Rational Design and Synthesis of HSF1-PROTACs for Anticancer Drug Development

PROTACs employ the proteosome-mediated proteolysis via E3 ligase and recruit the natural protein degradation machinery to selectively degrade the cancerous proteins. Herein, we have designed and synthesized heterobifunctional small molecules that consist of different linkers tethering KRIBB11, a HSF1 inhibitor, with pomalidomide, a commonly used E3 ligase ligand for anticancer drug development.


Introduction
Cancer continues to be one of the leading causes of morbidity and mortality worldwide, accounting for 10 million deaths in 2020 [1]. It has an immense economic impact, which is escalating, in addition to the huge loss of human lives. According to the WHO report, the total annual economic cost of cancer in 2010 was estimated at approximately USD 1.16 trillion [2]. The existing treatment by traditional chemotherapy, targeted therapy, and immunotherapy is fraught with the disadvantages of severe adverse reactions and toxicity, resistance to the treatment therapy, and excessive uncontrollable activity of the immune system [3][4][5][6]. Thus, new strategies to combat cancer are an urgent medical need.
Targeting proteins that contribute to cancer development for therapeutic purposes is challenging, and most of the proteome is still considered beyond reach that is deemed undruggable. Sakamoto et al. reported a novel chemical strategy in 2001, which can be used to rationally manipulate the intracellular levels of many proteins that are regulated by the molecular mechanism of ubiquitin-dependent proteolysis [7]. This technology opened new avenues in anticancer drug discovery and was also commercialized as PROTAC ® for targeted protein degradation [8]. Proteolysis-targeting chimeras are heterobifunctional molecules that can target any protein for ubiquitination and subsequent proteasomal degradation [9]. Further, lysosomal targeting with lysosomal-targeting chimeras (LYTACs), autophagy targeting with autophagy-targeting chimeras (AUTACs), and RNA targeting with ribonuclease-targeting chimeras (RIBOTACs) have been reported by recruiting alternative degradation pathways [10].
HSF1 is a master regulator of transcriptional responses to proteotoxic stress and an effective drug target for anticancer therapies including radiotherapy and thermotherapy [11]. HSF1 is known to be overactive or overexpressed in many cancer types including prostate cancer, pancreatic cancers, breast cancer, colorectal, lymphomas, melanoma, oral cancers, and so forth [12]. The activation of HSF1 essentially results in the over-expression of various heat shock proteins, which affects the outcome of the treatment.
The over-expression of heat shock proteins caused by HSF1 is reported as the main cause of resistance to chemotherapy, radiation therapy, and hypothermia used in cancer treatment [13]. Although HSF1 has been found to be a significant factor in the development and low prognosis of various chemotherapy cancer treatments, there are still very few effective inhibitors of HSF1 [14]. Most HSF1 inhibitors reported to date are indirect inhibitors through high signal disturbance in HSF1 rather than directly inhibiting HSF1. Continuing our effort of developing anticancer drug candidates [15,16], we report the rational design and synthesis of HSF1-PROTACs in an endeavor to create new chemical agents for the development of appropriate clinical candidates in anticancer drug discovery.

Results and Discussion
Our design rationale for making PROTAC involved tagging a known binder of HSF1 protein. A 3-nitropyridine derivative (N2-(1H-indazole-5-yl)-N6-methyl-3-nitropyridine-2,6-diamine) named KRIBB11 is a well-known HSF1 inhibitor used to treat tumor tissues without significant body weight loss [17]. The docking between the crystal structure HSF1 [18] and KRIBB11 revealed that the hydrogen bonding by the oxygen atoms of the nitro group of the inhibitor stabilizes the binding to HSF1 protein. The nitrogen atoms of the pyridine ring and the N-methyl group of the inhibitor form hydrogen bonding interactions with the amide group of Gln72 and the phenol group of Tyr76, respectively. The N-methyl moiety in KRIBB11 is projected away from the protein, as shown in Figure 1. Thus, we decided to link the E-3 ligase ligand to the N-methyl part of KRIBB11. We employed three different linkers. Phthalimide conjugation is the most effective strategy for proteasomemediated proteolysis [19]. Accordingly, we designed the PROTAC molecules by connecting KRIBB11 and pomalidomide as a ligand for E3 ligase. We began the synthesis for making the KRIBB11 part by treating 2,6-dichloro-3nitropyridine with 5-aminoindazole in the presence of triethylamine in ethanol, resulting in the formation of 3 in good yield [20]. Subsequent reaction with 1,4-diaminobutane in acetonitrile at room temperature yielded 4, as shown in Scheme 1. The pomalidomide part 9c and a precursor 9a was synthesized using the previously reported procedure, as illustrated in Scheme 2 [21]. The linkers were connected to the pomalidomide, as shown in Scheme 3. Firstly, the 4,7,10-Trioxatridecane-1,13-diamine 10a was mono-Boc protected into 10b using Boc anhydride and triethylamine in anhydrous dichloromethane. It was then treated with 9a in the presence of diisopropylethylamine in DMF to form 12a, which was further reacted with TFA to give 12b in good yield. The mono Boc-protected 4,7,10-Trioxatridecane-1,13-diamine 10b was also reacted with chloroacetyl chloride in the presence of Cs 2 CO 3 to give 11. It was then treated with 9c to form 13a, which, on subsequent TFA treatment, gave 13b. Finally, the HSF1-PROTACs were synthesized by nucleophilic aromatic substitution, as shown in Scheme 4. The PROTAC 14 was synthesized by heating 4 and 9a in DMF in the presence of diisopropylethylamine. The PROTACs 15 and 16 were synthesized by reacting 3 at room temperature with 12b and 13b, respectively, in the presence of triethylamine in acetonitrile. The copies of 1 H and 13 C spectra for all prepared compounds are given in Supplementary Figures S1-S19. PROTACs were purified by MPLC, and the purity was also determined by HPLC (Supplementary Figure S20).   We further evaluated the proteolysis effect of compounds 14-16 on HSF1 ( Figure 3). Our Western blotting data indicated that 14 showed a little degradation of HSF1. However, the results were not consistent despite several repetitions. We speculated that one reason for inconsistency could be due to the intrinsic formation of Hsp90-Hsp70-HSF1 multichaperone complex in the cancer cell, hampering compound 4 to interact with the binding site of HSF1. HSF1 is dynamically associated or dissociated from multi-chaperone complex under various stressed or non-stressed conditions. Hence, we decided to treat cancer cells with compounds 14-16, together with an Hsp90 inhibitor, which could induce a heat shock response (HSR) to dissociate HSF1 from the chaperone complex. Unfortunately, the results did not still show any meaningful degradation of HSF1 in MDA-MB-231 cells, shown in Figure 3B. To draw biological meaningful conclusions, our efforts are directed toward finding better PROTACs with new HSF1 ligands and E3 ligase ligands. The result will be reported in due course.

General Information
All reagents and solvents were purchased from commercial suppliers and used without further purification. All experiments dealing with moisture-sensitive compounds were carried out under argon atmosphere. Concentration or solvent removal under reduced pressure was carried out using rotary evaporator. Analytical thin layer chromatography was performed on precoated silica gel F254 TLC plates (E, Merck) with visualization under UV light or by staining using iodine. Column chromatography and medium-pressure liquid chromatography (MPLC) was conducted on silica (Merck Silica Gel 40-63 m) or performed by using a Biotage SP1 flash purification system with prepacked silica gel cartridges (Biotage, Uppsala, Sweden). NMR analyses were carried out using a JNM-ECZ500R (500 MHz) manufactured by Jeol resonance. Chemical shifts are reported in parts per million (δ). The deuterium lock signal of the sample solvent was used as a reference, and coupling constants (J) are given in hertz (Hz). The splitting pattern abbreviations are as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; td, triplet of doublet; and m, multiplet. The purities of all final compounds were confirmed to be higher than 95% by analytical HPLC performed with a dual-pump Shimadzu LC-6AD system equipped with a VP-ODS C18 column (4.6 mm × 250 mm, 5 µm, Shimadzu, Kyoto, Japan).

