Journal: International Journal of Molecular Sciences

Article Title: Antitumoral Activity of a CDK9 PROTAC Compound in HER2-Positive Breast Cancer

doi: 10.3390/ijms23105476

Figure Lengend Snippet: Evaluation of CDK9 protein expression and antiproliferative activity of SNS-032 and THAL-SNS-032 in breast cancer cell lines. Protein levels of CDK9 in all cell lines were analyzed by Western blot ( A ). Band quantification of CDK9 protein level referred to GAPDH as the loading control ( B ). THAL-SNS-032 EC50 obtained by MTT proliferation assays in breast cancer cell lines after 72 h of treatment ( C ). Heat map depicting the effect of different doses of THAL-SNS-032 in cell death in a breast cancer cell line panel ( D ). THAL-SNS-032 reduced cell viability more than SNS-032 in BT474, BT474-RH, BT474-TDM1R, and BT474-LAPA-R at the doses indicated (72 h). We used MTT assay ( E ). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The CDK-inhibitor SNS-032 was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and the CDK-PROTAC THAL-SNS-032 was purchased from Tocris Bioscience (Bio-Techne R&D Systems, S.LU, Minneapolis, MN, USA).

Techniques: Expressing, Activity Assay, Western Blot, Control, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Antitumoral Activity of a CDK9 PROTAC Compound in HER2-Positive Breast Cancer

doi: 10.3390/ijms23105476

Figure Lengend Snippet: Antitumoral effect of SNS-032 and THAL-SNS-032 in 3D-matrix and adhesion assays in breast cancer lines and CDKs protein level evaluation after treatments. SNS-032 or THAL-SNS-032 (100 nM) was applied for 72 h to BT474, RH, and TDM1R cells seeded in a Matrigel matrix. Results presented refer to control cells. Scale bar = 100 μm ( A ). Cell adhesion to fibronectin substrate after 24 h of exposure to SNS-032 or THAL-SNS-032 (100 nM) ( B ). Expression levels of CDK9, CDK7, CDK1, and CDK2 in BT474, RH, and TDM1R cells treated with SNS-032 and THAL-SNS-032 at the times indicated at 50 nM. Scale bar = 100 μm ( C ). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The CDK-inhibitor SNS-032 was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and the CDK-PROTAC THAL-SNS-032 was purchased from Tocris Bioscience (Bio-Techne R&D Systems, S.LU, Minneapolis, MN, USA).

Techniques: Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Antitumoral Activity of a CDK9 PROTAC Compound in HER2-Positive Breast Cancer

doi: 10.3390/ijms23105476

Figure Lengend Snippet: Cell cycle analyses of SNS-032 and THAL-SNS-032 in BT474 and BT474-derived cell lines representative of adaptive resistance. Bar graph showing populations generated by flow cytometry in each phase of cell cycle for BT474, RH, and TDM1R cells of SNS-032 (50 nM) and THAL-SNS-032 (50 nM) for 24 h ( A ). Expression of proteins involved in cell cycle in cell lines treated with SNS-032 (50 nM) and THAL-SNS-032 (50 nM) for 24 h. GAPDH was used as a loading control ( B ). * p < 0.05.

Article Snippet: The CDK-inhibitor SNS-032 was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and the CDK-PROTAC THAL-SNS-032 was purchased from Tocris Bioscience (Bio-Techne R&D Systems, S.LU, Minneapolis, MN, USA).

Techniques: Derivative Assay, Generated, Flow Cytometry, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Antitumoral Activity of a CDK9 PROTAC Compound in HER2-Positive Breast Cancer

doi: 10.3390/ijms23105476

Figure Lengend Snippet: Apoptosis process of SNS-032 and THAL-SNS-032 in BT474 and BT474-derived cell lines representative of adaptive resistance. Cell death following SNS-032 (50 nM) or THAL-SNS-032 (50 nM) treatment after 72 h in BT474 and BT474-derived cell lines representative of adaptive resistance cell lines and resistant cell lines was evaluated by flow cytometry with annexin V and propidium iodide staining ( A ). Expression of proteins involved in cell death was evaluated by Western blot in cells lines treated at 50 nM (SNS-032 and THAL-SNS-032) for 72 h. GAPDH was used as a loading control ( B ). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The CDK-inhibitor SNS-032 was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and the CDK-PROTAC THAL-SNS-032 was purchased from Tocris Bioscience (Bio-Techne R&D Systems, S.LU, Minneapolis, MN, USA).

