Introduction {#sec1}
============

Traditional drug development efforts are focused mainly on small molecules that target druggable protein classes such as enzymes and receptors.^[@ref1]^ The majority of drugs operate as inhibitors of protein function; however, because this mode of action utilizes a target occupancy paradigm requiring high drug concentrations to sustain the biological response, it also can lead to undesirable off-target effects. As an alternative, PROteolysis TArgeting Chimeras (PROTACs) hold great promise as a therapeutic modality since they require only a transient interaction with the target protein to promote its degradation.^[@ref2]−[@ref4]^ However, despite promoting the degradation of various proteins, PROTACs are limited to target engagement within the intracellular space for ubiquitination.^[@ref5]−[@ref9]^ PROTACs are therefore unable to act on secreted proteins, such as cytokines and chemokines, which exert their biological activity from the extracellular space.^[@ref10],[@ref11]^ These proteins bind to cell surface receptors and can initiate the aberrant signaling implicated in multiple diseases ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}A). While monoclonal antibodies can target secreted proteins or their cognate receptors to block signaling, efforts to develop small molecule inhibitors for secreted proteins have so far been less successful.^[@ref12],[@ref13]^ Given the functional importance of secreted proteins in diseases and the current limitations on inhibiting their activities, alternative technologies to efficiently target them are needed.

![ENDTAC technology. (A) In the absence of the ENDTAC, extracellular target protein (e.g., cytokine) binds to its cognate receptor (e.g., cytokine receptor) and activates downstream signaling leading to a cellular response (e.g., cell proliferation, apoptosis, and/or inflammation). (B) Upon ENDTAC addition, the target extracellular protein of interest (POI) is endocytosed via a decoy GPCR, CXCR7, and subsequently degraded by the lysosome. EE = early endosome, LE = late endosome. (C) An ENDosome TArgeting Chimera (ENDTAC) is a heterodimeric molecule consisting of an agonist ligand that binds to a cell surface receptor (e.g., CXCR7) coupled to a ligand that recruits the extracellular POI (e.g., cytokine).](oc-2019-00224s_0002){#fig1}

Receptor--ligand-mediated delivery systems have gained significant attention in the past few years.^[@ref14],[@ref15]^ Several studies have reported that cell surface receptors such as transferrin receptor, asialoglycoprotein (ASGPr), and folate receptor can be used to selectively deliver a wide range of therapeutic agents into cancer cells via receptor-mediated endocytosis.^[@ref16]−[@ref18]^ In addition, a recent study has shown that Cas9 could be selectively delivered into cells by harnessing the receptor--ligand interactions followed by endosomal escape.^[@ref19]^ Here we report a new approach to potentially target extracellular proteins for receptor-facilitated lysosomal degradation using chimeric molecules termed ENDosome TArgeting Chimeras (ENDTACs) ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}B). An ENDTAC is a heterodimeric molecule consisting of a small molecule (agonist) that binds to a plasma membrane-localized receptor of interest (e.g., a GPCR) while the other end, connected via a linker, of the ENDTAC binds and recruits the extracellular protein of interest (POI) (e.g., a cytokine) ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}C). Upon binding to the GPCR, the tethered extracellular protein can undergo receptor-mediated endocytosis and subsequently degradation by the lysosome ([Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"}B).

