Targeting androgen regulation of TMPRSS2 and ACE2 as a therapeutic strategy to combat COVID-19

Abstract

Epidemiological data showing increased severity and mortality of COVID-19 in men suggests a potential role for androgen in SARS-CoV-2 infection. Here, we present evidence for the transcriptional regulation of SARS-CoV-2 host cell receptor ACE2 and TMPRSS2 by androgen in mouse and human cells. Additionally, we demonstrate the endogenous interaction between TMPRSS2 and ACE2 in human cells and validate ACE2 as a TMPRSS2 substrate. Furthermore, camostat-a TMPRSS2 inhibitor-blocked the cleavage of pseudotype SARS-CoV-2 surface Spike without disrupting TMPRSS2-ACE2 interaction, thus providing evidence for the first time of a direct role of TMPRSS2 in priming the SARS-CoV-2 Spike, required for viral fusion to the host cell. Importantly, androgen-deprivation, anti-androgens, or camostat attenuated the SARS-CoV-2 S-mediated cellular entry. Together, our data provide a strong rationale for clinical evaluations of TMPRSS2 inhibitors and androgen-deprivation therapy/androgen receptor antagonists alone or in combination with antiviral drugs as early as clinically possible to prevent COVID-19 progression.

Keywords: Biological Sciences; Molecular Biology; Virology.

Graphical abstract

Figure 1

Effect of androgen deprivation by castration on the expression of TMPRSS2 and ACE2 in adult male mice (A) Schematic depicting the castration experiment in 8- to 9-week-old wild-type C57BL/6J mice. (B) Varying effect of systemic androgen deprivation on the transcription of TMPRSS2 and ACE2 in major organs. qRT-PCR analysis for TMPRSS2 and ACE2 transcripts in the indicated organs from mock, castrated male and normal females. Highly hormone-responsive seminal vesicles from the male mice served as a positive control for the effect of androgen deprivation on the target genes. (C) Immunoblot analysis showing the levels of TMPRSS2, ACE2, and AR proteins in the indicated organs from two separate mock versus castrated males. β-Actin served as a loading control. (D) Immunohistochemistry analysis of the indicated target protein in the prostate from mock and castrated males. Note the reduced TMPRSS2 staining in the castrated group and lack of/sparse ACE2 staining in both groups. (E) As in (D) for the indicated organs. Staining for AR was observed in all the tested organs. Note the reduced TMPRSS2 staining in the bronchial epithelium of lung, columnar epithelium of small intestine, and proximal convoluted tubules in the kidney, in the castrated group. Also, note the reduced ACE2 staining in alveolar epithelium of the lungs and columnar epithelium of small intestine and increased staining in the kidney from the castrated group. Also, see Figures S1 and S2. ∗p < 0.05 (Student's t test).

Figure 2

AR-mediated transcriptional regulation of TMPRSS2 and ACE2 in human prostate and lung cells (A) TMPRSS2 and ACE2 loci display enhanced AR binding upon testosterone stimulation. Shown are AR ChIP-seq peaks around TMPRSS2 and ACE2 loci (the red arrow indicates the TSS) in the vehicle and testosterone-treated conditions in mouse prostate tissue (GSE47192) and human prostate cell lines. (B-C) Androgen deprivation, anti-androgen, or AR degradation results in the loss of TMPRSS2 and ACE2 expression. Top, qRT-PCR analysis for TMPRSS2 and ACE2 transcripts in human prostate cells, LNCaP, grown in the indicated conditions; Reg. media: regular media with 10% serum, CSS: charcoal striped serum, Enza: Enzalutamide at 25 μM, ARD69: AR degrader at 250nM. Bottom, immunoblot analysis for the indicated protein. GAPDH was used the loading control. (D) Immunoblot showing the expression of AR, TMPRSS2, ACE2, and GAPDH proteins in a panel of human lung cell lines; LNCaP was used as the positive control. (E) qRT-PCR and immunoblot analysis as in (B and C) for human lung cell line H460. Also see Figure S3.

Figure 3

TMPRSS2 physically interacts with ACE2 in prostate and lung cells (A) Schematic showing the domain structure of human TMPRSS2 and ACE2 protein. The TMPRSS2 cleavage site on ACE2 is shown with red bar. Epitopes recognized by the antibodies used in the immunoprecipitation are indicated. (B) TMPRSS2 cleaves ACE2. HEK 293T ACE2 cells were transfected with TMPRSS2-encoding plasmid, and 6 h post transfection the cells were treated with DMSO or camostat (250 μM). 48h post-transfection, proteins were extracted and used for immunoblotting with anti-ACE2, anti-TMPRSS2, and anti-GAPDH antibody. (C) Chemical structure of camostat. (D) TMPRSS2 and ACE2 physically interact and are not disrupted by camostat, a TMPRSS2 inhibitor. Reciprocal immunoprecipitation using the indicated antibodies with LNCaP protein extracts. Cells were treated with DMSO or camostat (250 μM) for 24 h followed by protein extraction. Note, along with the full-length ~50-kDa TMPRSS2 protein, the presence of a novel approximately 38-kDa cleaved form detected in both N- and C-terminal TMPRSS2 antibody pull down and ACE2 pull down. IgG pull down served as a negative control; inputs were 5%–10%. (E) As in (D) with Calu-3 protein extracts. Also see Figure S4.

