HSF1
HSF1A targets TRiC/CCT (chaperonin containing TCP-1) complex (subunits including Tcp1/CCT1, Cct2, Cct3, Cct4, Cct5, Cct8); no IC50/Ki/EC50 values provided [1]
HSF1A targets TRiC/CCT complex; no IC50/Ki/EC50 values provided [2]
HSF1A targets HSF1 (heat shock transcription factor 1); no IC50/Ki/EC50 values provided [3]
HSF1A targets TRiC/CCT complex (CCT4/5 subunits); no IC50/Ki/EC50 values provided [4]

In vitro activity: HSF1A inhibits TRiC activity without interfering with ATP hydrolysis, binds TRiC subunits, and shields cells from stress-induced apoptosis. Human HSF1 is activated upon genetic inactivation or depletion of the TRiC complex, and the in vitro direct interaction between purified TRiC and HSF1 is inhibited by HSF1A. Moreover, HSF1A-FITC binds to a purified Tcp1 subunit of TRiC with an affinity of about 600 nM, according to fluorescence anisotropy experiments employing FITC coupled to HSF1A. Titration of purified Tcp1 into binding reactions containing 500 nM Biotin or HSF1A-Biotin serves as a qualitative validation of this[1]. Quantification using the count of aggregate-containing cells as a function of total cells shows that fewer aggregate-containing cells are seen at HSF1A concentrations as low as 2 µM. With pretreatment with 12 µM HSF1A, approximately 20% of the cells showed aggregates visible by fluorescence microscopy, as the fraction of cells containing aggregates decreased in a dose-dependent manner[2].

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1. HSF1A (50 μM) pretreated NIH3T3 cells for 15 h followed by 0.4 μg/ml tunicamycin for 15 h protects cells from stress-induced apoptosis; immunoblotting shows increased Hsp70 expression and reduced caspase-3 cleavage [1]
2. HSF1A (20 μM) pretreated INS 832/13 cells for 15 h before 0.5 mM palmitate/BSA treatment for 15 h protects cells from apoptosis, with elevated Hsp70 and reduced caspase-3 cleavage [1]
3. HSF1A (50 μM) pretreated NIH3T3 cells for 15 h before 5 mM homocysteine treatment for 15 h improves cell viability (Cell Titer Glo assay) and upregulates Hsp70 [1]
4. HSF1A (100 μM) binds TRiC subunits (Tcp1/CCT1, Cct2, Cct3, Cct4, Cct5, Cct8) in HeLa cell extracts (HSF1A-Biotin pull-down + immunoblotting); binds purified bovine TRiC and recombinant Tcp1 (fluorescence anisotropy, neutravidin-agarose pull-down) [1]
5. HSF1A (200 μM) inhibits TRiC-mediated actin refolding in vitro (DNaseI-agarose resin capture + autoradiography) without affecting TRiC ATP hydrolysis (ADP/ATP ratio measurement over time) [1]
6. HSF1A (200 μM) inhibits direct interaction between purified TRiC and HSF1 in vitro (cobalt-agarose resin pull-down + immunoblotting) [1]
7. HSF1A (10 μM) activates human HSF1 in yeast, promoting cell growth in glucose-containing medium (OD600 measurement); induces HSF1 multimerization (EGS cross-linking + SDS-PAGE + immunoblotting) [2]
8. HSF1A (30/50/80 μM) treatment of HSF1+/+ MEFs for 15 h upregulates Hsp70 and Hsp25 expression (immunoblotting); 80 μM HSF1A for 6 h increases Hsp70 mRNA levels (RNA blotting) [2]
9. HSF1A (80 μM) promotes HSF1 nuclear translocation in MEFs (nuclear/cytoplasmic fractionation + immunoblotting); synergizes with mild heat shock (40°C for 1 h) to enhance Hsp70 expression [2]
10. HSF1A (10 μM) pretreated HD-Q74 PC12 cells for 15 h followed by 1 μg/ml doxycycline for 48 h reduces httQ74-GFP aggregation (soluble/insoluble fraction immunoblotting, fluorescence microscopy; ~800 cells counted, p<0.01, p<0.001); 4 μM HSF1A pretreated PC12 cells for 15 h before doxycycline for 5 d improves cell viability (XTT assay, p<0.001) [2]
11. HSF1A (10 μM) pretreated H9c2 cells for 2 h followed by 1 μM DOX for 24 h stabilizes HSF1 expression, downregulates IGF-IIR and active caspase-3 levels (immunoblotting) [3]
12. HSF1A (100 μM) pretreated HeLa cells for 1 h inhibits TcdB (5 pM)-induced cell rounding (≥500 cells counted, p<0.001) and Rac1 glucosylation (immunoblotting with anti-Rac1 MAB102, p<0.05, p<0.01) [4]
13. HSF1A (50/100 μM) inhibits CCT4/5-mediated recovery of heat-treated TcdB^GT glucosyltransferase activity (UDP-[¹⁴C]glucose-based glucosylation assay of GST-RhoA, p<0.001) [4]