Techniques: Derivative Assay, Flow Cytometry, Staining, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Antitumoral Activity of a CDK9 PROTAC Compound in HER2-Positive Breast Cancer

doi: 10.3390/ijms23105476

Figure Lengend Snippet: THAL-SNS-032 therapeutic index exploration. Normal breast cancer cell line MCF10A was treated with 25, 50, and 100 nM of SNS-032 and THAL-SNS-032. Proliferation was evaluated by MTT assay after 72 h of treatment ( A ). Table summary of THAL-SNS-032 doses, frequency, and route of administration in in vivo studies ( B ). Mice engrafted with BT474 cells were treated with THAL-SNS-032 (at indicated doses, I.P.) and controls with excipient. Mean of tumor volume ± SEM at each point was represented. Day of dose administration and sacrifice are indicated in each graph ( C ). Mice weight during the in vivo experiments. Treated mice (specially 10 mg/kg and 5 mg/kg) lost weight after THAL-SNS-032 administration ( D ). Western blot showing the expression levels of CDKs in tumors treated or untreated with THAL-SNS-032 ( E ). Heat map of the dependency score obtained by DepMap to evaluate CDKs dependency after CRISPR (DepMap 21Q3 Public + Score, CERES) or RNAi (Achilles + DRIVE + Marcotte, DEMETER2) inhibition ( F ). Body map showing expression of CDK9, CDK7, CDK1, and CDK2 in normal tissues in Log2(Transcript Per Million + 1) Scale. Adapted from: GEPIA2 web server (Gene Expression Profiling Interactive Analysis. http://gepia2.cancer-pku.cn/ , accessed on 27 May 2021) ( G ). *** p < 0.001.

Article Snippet: The CDK-inhibitor SNS-032 was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and the CDK-PROTAC THAL-SNS-032 was purchased from Tocris Bioscience (Bio-Techne R&D Systems, S.LU, Minneapolis, MN, USA).

Techniques: MTT Assay, In Vivo, Western Blot, Expressing, CRISPR, Inhibition, Gene Expression

Journal: Oncotarget

Article Title: Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

doi:

Figure Lengend Snippet: (a) IC 50 values for CDK4/6 inhibitor, PD0332991 (palbociclib) and CDK2 inhibitors, SNS032 and dinaciclib in 10 ovarian cancer cell lines. Cell viability was measured using a luminometric assay following 7d (PD0332991) or 72 h (SNS032, dinaciclib) of exposure to increasing concentrations of drug. The IC 50 values displayed represent the mean plus SEM ( n = 2) ranked from top to bottom from the most sensitive (HEY) to the most resistant (OAW28). PD0332991-resistant cell lines have IC 50 values > 2 μM, while PD0332991-sensitive lines have IC 50 values < 0.5 μM. SKOV3 cells display intermediate sensitivity. In contrast, IC 50 values for SNS032 and dinaciclib fall along a continuum (0.05–0.25 μM for SNS032, 0.005–0.015 μM for dinaciclib). (b) CDKi-resistant cells avoid CDKi-mediated cell cycle arrest. Naïve HEY cells were treated with vehicle (control), 0.5 μM PD0332991, 0.2 μM SNS032 or 0.02 μM dinaciclib for 48 h and analyzed by BrdU/PI staining. HEY-PD R and HEY-SNS R cells were treated with 0.5 μM PD0332991 and 0.2 μM SNS032, respectively for 48 h. A representative FACS image is shown for each condition. (c) PD0332991 and SNS032 delay progression of HEY xenografts. HEY cells were injected into nude mice and treated with vehicle (control), 50 mg/kg PD0332991 or 15mg/kg SNS032 on day 8, 12, 15, 19 and 22 after injection. The data shown represent the mean tumor volume plus SEM ( n = 6). * p = 0.04 (SNS032-treated versus control) and p = 0.0002 (PD0332991-treated versus control). (d), (e) Downregulation of E2F target genes and ETS factors by CDKi. HEY cells were treated with vehicle (control), 0.5 μM PD0332991, 0.2 μM SNS032 or 0.02 μM dinaciclib for 48 h with subsequent RNA and protein isolation. Validation of candidate target genes by (d) qPCR and (e) Western blot analysis are shown. (f) Biochemical changes in CDKi-resistant cells. Lysates derived from two HEY-PD R clones (left) and two HEY-SNS R clones (right) were treated in the absence or presence of 0.2 μM SNS032 or 0.5 μM PD0332991, respectively, for 48 h and probed with the antibodies indicated. Naïve HEY cells were included as controls. SNS032 induces TP53 protein in naïve HEY but not in HEY-SNS R . Elevated cyclin E1 and reduced RB protein levels are present in HEY-PD R . (g) CDK2 inhibition sensitizes OVCAR3 cells to PD0332991. OVCAR3 cells were treated with 0.2 μM SNS032 in the absence or presence of 0.5 μM PD0332991. Cell viability was determined by crystal violet staining after 3 months.