Results and Discussion {#sec2}
======================

To validate the feasibility of the ENDTAC approach, we performed a proof-of-concept study using the decoy receptor, CXCR7 (ACKR3), as the cell surface receptor and an engineered HA-eGFP-HaloTag7 (eGFP-HT7) fusion protein as the extracellular target protein. The GPCR CXCR7 is constitutively endocytosed at a low level to degrade its cognate chemokines via transport to the lysosome.^[@ref20]^ A previous report has shown that two potent small molecule agonists **VUF11207** and **VUF11403** induce CXCR7 internalization ([Figure S1](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf)).^[@ref21]^ Since both these 3,4-dimethoxy and 3,4,5-trimethoxy styrene amides (**VUF11403** and **VUF11207**, respectively) exhibit similar agonist activity in various pairwise comparisons,^[@ref21]^ we incorporated the HT7-recruiting chloroalkane at their 5-position ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}A). Using a facile synthetic method ([Scheme S1](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf), in the Supporting Information), linkers ranging from a diethylene glycol to pentaethylene glycol were incorporated in both series (R1: H or OMe) to afford **ENDTACs-1--8** ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}A). Agonist activity for **ENDTACs-1--8** was evaluated by the Tango GPCR assay^[@ref22],[@ref23]^ ([Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}). Interestingly, the dimethoxy-containing ENDTAC series (**ENDTACs-1--4**; R~1~, OMe; R~2~, H) displayed only slightly reduced potency compared to the parent warhead **VUF11207** ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}B; [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}; left EC~50~ column), while the monomethoxy-containing ENDTACs (**ENDTACs-5--8**; R~1~, H; R~2~, H) exhibited greater than 10- to 40-fold reduction in activity compared to the respective warhead, **VUF11403** ([Figure S1](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf) and [Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}). Accordingly, we focused on the dimethoxy-containing ENDTAC series and measured the ability of the prereacted **ENDTAC**:eGFP-HT7 complex to activate the CXCR7 receptor. Purified eGFP-HT7 was used as the reporter POI ([Figures S2 and S3](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf)) to evaluate **ENDTACs-1--4**. The reaction between purified eGFP-HT7 and ENDTACs was confirmed by LC-MS analysis ([Figures S4 and S5 and Table S1](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf)).

![Characterization of ENDTACs. (A) Core structure of ENDTACs. (B) Characterization of agonist activity of **ENDTACs-1--4** using the Tango assay (*n* = 4). (C) Activity of **ENDTAC-1** and **ENDTAC-neg** after prereacting with eGFP-HT7 measured by Tango assay (*n* = 2). (D) Pulse chase Tango assay for warhead **VUF11207**, **ENDTAC-1**, and precomplexed **ENDTAC-1**:eGFP-HT7 (4 μM) (*n* = 2). Curves were fitted using GraphPad Prism 5. All data represent mean ± SEM.](oc-2019-00224s_0003){#fig2}

###### EC~50~ Values of **VUF11207** and CXCR7-Recruiting ENDTACs As Determined in the Tango Assay[a](#t1fn1){ref-type="table-fn"}

  entry   compound ID      *n*                                      EC~50~ (nM) compound alone   EC~50~ (nM) compound + eGFP -- HT7   EC~50~ (nM) compound + Nanoluc -- HT7
  ------- ---------------- ---------------------------------------- ---------------------------- ------------------------------------ ---------------------------------------
  1       **VUF11207**     N/A[b](#tbl1-fn1){ref-type="table-fn"}   68 ± 1.2                                                           
  2       **ENDTAC-1**     2                                        113 ± 1.3                    169 ± 1.5                            146 ± 1.3
  3       **ENDTAC-2**     3                                        111 ± 1.2                    251 ± 1.5                             
  4       **ENDTAC-3**     4                                        145 ± 1.1                    373 ± 2.0                             
  5       **ENDTAC-4**     5                                        158 ± 1.2                    287 ± 3.0                             
  6       **VUF11403**     N/A[b](#tbl1-fn1){ref-type="table-fn"}   76 ± 1.3                                                           
  7       **ENDTAC-5**     2                                        1360 ± 3.8                   ND                                    
  8       **ENDTAC-6**     3                                        2453 ± 1.1                   ND                                    
  9       **ENDTAC-7**     4                                        4705 ± 5.2                   ND                                    
  10      **ENDTAC-8**     5                                        1815 ± 1.4                   ND                                    
  11      **ENDTAC-neg**   2                                        ND                           ND                                    

ENDTACs (**1**--**4**) contain OMe at the R~1~ position and H at the R~2~ position whereas ENDTACs (**5**--**8**) contain H at both R~1~ and R~2~ positions. These ENDTACs are generated by adding a chloroalkane to **VUF11207** or **VUF11403** via varying linker lengths. The **ENDTAC-neg** structure is slightly different than **ENDTACs-1--4** where R~1~ is H, and R~2~ is OMe. **VUF11207** and **VUF11403** are known small molecule agonists of CXCR7. eGFP-HT7 is HA-eGFP-Halotag7 protein, and Nanoluc-HT7 is a HA-nanoluciferase-Halotag7 fusion protein. ND: not determined. All data represent mean ± SEM.

N/A: not applicable.