HSF1A reduces the cardiac damage caused by doxorubicin (DOX), stabilizes HSF1 expression, and increases HSF1 activity. WKY rats are given two challenges: 30 mg/kgw of DOX (accumulated dose) and 100 mg/kgw/day of DOX plus HSF1A. Heart function is considerably raised when HSF1A is supplemented, returning it to the control group's levels. In a model of neurodegenerative disease, HSF1A has been demonstrated to upregulate the nuclear translocation of human HSF1, increase the expression of protein chaperones, and reduce protein misfolding and cell death. According to the echocardiographic data, HSF1A also improves cardiac function impairments brought on by DOX[3].

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1. HSF1A (5 mM) supplemented in W¹¹¹⁸ fly food for 3 d upregulates Hsp70 expression (immunoblotting) [2]
2. HSF1A (400 μM) in food reduces eye morphological defects/depigmentation in UAS-MJDtrQ78 × gmr-GAL4 flies (polyQ-mediated neurodegeneration model) [2]
3. HSF1A administration to DOX-treated rats stabilizes HSF1 expression, downregulates IGF-IIR, Gαq, active caspase-3 and PARP levels in left ventricular heart tissue (immunoblotting, n=4 per group, p<0.05, p<0.01, p<0.001); reduces cardiac fibrosis (Masson's trichrome staining, n=4 per group, p<0.05) and improves fractional shortening (FS%, n=5 per group, p<0.001); decreases TUNEL⁺ cardiomyocytes (Ctrl:10.43±2.14; DOX:41.64±7.43; DOX+HSF1A:21.32±7.32, n=4 per group) [3]

Using biotin-binding buffer (20 mM HEPES, 5 mM MgCl₂, 1 mM EDTA, 100 mM KCl, 0.03% NP-40), in addition to protease inhibitors and 1% Trition-X100, protein extracts are produced from mammalian, yeast, and E. Coli cultures. After 4 hours at 4°C incubation with 100 μM HSF1A-Biotin, about 0.5 mg of protein extract is captured with NeutrAvidin Agarose Resin, corresponding with HSF1A-Biotin'sassociatedproteins. Once the proteins have been cleaned in the biotin binding buffer, they are resolved on a 4–20% SDS-PAGE and immunoblotted using 50 μL of biotin elution buffer (100 mM Tris, 150 mM NaCl, 0.1 mM EDTA, and 2 mM D-biotin). In order to analyze purified TRiC and Hsp70, 5 nM protein is incubated for 4 hours at 4°C in biotin-binding buffer+0.5% Triton X-100 with 100 μM biotin or 100 μM HSF1A-Biotin. The protein is then captured using NeutrAvidin Rinse. Different concentrations of Tcp1 (0.5 μM, 1 mM, 2 mM, 3 mM, and 4 mM) in 25 mM Hepes pH 7.5, 150 mM NaCl are incubated with 0.5 μM Biotin or HSF1A-Biotin for 4 hours at 4°C and then captured using NeutrAvidin Resin for NiNTA purified yeast Tcp1[1].

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1. TRiC-mediated actin refolding assay: Purified TRiC was treated with DMSO or HSF1A (200 μM), then incubated with denatured actin; folded actin was captured using DNaseI-agarose resin, resolved by SDS-PAGE and quantified by autoradiography to evaluate TRiC refolding activity inhibition by HSF1A [1]
2. TRiC ATP hydrolysis assay: Purified bovine TRiC was incubated with DMSO or HSF1A (200 μM) over time; ADP/ATP ratio was measured to assess whether HSF1A affects TRiC ATP hydrolysis activity [1]
3. TcdB^GT glucosyltransferase activity recovery assay: Heat-treated (48°C, 15 min) TcdB^GT was incubated with CCT4/5 (100 nM) in buffer containing 0.5 mM ATP for 1 h at 30°C, with/without HSF1A (50/100 μM); glucosylation of GST-RhoA was detected using UDP-[¹⁴C]glucose, and autoradiography was used to quantify TcdB^GT activity to evaluate HSF1A inhibition of CCT4/5-mediated TcdB^GT activity recovery [4]