Article Snippet: The following CDK inhibitors were used: PD0332991, SNS032 (Selleck Chemicals, Houston, TX) and dinaciclib (MedKoo Biosciences, Chapel Hill, NC).

Techniques: Luminescence Assay, Control, Staining, Injection, Isolation, Biomarker Discovery, Western Blot, Derivative Assay, Clone Assay, Inhibition

Journal: Oncotarget

Article Title: Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

doi:

Figure Lengend Snippet: CDKi-resistant cells restore expression of E2F target genes and ETS family genes Relative expression of known E2F targets ( a from Bracken et al ., 2004 [ 40 ]) and other select genes following exposure to 0.5 μM PD0332991 or 0.2 μM SNS032 in HEY cells for 48 h (left). Relative expression comparing HEY-SNS R to SNS-exposed HEY cells and HEY-PD R to PD0332991-exposed HEY cells (right). NS, not significant.

Article Snippet: The following CDK inhibitors were used: PD0332991, SNS032 (Selleck Chemicals, Houston, TX) and dinaciclib (MedKoo Biosciences, Chapel Hill, NC).

Techniques: Expressing

Journal: Oncotarget

Article Title: Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

doi:

Figure Lengend Snippet: (a) CDKi-resistant OV90, SKOV3 and OAW28 cells were generated by chronic exposure of parental cells to either 0.2 μM SNS032 alone or a combination of 0.2 μM SNS032 and 0.2 μM PD0332991. IC 50 values were determined using a luminometric viability assay. (b) Relative CCNE1 copy number across six ovarian cancer cell lines as determined by genomic qPCR and aCGH. (c) Recurrent gains and losses associated with CDKi resistance. Genomic DNA was isolated from parental and polyclonal populations of CDKi-resistant cells and subjected to aCGH analysis. Segmented log 2 values are shown for select genes. Red indicates de novo copy number gain, green indicates de novo copy number loss. *a focal amplification region close to the MYC locus is present in these cells. (d) Validation of aCGH results by genomic qPCR. Genomic DNA was isolated from OV90, SKOV3 and OAW28 parental and CDKi R cells. Relative DNA copy number for CCNE1 , RB1 , ERBB2 and MYC was determined by qPCR.

Article Snippet: The following CDK inhibitors were used: PD0332991, SNS032 (Selleck Chemicals, Houston, TX) and dinaciclib (MedKoo Biosciences, Chapel Hill, NC).

Techniques: Generated, Luminometric Viability Assay, Isolation, Amplification, Biomarker Discovery

Journal: Oncotarget

Article Title: Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

doi:

Figure Lengend Snippet: (a) HEY cells were stably transduced with retroviral pBABE-CCNE1 (red lines) or empty vector (black lines). Stable polyclonal populations were treated with 0.5 μM PD0332991 or vehicle over a course of 12 days and counted every four days. Ectopic cyclin E1 expression was confirmed by Western blot analysis and had no effect on proliferation in regular culture conditions (solid lines) but conferred partial resistance in the presence of PD0332991 (dotted lines). * p = 0.002 (b) Ectopic cyclin E1 expression rescues soft agar colony formation in PD0332991-treated HEY cells. Cells were seeded into soft agar followed by treatment with 0.2 μM PD0332991 or vehicle. Colonies were stained and counted after 2 weeks. Columns represent the relative mean plus SEM ( n = 3). * p = 0.01 (c) Depletion of cyclin E1 sensitizes OVCAR3 and (d) HEY cells to PD0332991-induced proliferation arrest. Two individual lentiviral shRNAs targeting CCNE1 were used to genetically deplete cyclin E1 in OVCAR3 and HEY cells. A shRNA targeting GFP was used as a control. After puromycin selection, cells were harvested to confirm CCNE1 knockdown by qPCR (relative CCNE1 expression is given as a percentage) and Western blot analysis (c, upper). Cells were seeded for viability using a luminometric assay in the absence or presence of 0.5 μM PD0332991 for 6d. Columns represent the relative mean plus SEM ( n = 3) * p ≤ 0.01 (c, lower). HEY-shGFP and HEY-shCCNE1 cells were seeded into soft agar and treated with vehicle or 0.2 μM PD0332991 (d, upper). Bar graph represents relative colony number compared to vehicle plus SD ( n = 3). * p ≤ 0.006. (e) Ectopic expression of mutant ERBB2 confers resistance to SNS032. OVCAR3 cells were stably transduced with retroviral pBABE-ERBB2 or empty vector. Stable polyclonal populations were treated with increasing concentrations of SNS032, and viability was assessed by a luminometric viability assay.

