Mechanistic studies of Sansalvamide A-amide: an allosteric modulator of Hsp90.
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ABSTRACT:
SUBMITTER: Vasko RC
PROVIDER: S-EPMC2922868 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description:RATIONALE:Diverse preclinical studies suggest the potential therapeutic utility of the modulation of the glutamatergic system in brain via metabotropic glutamate (mGlu) receptors. Lu AF21934, a positive allosteric modulator of the mGlu4 receptor, was previously shown to reverse behavioral phenotypes in animal models thought to mimic positive, negative, and cognitive symptoms of schizophrenia. OBJECTIVES:To begin elucidating the brain circuitry involved in mGlu4 receptor pharmacology and add mechanistic support to Lu AF21934-induced phenotypic responses, the potential involvement of 5-HT1A receptors in these antipsychotic-like effects was explored. The tests used were the following: MK-801-induced hyperactivity and 2,5-dimethoxy-4-iodoamphetamine (DOI)-induced head twitches in mice, for positive symptoms; MK-801-induced disruptions of social interactions for negative symptoms; and novel object recognition and spatial delayed alteration test for cognitive symptoms. The microdialysis studies in which the effect of Lu AF21934 on MK-801-induced dopamine and serotonin release was investigated. RESULTS:The effects caused by Lu AF2193 were inhibited by administration of the selective 5-HT1A receptor antagonist WAY100635 (0.1 mg/kg). That inhibition was observed across all models used. Moreover, the concomitant administration of sub-effective doses of Lu AF21934 and a sub-effective dose of the selective 5-HT1A receptor agonist tool compound (R)-(+)-8-hydroxy-DPAT hydrobromide (0.01 mg/kg) induced a clear antipsychotic-like effect in all the procedures used. Lu AF21934 (5 mg/kg) also inhibited MK-801-induced increase in dopamine and 5-HT release. CONCLUSIONS:The actions of Lu AF21934 are 5-HT1A receptor-dependent. Activation of the mGlu4 receptor may be a promising mechanism for the development of novel antipsychotic drugs, efficacious toward positive, negative, and cognitive symptoms.
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Project description:RG-7916 is a first-in-class drug candidate for the treatment of spinal muscular atrophy (SMA) that functions by modulating pre-mRNA splicing of the SMN2 gene, resulting in a 2.5-fold increase in survival of motor neuron (SMN) protein level, a key protein lacking in SMA patients. RG-7916 is currently in three interventional phase 2 clinical trials for various types of SMA. In this report, we show that SMN-C2 and -C3, close analogs of RG-7916, act as selective RNA-binding ligands that modulate pre-mRNA splicing. Chemical proteomic and genomic techniques reveal that SMN-C2 directly binds to the AGGAAG motif on exon 7 of the SMN2 pre-mRNA, and promotes a conformational change in two to three unpaired nucleotides at the junction of intron 6 and exon 7 in both in vitro and in-cell models. This change creates a new functional binding surface that increases binding of the splicing modulators, far upstream element binding protein 1 (FUBP1) and its homolog, KH-type splicing regulatory protein (KHSRP), to the SMN-C2/C3-SMN2 pre-mRNA complex and enhances SMN2 splicing. These findings underscore the potential of small-molecule drugs to selectively bind RNA and modulate pre-mRNA splicing as an approach to the treatment of human disease.
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Project description: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.
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Project description: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.
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Project description:Hsp90 is a molecular chaperone that facilitates the maturation of signaling proteins including many kinases and steroid hormone receptors. Through these client proteins, Hsp90 is a key mediator of many physiological processes and has emerged as a promising drug target in cancer. Additionally, Hsp90 can mask or potentiate the impact of mutations in clients with remarkable influence on evolutionary adaptations. The influential roles of Hsp90 in biology and disease have stimulated extensive research into the molecular mechanism of this chaperone. These studies have shown that Hsp90 is a homodimeric protein that requires ATP hydrolysis and a host of accessory proteins termed co-chaperones to facilitate the maturation of clients to their active states. Flexible hinge regions between its three structured domains enable Hsp90 to sample dramatically distinct conformations that are influenced by nucleotide, client, and co-chaperone binding. While it is clear that Hsp90 can exist in symmetrical conformations, recent studies have indicated that this homodimeric chaperone can also assume a variety of asymmetric conformations and complexes that are important for client maturation. The visualization of Hsp90-client complexes at high resolution together with tools to independently manipulate each subunit in the Hsp90 dimer are providing new insights into the asymmetric function of each subunit during client maturation.
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Project description:Canonical targeting of Polycomb Repressive Complex 1 (PRC1) to repress developmental genes is mediated by cell type-specific, paralogous chromobox (CBX) proteins (CBX2, 4, 6, 7 and 8). Based on their central role in silencing and their dysregulation associated with human disease including cancer, CBX proteins are attractive targets for small molecule chemical probe development. Here, we have used a quantitative and target-specific cellular assay to discover a potent positive allosteric modulator (PAM) of CBX8. The PAM activity of UNC7040 antagonizes H3K27me3 binding by CBX8 while increasing interactions with nucleic acids. We show that treatment with UNC7040 leads to efficient and selective eviction of CBX8-containing PRC1 from chromatin, loss of silencing and reduced proliferation across different cancer cell lines. Our discovery and characterization of UNC7040 not only reveals the most cellularly potent CBX8-specific chemical probe to date, but also corroborates a mechanism of Polycomb regulation by non-specific CBX nucleotide binding activity.
2022-01-03 | GSE182592 | GEO
Project description:Altered glutamatergic neurotransmission is linked to several neurological and psychiatric disorders. Metabotropic glutamate receptor 2 (mGlu?) plays an important role on the presynaptic control of glutamate release and negative allosteric modulators (NAMs) acting on mGlu?/? receptors are under assessment for their potential as antidepressants, neurogenics and cognitive enhancers. Two new potent mGlu?/? NAMs, RO4988546 and RO5488608, are described in this study and the allosteric binding site in the transmembrane (TM) domain of mGlu? is characterized.Site directed mutagenesis, functional measurements and ??-adrenoceptor-based modelling of mGlu? were employed to identify important molecular determinants of two new potent mGlu?/? NAMs.RO4988546 and RO5488608 affected both [³H]-LY354740 agonist binding at the orthosteric site and the binding of a tritiated positive allosteric modulator (³H-PAM), indicating that NAMs and PAMs could have overlapping binding sites in the mGlu? TM domain. We identified eight residues in the allosteric binding pocket that are crucial for non-competitive antagonism of agonist-dependent activation of mGlu? and directly interact with the NAMs: Arg³·²?, Arg³·²?, Phe³·³?, His(E2.52) , Leu?·?³, Trp?·??, Phe?·?? and Val?·?³. The mGlu? specific residue His(E2.52) is likely to be involved in selectivity and residues located in the outer part of the binding pocket are more important for [³H]-LY354740 agonist binding inhibition, which is independent of the highly conserved Trp?·?? residue.This is the first complete molecular investigation of the allosteric binding pocket of mGlu? and Group II mGluRs and provides new information on what determines mGlu? NAMs selective interactions and effects.
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Project description:Initialized from the scaffold of CGP7930, an allosteric agonist of GABAB receptors, a series of noncompetitive antagonists were discovered. Among these compounds, compounds 3, 6, and 14 decreased agonist GABA-induced maximal effect of IP3 production in HEK293 cells overexpressing GABAB receptors and Gqi9 proteins without changing the EC50. Compounds 3, 6, and 14 not only inhibited agonist baclofen-induced ERK1/2 phosphorylation but also blocked CGP7930-induced ERK1/2 phosphorylation in HEK293 cells overexpressing GABAB receptors. The results suggested that compounds 3, 6, and 14 are negative allosteric modulators of GABAB receptors. The representative compound 14 decreased GABA-induced IP3 production with IC50 of 37.9 ?M and had no effect on other GPCR Class C members such as mGluR1, mGluR2, and mGluR5. Finally, we showed that compound 14 did not bind to the orthosteric binding sites of GABAB receptors, demonstrating that compound 14 negatively modulated GABAB receptors activity as a negative allosteric modulator.
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