Drugs with prolonged, on-target residence time often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here, we demonstrate progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Utilizing an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrate biochemical residence times spanning from minutes to 7 days. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK more than 18 hours after clearance from the circulation. The inverted cyanoacrylamide strategy was further utilized to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating generalizability of the approach. Targeting noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates “residence time by design”, the ability to modulate and improve the duration of target engagement in vivo.

Androgen receptor (AR) is expressed in 90% of estrogen receptor alpha positive (ER+) breast tumors, but its role in tumor growth and progression remains controversial. Use of two anti-androgens that inhibit AR nuclear localization, enzalutamide and MJC13, revealed that AR is required for maximum ER genomic binding. Here, a novel global examination of AR chromatin binding found that estradiol induced AR binding at unique sites compared to dihydrotestosterone (DHT). Estradiol-induced AR binding sites were enriched for estrogen response elements and had significant overlap with ER binding sites. Furthermore, AR inhibition reduced baseline and estradiol-mediated proliferation in multiple ER+/AR+ breast cancer cell lines, and synergized with tamoxifen and fulvestrant. In vivo, enzalutamide significantly reduced viability of tamoxifen-resistant MCF7 xenograft tumors and an ER+/AR+ patient-derived model. Enzalutamide also reduced metastatic burden following cardiac injection. Lastly, in a comparison of ER+/AR+ primary tumors versus patient-matched local recurrences or distant metastases, AR expression was often maintained even when ER was reduced or absent. These data provide pre-clinical evidence that anti-androgens that inhibit AR nuclear localization affect both AR and ER, and are effective in combination with current breast cancer therapies. In addition, single agent efficacy may be possible in tumors resistant to traditional endocrine therapy, since clinical specimens of recurrent disease demonstrate AR expression in tumors with absent or refractory ER. Implications This study suggests that AR plays a previously-unrecognized role in supporting E2-mediated ER activity in ER+/AR+ breast cancer cells, and that enzalutamide may be an effective therapeutic in ER+/AR+ breast cancers.

No abstract

PlGF, one of the ligands for VEGFR-1, has been implicated in tumor angiogenesis. However, more recent studies indicate that genetic or pharmacological inhibition of PlGF signaling does not result in reduction of microvascular density in a variety of tumor models. Here we screened 12 human tumor cell lines and identified 3 that are growth inhibited by anti-PlGF antibodies in vivo. We found that efficacy of anti-PlGF treatment strongly correlates with VEGFR-1 expression in tumor cells, but not with antiangiogenesis. In addition, PlGF induced VEGFR-1 signaling and biological responses in tumor cell lines sensitive to anti-PlGF, but not in refractory tumor cell lines or in endothelial cells. Also, genetic ablation of VEGFR-1 signaling in the host did not affect the efficacy of PlGF blockade. Collectively, these findings suggest that the role of PlGF in tumorigenesis largely consists of promoting autocrine/paracrine growth of tumor cells expressing a functional VEGFR-1 rather than stimulation of angiogenesis.