PC12 cells seeded into a 96-well plate (5 ×10⁴ cells/well) are treated with increasing concentrations of HSF1A (2, 4, 8, and 12 μM) for 15 hours. Following this, httQ74-GFP expression is stimulated by incubating the cells for 5 days in the presence of 1 µg/mL Doxycycline. The XTT viability assay is used to evaluate cell viability[2].

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1. Apoptosis protection assay (NIH3T3): Cells were pretreated with HSF1A (50 μM) for 15 h, then exposed to 0.4 μg/ml tunicamycin for 15 h; total protein was extracted for immunoblotting to detect Hsp70, Bip, Erdj3 and cleaved caspase-3 (GAPDH as loading control) to evaluate anti-apoptotic effect [1]
2. Apoptosis protection assay (INS 832/13): Cells were pretreated with HSF1A (20 μM) for 15 h, then treated with 0.5 mM palmitate/BSA for 15 h; total protein was extracted for immunoblotting to detect Hsp70 and cleaved caspase-3 (GAPDH as loading control) [1]
3. Cell viability assay (NIH3T3): Cells were pretreated with HSF1A (50 μM) for 15 h, then treated with 5 mM homocysteine for 15 h; cell viability was measured using Cell Titer Glo assay [1]
4. HSF1A-TRiC binding assay (HeLa): Cell extracts were incubated with 100 μM HSF1A-Biotin, proteins were purified with neutravidin-agarose, resolved by SDS-PAGE and immunoblotted for TRiC subunits (Tcp1, Cct2, Cct3, Cct4, Cct5, Cct8) and HSF1 [1]
5. HSF1A-Tcp1 binding assay (E. coli/ purified protein): E. coli extracts expressing FLAG/His₆-tagged Tcp1 (full-length/fragments/mutants) were incubated with 100 μM HSF1A-Biotin (or Biotin as control) and purified with neutravidin-agarose; immunoblotting with anti-FLAG/His tag antibody was used to detect Tcp1-HSF1A binding; fluorescence anisotropy was used to assess affinity of HSF1A-FITC for purified recombinant Tcp1 [1]
6. HSF1 activation assay (yeast): Yeast cells expressing human HSF1 were treated with 10 μM HSF1A or DMSO and grown in 96-well plates for 4 d; growth was monitored by OD600 measurement; EGS cross-linking + SDS-PAGE + immunoblotting were used to detect HSF1 multimerization [2]
7. Hsp70 expression assay (MEFs): HSF1+/+ and HSF1⁻/⁻ MEFs were treated with increasing concentrations of HSF1A (30/50/80 μM) for 15 h or heat-shocked (42°C for 2 h + 15 h recovery); total protein was extracted for immunoblotting to detect Hsp70 and Hsp25 (SOD1 as loading control); total RNA was extracted for RNA blotting to detect Hsp70 mRNA [2]
8. HSF1 nuclear translocation assay (MEFs): HSF1+/+ MEFs were treated with 80 μM HSF1A for indicated times or heat-shocked (42°C for 2 h); nuclear/cytoplasmic fractions were prepared and immunoblotted for HSF1 (c-fos as nuclear marker, SOD1 as cytoplasmic marker) [2]
9. PolyQ aggregation inhibition assay (PC12): HD-Q74 PC12 cells were pretreated with 10 μM HSF1A for 15 h, then 1 μg/ml doxycycline was added for 48 h; soluble/insoluble fractions were extracted for immunoblotting with anti-GFP antibody to detect httQ74-GFP; fluorescence microscopy was used to count aggregate-containing cells (≈800 cells per group); XTT assay was used to measure cell viability after 4 μM HSF1A pretreatment + 5 d doxycycline treatment [2]
10. TcdB-induced cell rounding assay (HeLa): Cells were pretreated with HSF1A (0-100 μM) for 1 h, then intoxicated with TcdB (5 pM); cell rounding was observed under microscopy after 90 min, and ≥500 cells were counted to quantify the percentage of rounded cells [4]

Rats: Wistar Kyoto rats (WKY) aged ten weeks are employed. The rats live in housing that is consistently 22°C, with a 12-hour light/dark cycle, food, and tap water. Three groups of rats—the control group, DOX rats, and DOX rats treated with HSF1A—are used to house the animals. There are five animals in each group. The DOX group receives intraperitoneal injections of DOX (5 mg/kg) for 6 weeks in a row. This results in a cumulative dose of 30 mg/kg, which has been shown to cause cardiotoxicity. Intraperitoneal injection of the small molecular HSF1 activator HSF1A (100 mg/kg/day) is administered.

