Phosphoproteomics identifies potential downstream targets of the integrin α2β1 inhibitor BTT-3033 in prostate stromal cells

Bingsheng Li; Pan Li; Weiping Xia; Baiyang You; Qingfeng Yu; Bo Zhang; Ru Huang; Ruixiao Wang; Yuhan Liu; Zhi Chen; Yu Gan; Yao He; Martin Hennenberg; Christian G Stief; Xiang Chen

doi:10.21037/atm-21-3194

Phosphoproteomics identifies potential downstream targets of the integrin α2β1 inhibitor BTT-3033 in prostate stromal cells

Ann Transl Med. 2021 Sep;9(17):1380. doi: 10.21037/atm-21-3194.

Authors

Bingsheng Li^{1

2}, Pan Li³, Weiping Xia¹, Baiyang You^{4

5}, Qingfeng Yu⁶, Bo Zhang¹, Ru Huang², Ruixiao Wang², Yuhan Liu², Zhi Chen¹, Yu Gan¹, Yao He¹, Martin Hennenberg², Christian G Stief², Xiang Chen¹

Affiliations

¹ Department of Urology, Xiangya Hospital, Central South University, Changsha, China.
² Department of Urology, University Hospital Munich, LMU Munich, Munich, Germany.
³ Department of Pathology, LMU Munich, Munich, Germany.
⁴ National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, China.
⁵ Division of Cardiac Rehabilitation, Department of Physical Medicine and Rehabilitation, Xiangya Hospital, Central South University, Changsha, China.
⁶ Department of Urology and Guangdong Key Laboratory of Urology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

Abstract

Background: Integrin α2β1 inhibitor BTT-3033 (1-(4-fluorophenyl)-N-methyl-N-[4[[(phenylamino)carbonyl]amino]phenyl]-1H-pyrazole-4-sulfonamide) was recently reported to inhibit neurogenic and thromboxane A2-induced human prostate smooth muscle contraction, and thus represents a target with a different inhibition spectrum than that of α1-blockers in benign prostate hyperplasia (BPH) treatments. Clarifying the underlying mechanisms of the inhibition effects will provide insights into the role of integrin α2β1 in prostate contraction and enable new intracellular targets for smooth muscle contraction to be explored.

Methods: ProteomeHD was used to predict and enrich the top co-regulated proteins of integrin α2 (ITGA2). A phosphoproteomic analysis was conducted on human prostate stromal cells (WPMY-1) treated with 1 or 10 µM of BTT-3033 or solvent for controls. A clustering analysis was conducted to identify the intracellular targets that were inhibited in a dose-dependent manner. Gene ontology (GO) and annotation enrichments were conducted to examine any functional alterations and identify possible downstream targets. A Kinase-substrate enrichment analysis (KSEA) was conducted to identify kinases-substrate relationships.

Results: Enrichments of the actin cytoskeleton and guanosine triphosphatases (GTPases) signaling were predicted from the co-regulated proteins with ITGA2. LIM domain kinases, including LIM domain and actin-binding 1 (LIMA1), zyxin (ZYX), and thyroid receptor-interacting protein 6 (TRIP6), which are functionally associated with focal adhesions and the cytoskeleton, were present in the clusters with dose-dependent phosphorylation inhibition pattern. 15 substrates were dose-dependently inhibited according to the KSEA, including polo-like kinase 1 (PLK1), and GTPases signaling proteins, such as disheveled segment polarity protein 2 (DVL2).

Conclusions: In this study, we proposed that the mechanisms underlying the contractile and proliferative effects of integrin α2β1 are the LIM domain kinases, including the ZYX family, and substrates, including PLK1 and DVL2.

Keywords: BTT-3033; Integrin α2β1; benign prostatic hyperplasia (BPH); lower urinary tract symptoms (LUTS); phosphoproteomics.