CAVS—Novel in silico selection strategy of specific STAT inhibitory compounds

Highlights

• Specific STAT – targeting compounds of the phosphotyrosine (pTyr) SH2 interaction area.

• CAVS – five step comparative virtual screening tool.

• Selection criteria to identify specific STAT inhibitors.

• Developing of a general – purpose pipeline for different experiments and protein families in the future.

Abstract

Signal transducers and activators of transcription (STATs) are a family of proteins activated by different stimulating factors, including interferons, interleukins, growth factors and oncoproteins. Their role in many diseases has been proven, so there is a strong demand to find specific strategies for STAT inhibition. Our searches for specific STAT-targeting compounds focused on exploring the phosphotyrosine (pTyr) SH2 interaction area. Current selection strategies are insufficient, thus, we developed a new pipeline strategy. In this work we used our recently built 3D models for all human (h)STATs (1–4, 5A, 5B and 6). To select specific inhibitors for the STAT protein of interest, we designed and implemented a five step comparative virtual screening tool, which we named – CAVS (comparative approach for virtual screening). CAVS introduces the ‘comparative binding affinity value’ (CBAV) and ‘ligand binding pose variation’ (LBPV) as selection criteria to identify specific inhibitors of STATs. In a five-step approach, including pre-screen, primary filtering of inhibitors, re-screen, secondary filtering of inhibitors and graphical inspection and final selection, CAVS leads to selection of specific STAT inhibitory compounds. CAVS was tested on a small ligand library of 130 000 natural products and 5.7 million of clean leads for the selection of STAT1 or STAT3-specific inhibitors. With the use of a designed set of Python scripts for data managing and filtering, CAVS allows to convert the comparative virtual screening procedure into an automatic pipeline and to effectively analyze virtual screening results from standard Surflex-Dock 2.6 output files. We also adapted CAVS as a general-purpose pipeline, which will allow adapting written code to different experiments and protein families in the future.

Introduction

Signal transducers and activators of transcription (STATs) are 79–113 kDa proteins, whose activity is dependent on different stimulating factors, including interferons (IFNs), interleukins (ILs), growth factors like EGF and PDGF and oncoproteins like ABL and Src. In humans seven members have been identified in this family, including STAT1–STAT4, STAT5A, STAT5B and STAT6. Structurally they share five domains: an amino-terminal domain, a coiled-coil domain, a DNA-binding domain, an SH2 domain and a carboxyl-terminal transactivation domain (Fig. 1A) [1]. STATs are involved in the JAK/STAT signaling pathway, controlling programming gene expression in biological events as diverse as: embryonic development, programmed cell death, organogenesis, innate immunity, adaptive immunity and cell growth regulation [2], [3].

In general STAT activation is promoted by ligand binding to its receptor, which induces receptor phosphorylation and recruits the STAT proteins to the phosphorylated sites of the receptor. Phosphorylation of the critical tyrosine residue (Tyr) in the STAT protein is then initiated by tyrosine kinases (growth factor receptors, Janus kinases – JAKs and SRC family kinases). Two phosphorylated STAT monomers dimerize through reciprocal pTyr‑SH2 domain interactions (Fig. 1B), and the STAT homodimers translocate to the nucleus where they bind to specific STAT-response elements in the target gene promoters and regulate transcription [3].

For more than ten years STATs are of particular interest to the scientists. Accumulating evidence strongly implicates their role in diseases connected to immune system malfunction, e.g., infection and immune disorders (STAT1 and STAT2), cancer (STAT2, STAT3, STAT5A and STAT5B), cardiovascular diseases (STAT1), asthma and allergy (STAT6), systemic lupus erythematosus (STAT4) and chronic myelogenous leukemia (STAT5A and STAT5B) [4]. STAT inhibitors therefore could be valuable in treatment of these diseases and various STAT inhibitory strategies have been pursued, particularly for STAT3, due to its vast connection with nearly 70% of cancers [5].