NHD2-15, a novel antagonist of Growth Factor Receptor-Bound Protein-2 (GRB2), inhibits leukemic proliferation

• Published in: PloS one 2020-08, Vol.15 (8), p.e0236839-e0236839
• Main Authors: Lewis, Tina R, Smith, Jesse, Griffin, Kallie, Aguiar, Stephanie, Rueb, Kristen F, Holmberg-Douglas, Natalie, Sampson, Ellen M, Tomasetti, Skylar, Rodriguez, Sofia, Stachura, David L, Arpin, Carolynn C, Buday, Laszlo
• Format: Article
• Language: English
• Subjects: Adapter proteins; Adapters; Antagonists (Biochemistry); Apoptosis; Assaying; Autophagy; BCR protein; BCR-ABL protein; Binding; Biochemistry; Biology and Life Sciences; Cell growth; Cell proliferation; Cellulose; Cellulose nitrate; Chemistry; Chronic myeloid leukemia; Domains; Drug therapy; Fusion protein; Genetic aspects; Grb2 protein; Growth factors; Health aspects; Homology; Kinases; Leukemia; Medicine and Health Sciences; Mode of action; Myeloid cells; Myeloid leukemia; Neutrophils; Pathogens; Physiological aspects; Proteins; Quinoxaline; Quinoxalines; Receptors; Research and Analysis Methods; Surface plasmon resonance; Translocation; Zebrafish
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0236839

The majority of chronic myeloid leukemia (CML) cases are caused by a chromosomal translocation linking the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1), creating the mutant fusion protein BCR-ABL1. Downstream of BCR-ABL1 is growth factor receptor-bound protein-2 (GRB2), an intracellular adapter protein that binds to BCR-ABL1 via its src-homology-2 (SH2) domain. This binding constitutively activates growth pathways, downregulates apoptosis, and leads to an over proliferation of immature and dysfunctional myeloid cells. Utilizing novel synthetic methods, we developed four furo-quinoxaline compounds as GRB2 SH2 domain antagonists with the goal of disrupting this leukemogenic signaling. One of the four antagonists, NHD2-15, showed a significant reduction in proliferation of K562 cells, a human BCR-ABL1.sup.+ leukemic cell line. To elucidate the mode of action of these compounds, various biophysical, in vitro, and in vivo assays were performed. Surface plasmon resonance (SPR) assays indicated that NHD2-15 antagonized GRB2, binding with a K.sub.D value of 119 ± 2 [mu]M. Cellulose nitrate (CN) assays indicated that the compound selectively bound the SH2 domain of GRB2. Western blot assays suggested the antagonist downregulated proteins involved in leukemic transformation. Finally, NHD2-15 was nontoxic to primary cells and adult zebrafish, indicating that it may be an effective clinical treatment for CML.