Among these four ENDTACs, **ENDTAC-1** retained a similar potency compared to **VUF11207** after reacting with eGFP-HT7 ([Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}). On the basis of this initial activity profile, we selected **ENDTAC-1** for use in subsequent experiments. To ensure that eGFP-HT7 internalization is mediated via CXCR7 activity, we synthesized a negative control molecule, **ENDTAC-neg**, using a similar synthetic approach ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}A; [Scheme S2](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf)). The *para*-methoxy group on the styrene ring (R~1~, H; and R~2~, OMe) of **ENDTAC-neg** was previously identified to abrogate CXCR7 agonist activity.^[@ref21]^ Therefore, **ENDTAC-neg** was evaluated for activity in the absence or presence of eGFP-HT7. According to Tango assay data, prereacted **ENDTAC-neg**:eGFP-HT7 did not show any CXCR7 activation compared to **ENDTAC-1**:eGFP-HT7, supporting the use of **ENDTAC-neg** as a negative control. ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}C and [Figure S6](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf)). Furthermore, we also analyzed the saturation kinetics of the prereacted **ENDTAC-1**:eGFP-HT7 complex using the Tango assay. The warhead **VUF11207** and **ENDTAC-1** show fast saturation kinetics, whereas **ENDTAC-1**:eGFP-HT7 reaches saturation within 4 h ([Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}D). Given the similar agonistic activities of the warhead and corresponding ENDTAC, as well as favorable binding kinetics, we next proceeded to study ENDTAC-induced internalization of eGFP-HT7 protein.

CXCR7-expressing MCF7 cells were treated for 4 h with either **ENDTAC-1** or **ENDTAC-neg** conjugated to purified eGFP-HT7 (10 μM), and internalization was monitored by confocal microscopy. Internalized eGFP-HT7 was visualized as GFP-positive puncta (green), and we observed a greater uptake of eGFP-HT7 in the presence of 10 μM **ENDTAC-1** after 4 h ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}A). The quantitation of GFP-positive cells suggests **ENDTAC-1** induced internalization of eGFP-HT7, as compared to **ENDTAC-neg** ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}B). We also analyzed eGFP-HT7 internalization by immunostaining with HA antibody and observed an overlay between the GFP and HA puncta in the presence of **ENDTAC-1** ([Figure S7](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf)). eGFP-HT7 uptake was confirmed as being ENDTAC-dependent in CXCR7-expressing MCF7s and MIA PaCa-2 cells, where greater uptake is observed in the presence of 500 nM **ENDTAC-1**, compared to **ENDTAC-neg**, via immunoblotting ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}C). To corroborate this finding, we used purified Nanoluc-HT7 ([Figures S2 and S3](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf)) as the extracellular POI and evaluated Nanoluc-HT7 uptake by measuring luciferase activity in MCF7 and HTLA cells ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}D and [Figure S8](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf)). As was observed with the **ENDTAC-1**:eGFP-HT7 adduct, **ENDTAC-1** retained CXCR7 agonistic activity after reacting with Nanoluc-HT7 ([Table [1](#tbl1){ref-type="other"}](#tbl1){ref-type="other"}). We first incubated prereacted Nanoluc-HT7 + **ENDTAC-1** (1 μM) with MCF7 cells for 2 h and then changed to ENDTAC-free media and assayed for Nanoluc-HT7 uptake over 24 h. Consistent with previous data, we observed a 2.5-fold uptake of Nanoluc-HT7 in the presence of **ENDTAC-1** compared to **ENDTAC-neg** in the first 2--6 h ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}D, [Figure S8](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf)). In both MCF7 and HTLA cells, there is an observed reduction in Nanoluc-HT7 activity at 24 h, suggesting that internalized protein has been degraded in the lysosome.

![Internalization of eGFP-HT7 in the presence of **ENDTAC-1**. (A) Confocal microscopy analysis of internalized eGFP-HT7 (green puncta) with 10 μM **ENDTAC-1**. Nuclei are stained with Hoechst stain (blue). Scale bar: 5 μm. (B) eGFP-positive cells were quantified and presented as a percentage. Quantified data represent mean ± SEM, *n* = 3. \**p* \< 0.05. (C) Cellular uptake of eGFP-HT7 in MCF7 and MIA PaCa-2 cells was analyzed by immunoblotting after incubating for 4 h with **ENDTAC-neg**:eGFP-HT7 or **ENDTAC-1**:eGFP-HT7 (500 nM). (D) Cellular uptake of Nanoluc-HT7 in MCF7 cells was analyzed by evaluating luciferase activity. The relative luminescence units of **ENDTAC-1**:Nanoluc-HT7 were normalized to **ENDTAC-neg**:Nanoluc-HT7 and presented as the fold uptake using GraphPad Prism 5 (*n* = 6). Data represent mean ± SEM; \**p* \< 0.05; ns, not significant. (E) Cellular uptake of eGFP-HT7 in MCF7 cells was analyzed by immunoblotting after incubating for 4 h with DMSO:eGFP-HT7, **ENDTAC-neg**:eGFP-HT7, or **ENDTAC-1**:eGFP-HT7 (250 nM) in the absence or presence of excess warhead, **VUF11207** (25 μM).](oc-2019-00224s_0004){#fig3}

Given the possibility that bulk endocytosis could lead to nonspecific uptake of proteins, we performed a ligand competition assay with excess CXCR7 agonist **VUF11207** to probe that the **ENDTAC-1**-mediated HT7 uptake is CXCR7-dependent. We incubated MCF7 cells with DMSO:eGFP-HT7, **ENDTAC-neg**:eGFP-HT7, and **ENDTAC-1**:eGFP-HT7 in the absence or presence of excess warhead (**VUF11207**). The **ENDTAC-1**-mediated HT7 uptake was completely inhibited in the presence of the excess warhead ([Figure [3](#fig3){ref-type="fig"}](#fig3){ref-type="fig"}E), suggesting that the ternary complex formation between eGFP-HT7, **ENDTAC-1**, and CXCR7 is required to facilitate selective internalization of eGFP-HT7.

We next sought to determine whether the level of CXCR7 expression could play a key role in efficiency of the ENDTAC system. To probe the hypothesis, we first compared the uptake of eGFP-HT7 in CXCR7-overexpressing 293T cells. Upon incubation of **ENDTAC-1**:eGFP-HT7 with 293T cells, we observed an **ENDTAC-1**-dependent uptake of eGFP-HT7 compared to nontransfected cells ([Figure S9A](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf)), suggesting that increased expression of CXCR7 enhances the **ENDTAC-1**-mediated internalization of HT7. To further support this result, we compared the **ENDTAC-1**-dependent cellular uptake in MDA-MB-231 cells (CXCR7-negative) and MCF7 cells (CXCR7-positive). Compared to MDA-MB-231 cells, we observed an increased eGFP-HT7 uptake in **ENDTAC-1** treated MCF7 cells ([Figure S9B](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf)), suggesting that the level of CXCR7 surface expression is a key parameter that dictates ENDTAC efficiency.

Upon receptor-mediated endocytosis, we propose that the eGFP-HT7 protein traffics sequentially to the early endosome and the late endosome and subsequently is degraded in the lysosome. To evaluate this hypothesis, we treated MCF7 and MIA PaCa-2 cells with **ENDTAC-1**:eGFP-HT7 or **ENDTAC-neg**:eGFP-HT7 for 4 h, washed the cells, replaced the medium with fresh medium, and cultured the cells for another 3 h, to monitor the fate of internalized eGFP-HT7. We observed eGFP-HT7 uptake by the cells after a continuous 4 h **ENDTAC-1**:eGFP-HT7 incubation, followed by the disappearance of eGFP-HT7 after a 3 h chase ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}A,B). Therefore, it is possible that internalized eGFP-HT7 protein traffics to the lysosome and is therein degraded, given the mechanism of CXCR7 internalization following agonist treatment.^[@ref20]^ To evaluate if lysosomal-mediated eGFP-HT7 degradation occurs, we incubated cells with **ENDTAC-neg**:eGFP-HT7 or **ENDTAC-1**:eGFP-HT7 for 4 h, washed the cells, and cultured them in the absence or presence of the lysosome inhibitor, bafilomycin A1 (bafA1). Interestingly, eGFP-HT7 is degraded only in the absence of bafA1, suggesting that eGFP-HT7 degradation indeed occurs via the lysosomal pathway ([Figure [4](#fig4){ref-type="fig"}](#fig4){ref-type="fig"}C,D).

![Cellular uptake and lysosomal degradation of eGFP-HT7 in the presence of **ENDTAC-1**. (A) Immunoblot analysis of uptake and fate of internalized eGFP-HT7 after 4 h of incubation followed by a 3 h chase in **ENDTAC-neg/ENDTAC-1**:eGFP-HT7-free medium in MCF7 (*n* = 4) and (B) MIA PaCa-2 cells (*n* = 3). (C) MCF7 or (D) MIA PaCa-2 cells were incubated with **ENDTAC-neg**/**ENDTAC-1**:eGFP-HT7 (500 nM) for 4 h, released to ENDTAC-free medium, and chased for an additional 3 h in the absence or presence of bafilomycin A1 (bafA1) (100 nM) (*n* = 3).](oc-2019-00224s_0005){#fig4}

To further confirm endolysosomal accumulation of eGFP-HT7 in the presence of ENDTAC, we examined the uptake and degradation of eGFP-HT7 in MCF7 cells by confocal microscopy. MCF7 cells were treated with either Alexa Fluor 488-conjugated transferrin (TFN488), a positive control for endocytosis,^[@ref24]^ or the **ENDTAC-1/ENDTAC-neg**:eGFP-HT7 conjugate (10 μM) for 4 h, fixed, and analyzed by confocal microscopy. Interestingly, as similarly observed with TFN488 treatment, eGFP-HT7-positive cells were observed in the presence of **ENDTAC-1**:eGFP-HT7 ([Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}A--C). These eGFP-HT7 foci colocalize with the early endosome marker (EEA1) suggesting that internalized eGFP-HT7 traffics to the endosome ([Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}, panel C). Furthermore, we also observed that eGFP-HT7-positive foci partially colocalize with the lysosome marker (LysoTracker), suggesting that **ENDTAC-1** promotes receptor-mediated uptake and lysosomal degradation of eGFP-HT7 protein ([Figure [5](#fig5){ref-type="fig"}](#fig5){ref-type="fig"}D,E).

![Endolysosomal trafficking of eGFP-HT7 in the presence of **ENDTAC-1**. Confocal microscopy analysis of (A) AF488-conjugated transferrin (TFN488) was used as a positive control for endocytosis. (B--E) Trafficking of internalized eGFP-HT7 (10 μM) to the endolysosome compartment after 4 h. (B, C) EEA1, early endosome marker, and (D, E) LysoTracker, lysosome marker. Red, EEA1 and LysoTracker; green, eGFP-HT7; yellow, merged images; blue, Hoechst stain for nuclei. Scale bar: 5 μm.](oc-2019-00224s_0006){#fig5}

While PROTACs are widely used in the field to target many intracellular proteins,^[@ref9]^ a key limitation is their inability to target extracellular proteins. Here we show for the first time that chimeric molecules, which we designate as "ENDTACs", are capable of recruiting an extracellular protein (eGFP-HT7), for internalization and lysosomal degradation by hijacking a GPCR-mediated endocytosis pathway. Although we present a proof-of-concept approach using a covalent HaloTag-based system, we anticipate that the ENDTAC technology could be further optimized and modified for a noncovalent setting. In summary, the ENDTAC technology represents a new chemical biology tool to study extracellular proteins and has the potential for depleting disease-causing extracellular proteins in the future.

The Supporting Information is available free of charge on the [ACS Publications website](http://pubs.acs.org) at DOI: [10.1021/acscentsci.9b00224](http://pubs.acs.org/doi/abs/10.1021/acscentsci.9b00224).Experimental procedures and synthesis and characterization of ENDTACs ([PDF](http://pubs.acs.org/doi/suppl/10.1021/acscentsci.9b00224/suppl_file/oc9b00224_si_001.pdf))

Supplementary Material
======================

###### 

oc9b00224_si_001.pdf

The authors declare the following competing financial interest(s): C.M.C. is a consultant to and shareholder in Arvinas, which partially supports research in his lab.

This work was supported by the NIH (R35 CA197589). M.P. gratefully acknowledges the Swedish Research Council (Vetenskapsrådet) for international postdoctoral funding (2016-00294). HTLA (a HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene) cells were generously provided by Gilad Barnea (Brown University, RI). We thank the Alanna Schepartz lab for their technical help and John Hines for reviewing the manuscript.