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1. Drosophila feeding assay: W¹¹¹⁸ flies were raised on food supplemented with DMSO, 5 mM HSF1A or 0.15 mM geldanamycin for 3 d; total protein was extracted from flies for immunoblotting to detect Hsp70 (actin as loading control); UAS-MJDtrQ78 × gmr-GAL4 flies were fed food with DMSO, 400 μM HSF1A or 5 μM 17-AAG to evaluate eye morphological defects [2]
2. Rat DOX-induced cardiotoxicity assay: Rats were divided into control, DOX-treated, and DOX + HSF1A groups; HSF1A was administered to mitigate DOX-induced cardiac damage (administration route/frequency/dissolution formula not specified); echocardiography was used to assess fractional shortening (FS%); heart tissues were collected for hematoxylin-eosin (HE) staining, Masson's trichrome staining (cardiac fibrosis quantification), immunoblotting (HSF1, CHIP, IGF-IIR, Gαq, caspase-3, PARP) and TUNEL assay (TUNEL⁺ cardiomyocytes quantification) [3]

[1]. A direct regulatory interaction between chaperonin TRiC and stress-responsive transcription factor HSF1. Cell Rep. 2014 Nov 6;9(3):955-66.

[2]. Modulation of heat shock transcription factor 1 as a therapeutic target for small molecule intervention in neurodegenerative disease. PLoS Biol. 2010 Jan 19;8(1):e1000291.

[3]. Doxorubicin attenuates CHIP-guarded HSF1 nuclear translocation and protein stability to trigger IGF-IIR-dependent cardiomyocyte death. Cell Death Dis. 2016 Nov 3;7(11):e2455.

[4]. The chaperonin TRiC/CCT is essential for the action of bacterial glycosylating protein toxins like Clostridium difficile toxins A and B. Proc Natl Acad Sci U S A. 2018 Sep 18;115(38):9580-9585.

1. HSF1A is a small molecule activator of HSF1; it binds to the TRiC/CCT complex, inhibits the TRiC-HSF1 interaction, thereby activating HSF1 and upregulating Hsp70 to protect cells from proteotoxic stress [1]
2. The structure of HSF1A is different from other HSF1 activators; it does not inhibit Hsp90 (unlike geldamycin/redicillin/17-AAG) and has no adverse proteotoxic activity [2]
3. HSF1A can improve protein misfolding and cell death in polyQ-expressing neuronal progenitor cells and plays a protective role in polyQ-mediated neurodegenerative Drosophila models, preventing cytotoxicity [2]
4. HSF1A can stabilize HSF1 expression, thereby inhibiting the IGF-IIR apoptosis signaling pathway and minimizing DOX-induced cardiac damage in vitro and in vivo [3]
5. HSF1A blocks the cytotoxic effects of bacterial glycosylated toxins (TcdA/TcdB) by inhibiting TRiC/CCT-mediated toxin refolding/reactivation [4].

C21H19N3O2S2

409.52

409.092

C, 61.59; H, 4.68; N, 10.26; O, 7.81; S, 15.66

1196723-93-9

44472508

White to off-white solid powder

6.117

1

5

6

28

595

0

C1(S(NC2N(C3=CC=CC=C3)N=C(C3SC=CC=3)C=2)(=O)=O)=CC=C(CC)C=C1

KJTITGSAONQVPY-UHFFFAOYSA-N

InChI=1S/C21H19N3O2S2/c1-2-16-10-12-18(13-11-16)28(25,26)23-21-15-19(20-9-6-14-27-20)22-24(21)17-7-4-3-5-8-17/h3-15,23H,2H2,1H3

4-ethyl-N-(2-phenyl-5-thiophen-2-ylpyrazol-3-yl)benzenesulfonamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.10 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.10 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.10 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)