FAK-targeting PROTAC as a chemical tool for the investigation of non-enzymatic FAK function in mice

Animal models, most commonly mice, that lack a protein of interest play an important role in phenotypic and functional studies of a target gene, allowing researchers to answer various biological questions (Chaible et al., 2010). At present, a variety of tools act at the DNA or RNA level to enable researchers to model gene function (and thus protein) deficiency, including nucleic acid-based RNA interference (Elbashir et al., 2001), antisense oligonucleotides (Schoch and Miller, 2017), and genome editing-based CRISPR-Cas9 (Doudna and Charpentier, 2014) strategies. However, challenges remain. RNA and DNA-based technologies lack exquisite temporal control of the target gene at specified time points in an organism’s development, and they fail to realize acute and reversible target gene function (Chan, 2013). These shortcomings have garnered widespread concern in both fundamental research and drug development. Furthermore, gene knockout will often lead to embryonic lethality, precluding the study of post-embryonic pathophysiological functions of target genes and proteins of interest (Dhanjal et al., 2017). Proteolysis targeting chimera (PROTAC) is a novel chemical knockdown technology for the post-translational study of proteins of interest. PROTACs are hetero-bifunctional small molecules, which can drive E3 ubiquitin ligase to bind with the target protein, resulting in ubiquitination of the target protein and consequent proteasome-mediated degradation (Raina and Crews, 2010) (Fig. 1A). Unlike classic inhibitors, PROTAC eliminates rather than inhibits both enzymatic and non-enzymatic protein functions. Furthermore, unlike nucleic acid (e.g., siRNA) and genome editing-based (e.g., CRISPR-Cas9) strategies (Cong et al., 2013; Deng et al., 2014), the small molecule-based PROTAC approach is capable of degrading target proteins without requiring any genetic manipulation, guaranteeing the integrity and stability of the genome, which especially suitable for knockdown of embryonic lethal protein. Thus, PROTACs offer significantly broader therapeutic applicability than DNA or RNA-targeting strategies for protein knockdown in vivo. Focal adhesion kinase (FAK), an embryonic lethal protein, exerts kinase-dependent enzymatic functions and kinaseindependent scaffolding functions (Hall et al., 2011). Both functions are crucial in reproduction and early embryonic development (Gungor-Ordueri et al., 2014). Many essential non-enzymatic functions of FAK cannot be investigated with reported FAK kinase inhibitors. To the best of our knowledge, the PROTAC strategy has not been used to study the nonenzymatic function of FAK in vivo. It is also unknown whether FAK PROTACs will yield different phenotypes or reveal different FAK functions than kinase-dependent FAK inhibitors in vivo. For these reasons, we have chosen FAK as a target to demonstrate the potential utility of the PROTAC strategy for the study of non-enzymatic protein function in mouse reproductive system in vivo. Based on the previous studies of our laboratory, we synthesised the FAK-targeting PROTAC library with FAK ligand of PF562271, cereblon (CRBN)-based E3 ubiquitin ligase ligand of thalidomide, and a variable length of polyethylene glycol or alkyl linkers (Fig. S1). Firstly, we screened the degradation effect of FAK targeting PROTAC molecules in mice primary reproductive related cells. We separated and purified testis-related cells, including primary Sertoli cells and primary germ cells, from 6 dpp C57BL/6N mice, and tested whether degradation resulted from FAK PROTAC library molecules in these primary cells. A remarkable degradation effect, with a DC50 of 1.3 nmol/L for primary Sertoli cells and 0.4 nmol/L for primary germ cells, was observed from FAK-PROTAC library molecules, which we confirmed it is FC-11, a PROTAC from our previous reported work (Gao et al., 2019) (Fig. 1B–D). The optimized synthesis route of FC-11 was shown in Supplementary Materials (Scheme 1). Next, in order to overcome the defect of FAK knockdown in vivo caused by existing genetic tools and to clarify the effect of PROTAC tools on the non-enzymatic function of protein in the mouse reproductive system, a few critical issues need to be addressed: 1. Can FC-11 degrade FAK in vivo? 2. If it can, is there any different phenotypes between FAK PROTAC and FAK inhibitor? 3. Is the FAK protein degradation reversible?

