SGC-CAMKK2-1: A Chemical Probe for CAMKK2

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2

IDENTIFICATION AND TARGETING OF DOWNSTREAM AR-CAMKK2-MEDIATED MECHANISMS FOR THE TREATMENT OF ADVANCED PROSTATE CANCER

The androgen receptor (AR) is the primary driver of prostate cancer and, therefore, AR-regulated signaling events are essential for the development and progression of the disease. Despite their initial effectiveness, drugs targeting AR eventually fail as sustained inhibition of receptor activity remains a challenge. Previously, Ca2+/calmodulin-dependent protein kinase kinase 2 (CAMKK2) was identified as an essential downstream component of AR signaling in prostate cancer that correspondingly tracked with disease progression. While the importance of CAMKK2 in AR-mediated prostate cancer progression has been established, our understanding of the events downstream that promote the disease remains incomplete. Here, I elucidated two AR-CAMKK2-regulated kinase signaling cascades which are hypothesized to promote disease progression and therefore represent alternative therapeutic targets in castration-resistant prostate cancer (CRPC). First, I identified Unc-51 like autophagy activating kinase 1 (ULK1), an important autophagic initiator, as one of downstream effectors regulated by AR-CAMKK2-5’-AMP-activated protein kinase (AMPK) signaling. CAMKK2-induced protective autophagy is partially dependent on the phosphorylation of ULK1 at serine 555, which is required for prostate cancer cell growth. Accordingly, inhibition of CAMKK2-AMPK-ULK1 signaling by molecular, genetic and/or pharmacological inhibitors decreased autophagy and cell growth in CRPC tumor growth. Second, I investigated the role of AR-CAMKK2 on the activation of the transcription factor cyclic-AMP response element-binding protein (CREB). Cancer cell-intrinsic CAMKK2 signaling promoted CRPC in part through increasing the activity of CREB. Molecularly, this was shown to occur through the calcium/calmodulin-dependent protein kinase I-mediated phosphorylation of CREB on its serine 133 activation site. I also determined a functional redundancy between two CREB family members, CREB1 and Activating Transcription Factor 1. Deletion of both genes impaired transcriptional activity and maximal prostate cancer cell proliferation in tissue culture and tumor initiation as well as growth in CRPC. Therapeutically, pharmacological targeting of CREB by 666-15 effectively blocked cell cycle and inhibited cell/tumor growth without significant toxicity and was able to decrease the growth of CRPC tumors resistant to second-generation antiandrogens. Collectively, I have leveraged orthogonal molecular, genetic and pharmacological approaches to delineate how AR-CAMKK2 signaling drives prostate cancer progression and define potential new therapeutic strategies for targeting this oncogenic cascade in CRPC

Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes

CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934 (CAMKK2 IC50 = 3 nM), a total of 32 compounds, composed of single ring, 5,6-, and 6,6-fused heteroaromatic cores were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. These compounds were evaluated in vitro in biochemical and cellular assays for CAMKK2 inhibition. Compared to GSK650394 and STO-609, thirteen of our compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had greatly improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs aimed at the identification of CAMKK2 chemical probes and clinical candidates</div

Transcriptional regulation of core autophagy and lysosomal genes by the androgen receptor promotes prostate cancer progression

<p>AR (androgen receptor) signaling is crucial for the development and maintenance of the prostate as well as the initiation and progression of prostate cancer. Despite the AR's central role in prostate cancer progression, it is still unclear which AR-mediated processes drive the disease. Here, we identified 4 core autophagy genes: <i>ATG4B, ATG4D, ULK1</i>, and <i>ULK2</i>, in addition to the transcription factor <i>TFEB</i>, a master regulator of lysosomal biogenesis and function, as transcriptional targets of AR in prostate cancer. These findings were significant in light of our recent observation that androgens promoted prostate cancer cell growth in part through the induction of autophagy. Expression of these 5 genes was essential for maximal androgen-mediated autophagy and cell proliferation. In addition, expression of each of these 5 genes alone or in combination was sufficient to increase prostate cancer cell growth independent of AR activity. Further, bioinformatic analysis demonstrated that the expression of these genes correlated with disease progression in 3 separate clinical cohorts. Collectively, these findings demonstrate a functional role for increased autophagy in prostate cancer progression, provide a mechanism for how autophagy is augmented, and highlight the potential of targeting this process for the treatment of advanced prostate cancer.</p

SGC-CAMKK2-1: A Chemical Probe for CAMKK2

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2

Hinge binder scaffold hopping identifies potent calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) inhibitor chemotypes

CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934, a total of 32 compounds, composed of single-ring, 5,6-, and 6,6-fused heteroaromatic cores, were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. Compared to GSK650394 and STO-609, 13 compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge-binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs