Dear Editor,
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.
Figure 1
Rapid and reversible FAK knockdown by FAK targeting PROTAC in male mice reproductive
organs. (A) Schematic depiction of the PROTAC strategy. FAK-PROTAC tool can act on
both enzymatic and non-enzymatic functions of FAK, while FAK inhibitor only act on
the enzymatic function of FAK. TP, Target Protein. (B) Chemical structure of FAK PROTAC,
as shown in the upper portion. Binding mode of PROTAC (ball stick), FAK (PDB 5TOB,
green) and CRL4-CRBN (PDB 2HYE and 4CI3, gray) was simulated by Pymol. (C and D) FAK
protein degradation in mice primary Sertoli cells and primary Germ cells, the cells
were treated at the indicated doses of FC-11 for 8 h. (E) Schematic depiction of FC-11
treated mice for FAK degradation and recovery. (F) FC-11 leads to more extensive FAK
degradation in testis, epididymis, seminal vesicle and preputial gland, respectively.
Each lane represented a single mouse (n = 4 or 5). (G) The recovery ability of FAK
in testis, epididymis, seminal vesicle and preputial gland in the indicated days after
withdraw administration (n = 6). All western blots are the representatives from at
least 3 experiments
Focal adhesion kinase (FAK), an embryonic lethal protein, exerts kinase-dependent
enzymatic functions and kinase-independent 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 non-enzymatic 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?
Encouraged by the results from primary cells, we continued to test FC-11 induced FAK
degradation in the reproductive tissues (testis, epididymis, seminal vesicle and preputial
gland) of male mice in vivo (Fig. S2). Ten-week-old male C57BL/6N mice were administered
intraperitoneal injections of FC-11 (20 mg/kg, twice daily [BID]), PF562271 (10 mg/kg,
BID), or vehicle control over a 5 day period (Fig. 1E). After 5 days treatment, all
FC-11 treated mice exhibited a more than 90% reduction of FAK and phosphor FAKtyr397
in the tested reproductive tissues, while PF562271 had no effect on the level of FAK
protein, but had an inhibitory effect on the phosphor FAKtyr397 levels (Figs. 1F and
S3). These results demonstrated that FC-11 can rapidly and efficiently degrade FAK
in the reproductive tissues of male mice. In addition, the location and expression
of FAK in the testis were detected by immunofluorescent. Immunostaining revealed that
FAK was mainly localized to the basal compartment of seminiferous tubules, which was
consistent with previously published data (Siu et al., 2009) (Fig. S4). As above,
FC-11 treatment significantly decreased the cytoplasmic expression of FAK, while PF562271
treatment had no effect on FAK levels, again demonstrating the totally different mechanisms
of action of the FAK-PROTAC protein degrader FC-11 and the FAK inhibitor PF562271.
Whether FAK protein could be recovered to normal levels after withdrawal of treatment?
The mice were raised normally for 2, 4, 6, 9, and 14 days, after withdrawing drug
treatment (Fig. 1E). FAK levels recovered gradually over time after FC-11 withdrawal.
Except in the preputial gland, the level of FAK in mouse reproductive organs (testis,
epididymis and seminal vesicle) was almost normal at 14 days after FC-11 withdrawal.
FAK levels in the preputial gland only recovered about 40% in 14 days (Fig. 1G). The
above results indicate that FC-11 can achieve reversible regulation of FAK in mice.
Based on the above results, FAK-PROTAC showed a potent and reversible FAK degradation
in mouse reproductive tissues of male mice, which indicated that FC-11 can be used
as a biological tool for FAK knockdown in vivo. Next, the possible functional consequence
differences between FAK PROTAC and FAK inhibitor in mouse reproductive system of male
mice were further observed. We administered FC-11 (20 mg/kg, BID), PF562271 (10 mg/kg,
BID), and vehicle control to 10 week old male C57BL/6N mice via intraperitoneal injections
for 13 days. After 13 days treatment, all FC-11 treated males exhibited a significant
reduction in the weight or size of the testis, epididymis, seminal vesicle, and preputial
gland of 24.2%, 37.5%, 51.6% and 90% of vehicle control, respectively. No reduction
was observed in PF562271-treated mice compared to the vehicle-treated group (Figs.