Article Snippet: The following CDK inhibitors were used: PD0332991, SNS032 (Selleck Chemicals, Houston, TX) and dinaciclib (MedKoo Biosciences, Chapel Hill, NC).

Techniques: Stable Transfection, Transduction, Retroviral, Plasmid Preparation, Expressing, Western Blot, Staining, shRNA, Control, Selection, Knockdown, Luminescence Assay, Mutagenesis, Luminometric Viability Assay

Journal: Oncotarget

Article Title: Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

doi:

Figure Lengend Snippet: (a) SKOV3-PD/SNS R release cells are hyperproliferative. CDKi-resistant SKOV3 cells were generated by chronic exposure to either 0.2 μM SNS032 alone or a combination of 0.2 μM PD0332991 and 0.2μM SNS032 for up to two years. SKOV3-PD/SNS R release cells were analyzed after 6 months in the absence of CDKi following two years of exposure. Proliferation rates of SKOV3-PD/SNS R , release and parental cells were determined using a luminometric viability assay. * p = 5.36E-06. (b) Western blot analysis of CDKi-resistant SKOV3 cells using antibodies against RB, RTK and PI3K signaling markers. CDKi-resistant cells hyperactivate ERBB2 and its downstream effectors MAPK and AKT, resulting in increased ETV5 protein expression. (c) ETV4 and ETV5 are upregulated in CDKi-resistant SKOV3 cells. Gene expression in SKOV3-PD/SNS R and parental SKOV3 cells was determined by qPCR. (d) Increased binding of E2F1 at the ETV5 promoter in CDKi-resistant cells. Chromatin from parental and CDKi-resistant SKOV3 cells was isolated and used in a ChIP assay with an E2F1 antibody or IgG control. Relative enrichment (E2F1/IgG) at the ETV5, CCND1 (left) and BRCA1 , BRCA2 (right) promoters is shown for naïve, early and late SKOV3-PD/SNS R cells. (e) ETV5 expression correlates with KRAS and CCNE1 DNA copy number in primary HGSOC. Using the TCGA dataset, expression of KRAS , ETV5 , CCNE1 and BRCA1 (y-axis) were plotted relative to KRAS (x-axis) and CCNE1 (z-axis) copy number status. ETV5 expression is positively correlated with KRAS copy number and inversely correlated with CCNE1 copy number.

Article Snippet: The following CDK inhibitors were used: PD0332991, SNS032 (Selleck Chemicals, Houston, TX) and dinaciclib (MedKoo Biosciences, Chapel Hill, NC).

Techniques: Generated, Luminometric Viability Assay, Western Blot, Expressing, Gene Expression, Binding Assay, Isolation, Control

Journal: International Journal of Molecular Sciences

Article Title: Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity

doi: 10.3390/ijms222312858

Figure Lengend Snippet: THAL-SNS032 induces degradation of CDKs in HFFs. ( A ) Chemical structures of SNS032 (upper panel) and THAL-SNS032 (lower panel). ( B ) Parallel cultures of HFFs were treated with 100, 10, or 1 nM of THAL-SNS032 and cells were harvested at 40 min, 2, 6, and 24 h. After 24 h of treatment, a removal and washout of THAL-SNS032 was performed and cells were then harvested at additional 40 min, 2, 6, and 24 h. ( C ) Cell lysates were used in Western blot analyses to detect the indicated CDKs; β-actin served as loading control.

