{"id":588,"date":"2019-09-15T15:09:57","date_gmt":"2019-09-15T19:09:57","guid":{"rendered":"https:\/\/www.lifetein.com\/blog\/?p=588"},"modified":"2024-11-16T21:42:03","modified_gmt":"2024-11-17T02:42:03","slug":"braftide-a-10mer-peptide-synthesized-at-lifetein-a-potent-allosteric-inhibitor-of-braf-dimer-for-cancer-therapy","status":"publish","type":"post","link":"https:\/\/www.lifetein.com\/blog\/braftide-a-10mer-peptide-synthesized-at-lifetein-a-potent-allosteric-inhibitor-of-braf-dimer-for-cancer-therapy\/","title":{"rendered":"Braftide, a 10mer peptide synthesized at LifeTein, a potent allosteric inhibitor of BRAF dimer for cancer therapy"},"content":{"rendered":"\n
\"\"
Dabrafenib <\/figcaption><\/figure>\n\n\n\n


BRAF is an RAF kinase. It is a core component of the RAS\/RAF\/MEK\/ERK signaling cascade, known as mitogen-activated protein kinase (MAPK) pathway. It is one of the major effectors of oncogene RAS, and is often mutated in human cancer cells.<\/p>\n\n\n\n

LifeTein’s Braftide & Cancer Therapy<\/h2>\n\n\n\n


Two FDA approved drugs, Dabrafenib, and vemurafenib, effectively inhibit the most common BRAF variant V600E, a monomeric BRAF. But, the non-V600E BRAF mutations are intrinsically resistant to these drugs. These drugs may also paradoxically stimulate the pathway when the tumor cells contain wild-type BRAF and oncogenic RAS, causing secondary malignancies.
The researchers tried to tackle the dimeric BRAF. The dimeric BRAF, such as the wild type and G469A, a most prevalent non-V600E variant in lung cancer cells, hinges on dimer interface (DIF), a 20aa span near the tail end of the alpha-C helix of BRAF. The researchers designed Braftide using computational modeling, aiming to block the dimerization. They tested the functionality in vitro, in HEK263 cells and colon cancer cell lines. <\/p>\n\n\n\n

LifeTein synthesized Braftide (TRHVNILLFM), Null-Braftide (THHVNILLFM), Cy3-Braftide (TRHVNILLFM-Cy3), TAT-Braftide (GRKKRRQRRRPQ-PEG-TRHVNILLFM), and TAT (GRKKRRQRRRPQ). We reviewed here some of the assays that helped support Braftide as an allosteric inhibitor of BRAF dimer and down-regulator of MAPK signaling pathway for cancer therapy.<\/p>\n\n\n\n


1) Cell-free in vitro assay: dose-response curve. First of all, the researchers show that Braftide has a sub-micromolar IC50 for dimeric BRAF. Full-length dimeric BRAF-WT and BRAF-G469A (from HEK293F cells) were used for dose-response curves, and the BRAF activity was probed by pMEK production.<\/p>\n\n\n\n


2) Cell-free in vitro assay: Saturation binding assay. The researchers used Cy3-labeled Braftide (Cy3-Braftide) to characterize (KD) the binding of Braftide with dimeric BRAF-WT using fluorescence quantification.<\/p>\n\n\n\n


3) Cell-free in vitro assay: Immunoprecipitation (IP). The purpose of IP was to show Braftide disrupted the BRAF dimerization. Braftide was added to HEK293 cell lysate coexpressing V5- and FLAG-tagged BRAF-WT. FLAG-tagged BRAF was pulled down by FLAG antibody-conjugated resin, which was further probed for V5-tagged BRAF. Braftide indeed reduced homodimer BRAF.<\/p>\n\n\n\n


4) Delivery of Braftide into HEK cell for BRAF inhibition. Braftide was tagged with cell-penetrating peptide TAT. TAT-Braftide (and its negative control TAT alone) was used to treat HEK293 cells transiently transfected with BRAF-WT and BRAF-G469A. Four hours of treatment resulted in reductions of BRAF, pMEK, MEK (i.e. the MAPK pathway), which were analyzed with respective antibodies by immunoblotting.<\/p>\n\n\n\n


5) Delivery of Braftide into cancer cells for BRAF inhibition and cell proliferation inhibition. Two colon cancer cell lines (KRAS-G13D-colon carcinoma) were treated with cell-penetrating TAT-Braftide and assayed for the inhibition of BRAF activity, down-regulation of MAPK signaling, and cell proliferation. All were shown positive, while the negative control TAT alone were negative. <\/p>\n\n\n\n

https:\/\/pubs.acs.org\/doi\/abs\/10.1021\/acschembio.9b00191<\/a><\/p>\n\n\n\n

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BRAF is an RAF kinase. It is a core component of the RAS\/RAF\/MEK\/ERK signaling cascade, known as mitogen-activated protein kinase (MAPK) pathway. It is one of the major effectors of oncogene RAS, and is often mutated in human cancer cells. … Continue reading →<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":712,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"footnotes":""},"categories":[4],"tags":[],"class_list":["post-588","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-peptide_synthesis"],"aioseo_notices":[],"aioseo_head":"\n\t\t\n\t\n\t\n\t\n\t\n\t\n\t\n\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\t\t\n\n","aioseo_head_json":{"title":"Braftide, a 10mer peptide synthesized at LifeTein, a potent allosteric inhibitor of BRAF dimer for cancer therapy | LifeTein Peptide Blog","description":"BRAF is an RAF kinase. 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