Figure 4

TMPRSS2 in complex with ACE2 cleaves SARS-CoV-2 Spike protein (A) Schematic representation of the expression construct of full-length SARS-CoV-2 spike (S0) protein that has S1 and S2 segments involved in virus attachment and fusion, respectively. Segments of S1 include RBD - receptor-binding domain, and S1/S2 cleavage site; S2 has an S2′ cleavage site. Tree-like symbol denotes glycans, and the C terminus contains 2x Strep-tag. The TMPRSS2 cleavage site on S1/S2 and S2′ site is indicated with the resulting length of the S2 and S2′ protein. Cleaved S2 and S2′ fragment lengths are shown. (B) TMPRSS2 cleaves glycosylated SARS-2-S protein. HEK293T cells were transfected with SARS-2-S plasmid with or without TMPRSS2-encoding plasmid. 48 h post-transfection, proteins were extracted and used for immunoblotting with anti-streptavidin, anti-TMPRSS2, and anti-GAPDH antibody. The open arrow and ∗ indicate the glycosylated and non-glycosylated uncleaved S0 protein, respectively. The filled black arrow indicates TMPRS2-cleaved S2 and S2’. The filled gray arrow indicates TMPRSS2-cleaved potential S2 and S2′ fragments arising from non-glycosylated S0. (C) Mass spectrometric (MS) analysis of pseudotype-incorporated S0 protein. SARS-2-S pseudovirus was directly subjected to streptavidin immunoprecipitation (IP), followed by MS analysis for sequencing. Histograms show peptide coverage for the two prominent bands detected with in IP-western (See Figure S4). (D) Pseudotype SARS-CoV-2 Spike is cleaved by TMPRSS2, which is blocked by camostat. HEK293T ACE2-overexpressing cells were transfected with TMPRSS2 plasmid; 48 h later cells were pretreated with 500 μM camostat for 4 h followed by pseudovirus inoculation by spinoculation for 1 h. Next, 4 h post-spinoculation recovery total lysates were prepared and used for immunoblotting with the indicated antibody. VSV-G-containing pseudotype virus served as a negative control, and HIVp24 served as a positive control for viral inoculation. (E) Camostat blocks TMPRSS2-mediated cleavage of S0 without disrupting the interaction between TMPRSS2-ACE2-Spike S complex. Lysate from (C) was used to immunoprecipitate TMPRSS2 using N-terminal antibody followed by immunoblotting for the indicated target. Note the increased pull-down of uncleaved Spike S0 by the TMPRSS2 and the complete absence of S2, S2′, and other cleaved fragments in both DMSO and camostat lane. (F) Camostat reverses the endogenous TMPRSS2-mediated Spike cleavage in human prostate and lung cells. The LNCaP and Calu-3 cells were pretreated with camostat (500 μM) for 4 h followed by pseudotype VSV-G or SARS-CoV-2 particle spinoculation for 1 h and processed as in (D). (G) Camostat blocks TMPRSS2-mediated cleavage of S0 and S2 without disrupting the interaction between TMPRSS2-ACE2-Spike S complex in LNCaP and Calu-3 cells. Lysate from (C) was used to immunoprecipitate TMPRSS2 using N-terminal antibody followed by immunoblotting for the indicated target. Note the increased pull-down of Spike S0 by the TMPRSS2 and the lack of S2, S2′, and other fragments in the camostat lane. (H) Schematic showing the interaction between Spike S, ACE2, and TMPRSS2 leading to cleavage of Spike S into S1, S2, and S2′ segments. S0/S1 continues to bind tightly through its RBD to ACE2, which is in complex with TMPRSS2, whereas upon cleavage the S2 and S2′ get dissociated from the TMPRSS2-ACE2 complex. Also see Figure S5.

Figure 5

AR-targeted therapies in combination with camostat attenuates the entry of pseudotype SARS-CoV-2 into the host cells (A) Schematic depicting the SARS-CoV-2 Spike S pseudovirus entry assay. The pseudotype consists of Spike S and nano-luciferase reporter. (B) Androgen deprivation attenuates pseudotype entry. LNCaP cells grown in androgen-deprived serum-containing media for 3 days were pretreated with DMSO, DHT (10 nM), or camostat (300 μM) for 1 h followed by inoculation with SARS-CoV-2 Spike S pseudovirus; 24 h post-inoculation, the pseudovirus entry efficiency was measured by means of nano-luciferase signal accompanying entry. The entry efficiency in the DHT-treated cells was taken as 100%. Error bar indicates SEM (n = 5). (C) Anti-androgens or AR degraders with or without camostat attenuate pseudovirus entry. LNCaP cells grown in complete media were pretreated with enzalutamide (10 μM) or ARD-69 (500 nM) alone, or in combination with camostat (300 μM) for 1 h followed by inoculation with pseudovirus; 24 h post-inoculation reporter activity measured as in (D). (D and E) As in (C and D) with AR-positive H460 lung cells. (F) AR-negative Calu-3 cells do not respond to anti-androgens. Calu-3 cells are pretreated with enzalutamide, ARD-69, or camostat followed by pseudovirus inoculation; 24 h later reporter signal characterizes the pseudovirus entry efficiency. Error bar indicates SEM (n = 5). ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005 (Student's t test). (G) Schematic depicting the role of TMPRSS2 in SARS-CoV-2 Spike cleavage, and androgen-mediated expression of ACE2 and TMPRSS2 that could potentially be targeted by AR-directed therapies. Also see Figure S6.