Article Snippet: Stock aliquots of THAL-SNS032 and SNS032 (both Tocris, Wiesbaden-Nordenstadt, Germany), SEL 120 (MedChemExpress, Monmouth Junction, NJ, USA), roscovitine (Millipore-Calbiochem) and R22 (GPC Biotech AG, Martinsried, Germany) were prepared in DMSO and stored at −20 °C.

Techniques: Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity

doi: 10.3390/ijms222312858

Figure Lengend Snippet: THAL-SNS032 induces degradation of CDKs in HCMV-infected HFFs. HFFs were inoculated with HCMV-AD169 at a multiplicity of infection (MOI) of 2, and treated with THAL-SNS032 or controls (CDK9 inhibitor R22 [1 µM], solvent control DMSO) immediately after infection. After 3 h, 1 day or 3 days, cells were harvested and subsequently analyzed for their CDK steady-state levels by Western blot stainings; β-actin served as loading control.

Article Snippet: Stock aliquots of THAL-SNS032 and SNS032 (both Tocris, Wiesbaden-Nordenstadt, Germany), SEL 120 (MedChemExpress, Monmouth Junction, NJ, USA), roscovitine (Millipore-Calbiochem) and R22 (GPC Biotech AG, Martinsried, Germany) were prepared in DMSO and stored at −20 °C.

Techniques: Infection, Solvent, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity

doi: 10.3390/ijms222312858

Figure Lengend Snippet: Comparative analysis of the anti-HCMV activity exerted by THAL-SNS032 and SNS032. THAL-SNS032 and SNS032 were assayed for their antiviral activity in a GFP-based replication assay using HCMV AD169-GFP for the infection of HFFs. Antiviral compounds were added immediately p.i., starting at 1 µM followed by five three-fold dilution steps. Cells were lysed; 7 d p.i. to perform quantitative GFP fluorometry. Cell viability was determined by performing NRA in parallel uninfected cells. Values represent mean ± SD of triplicate (NRA) or quadruplicate (GFP) determinations. One representative experiment of at least four independent replicates is depicted.

Article Snippet: Stock aliquots of THAL-SNS032 and SNS032 (both Tocris, Wiesbaden-Nordenstadt, Germany), SEL 120 (MedChemExpress, Monmouth Junction, NJ, USA), roscovitine (Millipore-Calbiochem) and R22 (GPC Biotech AG, Martinsried, Germany) were prepared in DMSO and stored at −20 °C.

Techniques: Activity Assay, Infection

Journal: International Journal of Molecular Sciences

Article Title: Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity

doi: 10.3390/ijms222312858

Figure Lengend Snippet: Analysis of the THAL-SNS032 drug interaction with LDC4297 using the Loewe additivity fixed-dose assay. ( A ) HCMV AD169-GFP-infected HFFs are treated with the indicated concentrations of THAL-SNS032 and LDC4297, either as a single-compound treatment or as a combination of both. Inhibition of viral replication was determined by quantitating the cell-associated GFP fluorescence of compound-treated cells relative to the solvent control (DMSO). ( B ) The generated data were used to determine CI values at 50%, 75%, 90%, and 95% of virus inhibition. Subsequently, the weighted CI value (CI wt ) was calculated as: (1 × CI 50 + 2 × CI 75 + 3 × CI 90 + 4 × CI 95 )/10. ( C ) The drug interaction between THAL-SNS032 and the CDK8 inhibitor SEL120 was analogously determined and the respective CI and CI wt values were calculated. Data represent means ± SD of three experimental replicates.

Article Snippet: Stock aliquots of THAL-SNS032 and SNS032 (both Tocris, Wiesbaden-Nordenstadt, Germany), SEL 120 (MedChemExpress, Monmouth Junction, NJ, USA), roscovitine (Millipore-Calbiochem) and R22 (GPC Biotech AG, Martinsried, Germany) were prepared in DMSO and stored at −20 °C.

Techniques: Infection, Inhibition, Fluorescence, Solvent, Control, Generated, Virus

Journal: International Journal of Molecular Sciences

Article Title: Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity

doi: 10.3390/ijms222312858

Figure Lengend Snippet: THAL-SNS032 inhibits HCMV replication in MRC-5 cells and human first-trimester extravillous trophoblast (TEV-1) cells. Merlin-infected MRC-2 and TEV-1 cells were with various concentrations of THAL-SNS032. Viral replication was quantitated by HCMV-specific qPCR using cell culture supernatants collected at 7 p.i. Cell viability was determined in parallel by a Neutral Red uptake assay using uninfected MRC-5 and TEV-1 cells.