Sertoli cell and germ cell cultures. C57BL/6N mice at 6 days of age were used for primary undifferentiated germ cells and Sertoli cells isolation. The method was as previously described, with minor modifications (Kanatsu-Shinohara et al., 2003;Kokkinaki et al., 2009;Wang et al., 2015). Briefly, testis were decapsulated with tweezers under the dissection microscope, and then seminiferous tubules were detached gently and washed with DPBS 3 times. The tubules were digested with 1 mg/mL collagenase IV for 5 min at 37°C, then washed 3 times with F12-DMEM and further digested with 0.25% trypsin for 5 min at 37°C. The digested product, containing primarily Sertoli cells and undifferentiated germ cells, was seed in Animals. The experiment animals (C57BL/6N, 10 weeks of age) used in this study were approved by the Tsinghua University Institutional Animal Care and Use Committee (protocol number 18-RY1). Mice were housed in a SPF (specific pathogen-free) unit on a 12 h light/dark cycle (lights off at 19:00) with the ambient temperature is 24 ± 2°C and the humidity is 50 ± 5%, free access to food and water.
Six mice were raised in one polyacrylic cage and all the animals were housed for one week under controlled conditions before the experiments. During the period of the study, the care and use of animals were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
After drug administration, the mice were euthanized by CO2 asphyxiation using slow (20%-30%/min) displacement of chamber air with compressed CO2 at specified time points. Testis, epididymis, seminal vesicle and preputial gland in male mice were removed immediately. The whole testis was fixed with 4% paraformaldehyde (Santa Cruz Biotechnologies, sc-281692), then embedded in paraffin for hematoxylin and eosin staining (H&E) and immunofluorescence (IF) analysis. Other tissues were snapped frozen in liquid nitrogen for western blot analysis. Each time point had at least 3 mice, including controls.

FAK knockdown and recovery in vivo.
FC-11 was dissolved in DMSO at 40 mg/mL, diluted with Cremophor-EL (C5135, Sigma) and PBS solution in the ratio of 1:2:17 (Drug:Cremophor-EL:PBS) to the work dose of 2 mg/mL. PF562271 was dissolved in DMSO at 20 mg/mL, diluted with the same solvent as FC-11 to 1 mg/mL.
The vehicle was DMSO dissolved with Cremophor-EL and PBS in the ratio of 1:2:17.
The drug mixture was injected into mice through intraperitoneal (i.p.) injection with the volume of 10 mL/kg, twice a day (BID). At the end of the treatment, take the related tissues immediately and snap frozen in liquid nitrogen for Western blot analysis. All the mice in these studies were weighed daily before injections and fed ad libitum.
For the recovery studies, the mice were treated as the above description for 5 days, then stopped the drug treatment, and removed the testis, epididymis, seminal vesicle and preputial gland immediately at the indicated days (0, 2, 4, 6, 9 and 14 days post-drug treatment), then snap frozen in liquid nitrogen for FAK protein levels analysis.
Caudal epididymis spermatozoa from one male were prepared in 1 mL of prewarmed Quinn's Advantage® Fertilizationmedia, and incubated at 37°C for 30 min in a 5% CO2 incubator prior to sperm number counting and sperm motility analysis. After incubation, sperm samples were diluted with the ratio of 1:100 in 1 mL warmed HTF media (contained with 90% Trypan blue for viable cells staining, Gibco, 15250061) and spotted onto a glass slide, covered with a 22 × 22 mm cover slip.
When the sperm settled down after about 2 min, sperms were counted under the differential interference contrast (DIC) optics with a 20 objective.

Sperm motility percentages and velocities (average path velocity) analyzed with
Hamilton Thorne's CEROS II system (Ceros, Hamilton-Thorne, Beverly, MA). The procedure was repeated three times for each sample and averaged as described previously (Mortimer et al., 1997;Roy et al., 2007). FITC-phalloidin (40735ES75, Yeasen) and TUNEL (G3250, Promega) were stainned according to the protocol of the product from the manufactory.
In vitro fertilization. In vitro fertilization was performed with some modifications based on previous described (Guan et al., 2014). 10-week-old adult male mice were given daily intraperitoneal injections of FC-11 (20 mg/kg, BID), PF562271 (10 mg/kg, BID), or vehicle control over a 13 day period. After treatment, mice were sacrificed and sperm were immediately taken from the caudal epididymis for IVF. Collect mature sperms from caudal epididymis of FC-11 treated males, and added to the HTF medium droplet to capacitation for 1 h at 37°C, 5% CO2 incubator. Then, collect the same number of cumulus-intact oocytes from superovulated WT female mice (4-week-old), pooled the oocytes and divided into several groups to the fertilization droplet. Put the capacitated sperm to the fertilization droplet containing the eggs to fertilization at 37°C, 5% CO2 incubator. After 4 to 6 hours incubation, the eggs were washed to remove unbound sperm and transferred to new fertilization droplets. Fertilization rates were evaluated by recording the number of zygote numbers in each group. The development of embryo were evaluated by recording the number of two-cell embryo at twenty-four hours, the morula at seventy-two hours and the expanded blastocyst at ninety-six hours.
Statistical analysis. All data are presented as the mean ± SD, and repeated at least three times. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test was used as a calculated statistical method for data comparison among different treatment groups with Graphpad Prism Version 6. All statistics are representative of biological replicates ( * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significant).