2A and S5). In addition, the number of viable sperm from the caudal epididymis was
markedly decreased in FC-11 treated mice. Analysis of sperm motility in FC-11 treated
males also revealed a significant (more than five fold) reduction compared to vehicle
males. However, there was no significant decrease in sperm viability in PF562271 treated
mice compared to vehicle control mice (Fig. 2B and 2C).
Figure 2
FAK PROTAC and FAK inhibitor showed different effects on reproductive tissues and
sperm phenotypes in male mice. (A) Organ index of each group mice (Organ index % =
organ weight/body weight × 100, n = 6). (B and C) Characteristics of sperm phenotype
(viable sperms and sperm motility) from each group (n = 6). (D–G) Statistical analysis
of in vitro fertilization rate of spermatozoa and the development of pre-implantation
mouse embryos in each group mice, the image represents the morphology of pre-implantation
mouse embryos in various developmental stages (n = 6). (H) TUNEL staining of testis
sections from each group mice (bar, 50 μm). (I) Statistic data of apoptosis cells
in seminiferous tubules of testis by TUNEL staining, about 160 seminiferous tubules
are selected for statistic in each group. The graphs depict mean ± SD of six mice,
ns: no significant, *
P < 0.05, **
P < 0.01, ***
P < 0.001 vs. vehicle group
In addition, the fertilization rate and the development of 2-cell, morula, and expanded
blastocyst embryos from FC-11 treated mouse sperm was strongly impaired compared to
embryos fertilized by sperm from vehicle mice, while the fertilization rate and embryos
fertilized by sperm from PF562271-treated mice displayed normal or only slightly impaired
embryonic development in vitro (Figs. 2D–G and S6). The observed decrease in sperm
fertility and impaired embryo development in FC-11 treated males imply that FC-11
has the potential to modulate the fertility of male mice. Furthermore, histopathology
and apoptosis analysis of the seminiferous tubules of the testis showed that FC-11,
but not PF562271, induced a significant increase in apoptosis of germ cells close
to the base membrane of seminiferous tubules compared to vehicle (Figs. 2H, 2I and
S7).
In summary, we have described developing a FAK-targeting PROTAC probe for chemical
biology study of related non-enzymatic function of FAK in murine reproductive system.
This novel strategy has an advantage over current FAK small molecule inhibitors because
inhibitors are only applicable to the study of enzymatic functions, not the study
of both enzymatic and non-enzymatic function. In this study, the result showed that
FAK can be degraded by more than 90% after representative degarder FC-11 treatment,
and that it can be recovered to normal levels within two weeks after withdrawing treatment
in vivo. In contrast to FAK inhibitor PF562271-treated mice, which exhibit an intact
reproductive system, FC-11-treated FAK knockdown mice exhibit low sperm viability
and motility, and subsequent decreased fertility and poor embryo development due to
the impairment of non-enzymatic FAK functions.
Furthermore, in order to make better use of the FAK PROTAC tool in mice, we also detected
the general distribution of FC-11 in mice with 20 mg/kg of FC-11 (BID) in 10 week
old male C57BL/6N mice through intraperitoneal injections for 5 days. The result displayed
that FC-11 can not penetrate the blood–brain barrier, and we did not detect the FAK
knockdown in brain of mice. However, FC-11 can work in other mice organs such as liver,
spleen, lung, and kindey with different degradation degree, which may further broaden
the potential application of FC-11 in other biological studies. In addition, from
the celluar FAK selectivity analysis, we also found that FC-11 did not degrade the
off-targets of PF562271 such as CDK1, CDK2, CDK7 and FLT3 (Roberts et al., 2008),
but FC-11 showed a very slight protein degradation in proline-rich tyrosine kinase
2 (Pyk2) in SRD15 cell line, the homolog of FAK, which have a highly consistent in
structure with FAK (45% homology with FAK in amino acid sequence and 61% homology
in catalytic domain) (Fig. S8) (Zheng et al., 1998).
Overall, our findings indicate that PROTACs can be used as chemical knockdown tools
to study the non-enzymatic functions of proteins, shedding the constraints of traditional
small molecule inhibitors. Unlike DNA- or RNA-based protein knockout technology, the
PROTAC strategy knocks down target proteins directly, rather than acting on the genome
or nucleic acid level, and is suitable for the functional study of embryonic-lethal
proteins in adult organisms. Finally, PROTAC probes also provide exquisite temporal
control, allowing the knockdown of a target protein of interest at specific developmental
time points and enabling the recovery of the target protein after withdrawal of drug
treatment.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary file1 (PDF 1397 kb)