Article Snippet: Stock aliquots of THAL-SNS032 and SNS032 (both Tocris, Wiesbaden-Nordenstadt, Germany), SEL 120 (MedChemExpress, Monmouth Junction, NJ, USA), roscovitine (Millipore-Calbiochem) and R22 (GPC Biotech AG, Martinsried, Germany) were prepared in DMSO and stored at −20 °C.

Techniques: Infection, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity

doi: 10.3390/ijms222312858

Figure Lengend Snippet: Comparable analysis of anti-MCMV activity of THAL-SNS032 and SNS032. Bot compounds were analyzed for their antiviral activity in a GFP-based replication assay by titration on MCMV Smith-GFP-infected MEFs. A concentration of 1 µM was used as the starting point followed by six three-fold dilution steps. The cell-associated GFP fluorescence was used to quantitate viral replication 5 d p.i. Cell viability was determined in parallel using uninfected MEFs incubated with a compound for 5 d. Values represent means ± SD of biological triplicates (NRA) or quadruplicates (GFP).

Article Snippet: Stock aliquots of THAL-SNS032 and SNS032 (both Tocris, Wiesbaden-Nordenstadt, Germany), SEL 120 (MedChemExpress, Monmouth Junction, NJ, USA), roscovitine (Millipore-Calbiochem) and R22 (GPC Biotech AG, Martinsried, Germany) were prepared in DMSO and stored at −20 °C.

Techniques: Activity Assay, Titration, Infection, Concentration Assay, Fluorescence, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Development of a PROTAC-Based Targeting Strategy Provides a Mechanistically Unique Mode of Anti-Cytomegalovirus Activity

doi: 10.3390/ijms222312858

Figure Lengend Snippet: Anti-SARS-CoV-2 activity of THAL-SNS032 and SNS032 in a permissive human cell line. Caco-2 cells were cultivated in 96-well plates at 25,000 cells/well, infected with the recombinant SARS-CoV-2 d6-YFP reporter virus at an MOI of 0.003 and treated with the indicated concentrations of THAL-SNS032 and SNS032. At 30 h p.i., cells were harvested and viral replication was determined by quantitative fluorescence detection of virus-driven YFP expression using the fixed cells. Cell viability was determined from parallel cultures of uninfected Caco-2 cells by determination of release LDH after 48 h of treatment. Values represent means ± SD of biological quadruplicates (replication) or triplicates (LDH release). One representative experiment out of three is depicted.

Article Snippet: Stock aliquots of THAL-SNS032 and SNS032 (both Tocris, Wiesbaden-Nordenstadt, Germany), SEL 120 (MedChemExpress, Monmouth Junction, NJ, USA), roscovitine (Millipore-Calbiochem) and R22 (GPC Biotech AG, Martinsried, Germany) were prepared in DMSO and stored at −20 °C.

Techniques: Activity Assay, Infection, Recombinant, Virus, Fluorescence, Expressing

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of dCDK9-202 as a Highly Potent and Selective PROTAC CDK9 Degrader with Strong In Vivo Antitumor Activity

doi: 10.1021/acs.jmedchem.5c01111

Figure Lengend Snippet: Antitumor activity of 14 (dCDK9-202) in the TC-71 xenograft model in immunocompromised mice. (A) Changes in tumor volume during dCDK9-202, 6 (SNS032), or 10 (THAL-SNS032) treatments (10 mg/kg, i.v., qod). (B) Tumor weight at the end point of experiment. (C) Image of subcutaneous TC-71 xenograft tumors harvested at the end point of experiment. (D) Body weight of recipient mice during treatments. (E) Western blot analysis of CDK9 protein levels in tumor lysates harvested 4 h after final treatment (representative samples shown). Data in panels A, B, and D are presented as mean ± SD and were analyzed by one-way ANOVA (significance denoted as n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Article Snippet: Apart from those synthesized in-house, additional chemicals used in culture assays included DMSO (Solarbio), SNS032 (MedChemExpress), THAL-SNS032 (MedChemExpress), thalidomide (MedChemExpress), (R)-MG132 (Targetmol), and MLN4924 (Targetmol).

Techniques: Activity Assay, Western Blot