updated guidelines for use of pvs1 and pp3 criteria  (ClinGen Resource)

Journal: bioRxiv

Article Title: The c-Src inhibitor eCF506 diminishes opioid tolerance creating bias against β-arrestin2 recruitment

doi: 10.1101/2025.07.01.662507

Figure Lengend Snippet: Concentration-response relationships of ( A ) DAMGO as a recruiter of β-arrestin2 to human μ receptors in PathHunter CHO cells and ( B ) DAMGO as an inhibitor of forskolin (30 μM) stimulated cAMP accumulation in the same cell line transiently expressing the pGloSensor-22F protein following 16 h exposure to 1 μM PP2, PP3, eCF506 or an equal volume of DMSO. (A) Data are expressed as a percentage of maximum luminescence (% Max RLU) produced by DAMGO in DMSO exposed cells present in duplicate on each 96-well plate. The insert demonstrates c-Src phosphorylation at Y416 (upper panel) and total c-Src (lower panel) expression assessed using western blot in each cell lysate following overnight exposure and confirms inhibition of c-Src phosphorylation. (B) Data are expressed as a percentage of luminescence in each well measured prior to the addition of agonist (% control). Data from individual replicates were plotted and fitted with logistics functions to derive efficacy (E MAX ) and potency (EC 50 /IC 50 ) parameters, presented in . Data are the mean ± SEM of 5 replicates (A) and 8 replicates (B).

Article Snippet: DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) and forskolin (HelloBio, UK), morphine sulfate salt pentahydrate (#M8777), fentanyl citrate (#F3886), oxycodone hydrochloride (#BP1068; all from Merck, UK), TRV130 (#MBS3601671; CliniSciences, UK), buprenorphine hydrochloride (#2808), β-FNA (#0926), PP2 (#1407), PP3 (#2794), caffeic acid pYEEIE (#1935; all from Tocris, UK), DAS-5-oCRBN (#HY-163144; Cambridge Biosciences, UK) were prepared as 10 mM (DAMGO, morphine, fentanyl, buprenorphine, TRV130, PP2, PP3, DAS-5-oCRBN, caffeic acid-pYEEIE and eCF506), 20 mM (oxycodone), 25 mM (forskolin) or 100 mM (β-FNA) stock solutions in DMSO (DAMGO, forskolin, buprenorphine, β-FNA, PP2, PP3, DAS-5-oCRBN, caffeic acid-pYEEIE and eCF506) or water (fentanyl, morphine, oxycodone).

Techniques: Concentration Assay, Expressing, Produced, Phospho-proteomics, Western Blot, Inhibition, Control

Journal: bioRxiv

Article Title: The c-Src inhibitor eCF506 diminishes opioid tolerance creating bias against β-arrestin2 recruitment

doi: 10.1101/2025.07.01.662507

Figure Lengend Snippet:

Article Snippet: DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) and forskolin (HelloBio, UK), morphine sulfate salt pentahydrate (#M8777), fentanyl citrate (#F3886), oxycodone hydrochloride (#BP1068; all from Merck, UK), TRV130 (#MBS3601671; CliniSciences, UK), buprenorphine hydrochloride (#2808), β-FNA (#0926), PP2 (#1407), PP3 (#2794), caffeic acid pYEEIE (#1935; all from Tocris, UK), DAS-5-oCRBN (#HY-163144; Cambridge Biosciences, UK) were prepared as 10 mM (DAMGO, morphine, fentanyl, buprenorphine, TRV130, PP2, PP3, DAS-5-oCRBN, caffeic acid-pYEEIE and eCF506), 20 mM (oxycodone), 25 mM (forskolin) or 100 mM (β-FNA) stock solutions in DMSO (DAMGO, forskolin, buprenorphine, β-FNA, PP2, PP3, DAS-5-oCRBN, caffeic acid-pYEEIE and eCF506) or water (fentanyl, morphine, oxycodone).

Techniques: Inhibition, Concentration Assay, cAMP Assay

Journal: bioRxiv

Article Title: The c-Src inhibitor eCF506 diminishes opioid tolerance creating bias against β-arrestin2 recruitment

doi: 10.1101/2025.07.01.662507

Figure Lengend Snippet: Concentration-response relationships of DAMGO to recruit β-arrestin2 to human μ receptors in PathHunter CHO cells transiently overexpressing ( A ) full-length (residues 1-536; WT), ( B ) C-terminally truncated (residues 1-249) or ( C ) N-terminally truncated and catalytically inactive (residues 250-536(K298M)) human c-Src following 16-h exposure to PP2, PP3, eCF506 (300 nM) or an equal volume of DMSO. Data are expressed as a percentage of maximum luminescence (% Max RLU) produced by DAMGO in DMSO exposed cells present in duplicate on each plate i.e., cells overexpressing (A) WT, (B) Src 1-249 or (C) Src 250-536(K298M) are each expressed relative to the maximum β-arrestin2 recruitment of each construct caused by DAMGO. Data from individual replicates were plotted and fitted with logistics functions to derive efficacy (E MAX ) and potency (EC 50 ) parameters, presented in Supplementary Table 3. Data are the mean ± SEM of 5-6 replicates.

Article Snippet: DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) and forskolin (HelloBio, UK), morphine sulfate salt pentahydrate (#M8777), fentanyl citrate (#F3886), oxycodone hydrochloride (#BP1068; all from Merck, UK), TRV130 (#MBS3601671; CliniSciences, UK), buprenorphine hydrochloride (#2808), β-FNA (#0926), PP2 (#1407), PP3 (#2794), caffeic acid pYEEIE (#1935; all from Tocris, UK), DAS-5-oCRBN (#HY-163144; Cambridge Biosciences, UK) were prepared as 10 mM (DAMGO, morphine, fentanyl, buprenorphine, TRV130, PP2, PP3, DAS-5-oCRBN, caffeic acid-pYEEIE and eCF506), 20 mM (oxycodone), 25 mM (forskolin) or 100 mM (β-FNA) stock solutions in DMSO (DAMGO, forskolin, buprenorphine, β-FNA, PP2, PP3, DAS-5-oCRBN, caffeic acid-pYEEIE and eCF506) or water (fentanyl, morphine, oxycodone).

Techniques: Concentration Assay, Produced, Construct

Journal: PLOS Genetics

Article Title: Leveraging cancer mutation data to inform the pathogenicity classification of germline missense variants

doi: 10.1371/journal.pgen.1011540

Figure Lengend Snippet: (A) The presence of either gain-of-function or loss-of-function mutations in cancer driver genes can lead to cancer (left) or rare Mendelian disorders (right) in different contexts. Most cancers result from somatic mutations that accumulate in a tissue-specific manner, whereas germline mutations are present in all cells of the body and cause a type of rare Mendelian disorder (e.g., neurodevelopmental disorder). (B) The HRAS Q61K mutation is an example of a known cancer mutation that drives different types of cancers that also causes Costello syndrome, a developmental disorder, when observed as a germline variant. (C) Workflow for extracting cancer mutations from Cancer Hotspots. Recurrent cancer mutations were filtered to 2,447 missense mutations. See main text for details. REVEL scores thresholds correspond to supporting evidence for pathogenicity (PP3) and for benign-ness (BP4). Created with Lucidchart. Created with BioRender.

Article Snippet: We grouped these CH mutations by REVEL scores using the ClinGen-proposed PP3/BP4 score thresholds ( ) [ ].

Techniques: Mutagenesis, Variant Assay