins. To date, a majority of Hsp90 inhibitors that have been discovered are macrocycles. The relatively rigid conformation provided by the macrocyclic scaffold allows for a selective interaction with a biological target such as Hsp90. This review highlights the discovery and development of nine macrocycles that inhibit the function of Hsp90, detailing their potency and the client proteins affected by Hsp90 inhibition.

Evaluation of di-sansalvamide A derivatives: synthesis, structure-activity relationship, and mechanism of action.

Described is the SAR of 18 di-sansalvamide A derivatives and the mechanism of action of the most potent compound. We show that this scaffold is a promising lead in the development of novel cancer therapeutics because it is cytotoxic at nanomolar potency, inhibits a well-established oncogenic target (Hsp90), and does not share structural motifs with current drugs on the market.

Conformationally based design of macrocycles as antitumor agents.

A key feature for any chemotherapeutic agent is a favorable conformation when it is presenting itself to its intended target. Numerous macrocycles have been identified as having antitumor activity and have been a source of lead compounds in anticancer research. The macrocyclic scaffold restricts bond rotation, therefore macrocycles maintain a relatively rigid conformation compared to their linear counterparts. This review discusses recent progress in the development of macrocyclic versions of linear compounds with known antitumor activity, and describes how restrictions in molecular conformation affect tumor inhibition.