Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation.

BACKGROUND: Heat shock protein 90 (HSP90) has a key role in the maintenance of the cellular proteostasis. However, HSP90 is also involved in stabilisation of oncogenic client proteins and facilitates oncogene addiction and cancer cell survival. The development of HSP90 inhibitors for cancer treatment is an area of growing interest as such agents can affect multiple pathways that are linked to all hallmarks of cancer. This study aimed to test the hypothesis that targeting cysteine residues of HSP90 will lead to degradation of client proteins and inhibition of cancer cell proliferation. METHODS: Combining chemical synthesis, biological evaluation, and structure-activity relationship analysis, we identified a new class of HSP90 inhibitors. Click chemistry and protease-mass spectrometry established the sites of modification of the chaperone. RESULTS: The mildly electrophilic sulphoxythiocarbamate alkyne (STCA) selectively targets cysteine residues of HSP90, forming stable thiocarbamate adducts. Without interfering with the ATP-binding ability of the chaperone, STCA destabilises the client proteins RAF1, HER2, CDK1, CHK1, and mutant p53, and decreases proliferation of breast cancer cells. Addition of a phenyl or a tert-butyl group in tandem with the benzyl substituent at nitrogen increased the potency. A new compound, S-4, was identified as the most robust HSP90 inhibitor within a series of 19 derivatives. CONCLUSION: By virtue of their cysteine reactivity, sulphoxythiocarbamates target HSP90, causing destabilisation of its client oncoproteins and inhibiting cell proliferation.

Different regulation of rat 5-HT(2A) and rat 5-HT(2C) receptors in NIH 3T3 cells upon exposure to 5-HT and pipamperone.

The 5-HT(2A) and 5-HT(2C) receptors belong to the same subtype of the G-protein coupled receptor family and have several agonist and antagonist ligands in common. To gain more insight into the differences in the regulation of the two receptors, we studied the effect of agonist and antagonist pre-treatment on radioligand receptor binding and 5-HT-induced inositol phosphate formation on rat 5-HT(2A) and rat 5-HT(2C) receptors stable expressed in NIH 3T3 cells. We compared short (15 min) and prolonged (48 h) pre-treatment of the cells with the natural agonist, 5-HT and with the antagonist pipamperone, which can be readily washed out. The rat 5-HT(2C) receptor showed an agonist-induced down-regulation (decrease in B(max) of labelled agonist and antagonist binding) and desensitisation (decrease in 5-HT-induced inositol phosphate formation and potency of 5-HT). Antagonist pre-treatment induced an increase in rat 5-HT(2C) receptor-mediated inositol phosphate formation as well as increased agonist and antagonist radioligand binding. These findings are consistent with the classical model of G-protein coupled receptor regulation. In contrast, the rat 5-HT(2A) receptor expressed in the same host cell behaved differently, unlike the classical model. Pre-treatment with 5-HT for 15 min and 48 h did not change receptor levels measured by radioligand binding, but the signal transduction response (inositol phosphate formation) was significantly reduced. Pre-treatment with the antagonist pipamperone for 15 min and 48 h caused an increase in antagonist radioligand binding but a reduction in agonist radioligand binding and a decrease in inositol phosphate formation and potency of 5-HT. Hence, the rat 5-HT(2A) receptor apparently undergoes agonist desensitisation without down-regulation of the total receptor number. Antagonist pre-treatment causes a paradoxical desensitisation, possibly by uncoupling of the receptor from G-proteins. The uncoupled receptor does not bind 5-HT in the nanomolar range but retains its antagonist binding properties. Paradoxical antagonist-induced desensitisation of rat 5-HT(2A) receptors has also been observed in vivo.