Synthesis of 9c
A mixture of 36 mL glacial acetic acid and (0.308 g, 3.75 mmol) anhydrous sodium acetate was added to (0.58 g, 3 mmol) of 3-nitrophthalic anhydride (0.57 g, 2.5 mmol) of compound 7. The reaction mixture was heated at 140 • C for 18 h. After completion of the reaction, the reaction mixture was cooled to rt, water was added to the reaction mixture, and it was stirred for another 30 min. The solid was filtered and washed with water. The product was dried under vacuum to obtain 9b in 44.5% yield. The compound 11 (0.14 g, 0.8 mmol) and (0.108 g, 0.96 mmol) chloroacetyl chloride in Acetonitrile (2 mL) was treated with cesium carbonate (0.18 g, 1.4 mmol) at rt for 3 h. The reaction mixture was filtered, and the solvent was removed under reduced pressure. The crude product was purified by column to give compound 13a in 33% yield. 1 H-NMR poses of the ligand inside a known or predicted binding site utilizing the Lamarckian version of the genetic algorithm, where the changes in conformations adopted by molecules after in situ optimization are used as subsequent poses for the offspring. In the docking experiments carried out, Gasteiger charges were placed on the X-ray structure of HSF1 along with KRIBB11, using tools from the AutoDock suite [23]. A grid box centered on the substrate binding pocket of HDACs enzyme with definitions of 50 × 50 × 50 points and 0.375 Å spacing was chosen for ligand docking experiments. The docking parameters consisted of setting the population size to 150, the number of generations to 27,000, and the number of evaluations to 2,500,000;the number of docking runs was set to 100, with a cutoff of 1 Å for the root-mean-square tolerance for the grouping of each docking run. The docking pose of HSF1 with compound KRIBB11 was depicted in Figure 1, and rendering of the picture was generated using PyMol (DeLanoScientific, South San Francisco, CA, USA) [24].

MTS Colorimetric Assay
MDA-MB-231 cells were seeded at 2 × 103 cells per well in a clear 96-well plate, the medium volume was brought to 100 µL, and the cells were allowed to attach overnight. The cells were then incubated with the indicated concentration of compound 14, 15, or 16 at 37°C for 24 h. Cell viability was determined using the Promega Cell Titer 96 Aqueous One Solution cell proliferation assay. Absorbance at 490 nm was read on Tecan Infinite F200 Pro plate reader, and values were expressed as percent of absorbance from cells incubated in DMSO alone.

Western Blot
Cells were seeded in 100 mm culture dishes (1 × 106 cells/dish) and allowed to attach overnight. The compound was added at varying concentrations, and the cells were incubated for an additional 24 h. For comparison, cells were also incubated with DMSO (0.5%) for 24 h. The cells were harvested in ice-cold lysis buffer (23 mM Tris-HCl pH 7.6, 130 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS), and 30 µg of lysate per lane was separated by SDS-PAGE, which was transferred to a PVDF membrane (Bio-Rad). The membrane was blocked with 5% skim milk in TBST and then incubated with the corresponding antibody (HSF-1 or β-actin). After binding of an appropriate secondary antibody coupled to horseradish peroxidase, proteins were visualized by ECL chemiluminescence according to the instructions of the manufacturer (GE healthcare, Chicago, IL, USA).

Conclusions
Keeping in mind the importance of HSF1 as a master regulator of heat shock protein and as a potential target for the development of anticancer drugs, we designed and synthesized new HSF1-based proteolysis-targeting chimeric molecules HSF1-PROTACs, in which the pomalidomide is tethered with HSF1 binder KRIBB11 to unlock the untapped potential and expand the repertoire of target warhead that can be recruited by PROTACs for anticancer drug development. Further evaluation to find better PROTACs with newer targets is underway in our lab and will be published in a due course of time.