Sophisticated genetic tools have made possible the identification of the genes responsible for most well-described immunodeficiencies in the past 15 years. Mutations in Btk, components of the pre-B cell and B cell receptor (lambda5, Igalpha, Igbeta), or the scaffold protein BLNK account for approximately 90% of patients with defects in early B cell development. Hyper-IgM syndromes result from mutations in CD40 ligand, CD40, AID, or UNG in 70-80% of affected patients. Rare defects in ICOS or CD19 can result in a clinical picture that is consistent with common variable immunodeficiency, and as many as 10% of patients with this disorder have heterozygous amino acid substitutions in TACI. For all these disorders, there is considerable clinical heterogeneity in patients with the same mutation. Identifying the genetic and environmental factors that influence the clinical phenotype may enhance patient care and our understanding of normal B cell development.

Bone Morphogenetic Proteins (BMPs) are morphogens that play a major role in regulating development and homeostasis. Although BMPs are used for the treatment of bone and kidney disorders, their clinical use is limited due to the supra-physiological doses required for therapeutic efficacy causing severe side effects. Because recombinant BMPs are expensive to produce, small molecule activators of BMP signaling would be a cost-effective alternative with the added benefit of being potentially more easily deliverable. Here, we report our efforts to identify small molecule activators of BMP signaling. We have developed a cell-based assay to monitor BMP signaling by stably transfecting a BMP-responsive human cervical carcinoma cell line (C33A) with a reporter construct in which the expression of luciferase is driven by a multimerized BMP-responsive element from the Id1 promoter. A BMP-responsive clone C33A-2D2 was used to screen a bioactive library containing ∼5,600 small molecules. We identified four small molecules of the family of flavonoids all of which induced luciferase activity in a dose-dependent manner and ventralized zebrafish embryos. Two of the identified compounds induced Smad1, 5 phosphorylation (P-Smad), Id1 and Id2 expression in a dose-dependent manner demonstrating that our assays identified small molecule activators of BMP signaling.

Although null mutations in Igα have been identified in patients with defects in B cell development, no mutations in Igβ have been reported. We recently identified a patient with a homozygous amino acid substitution in Igβ, a glycine to serine at codon 137, adjacent to the cysteine required for the disulfide bond between Igα and Igβ. This patient has a small percentage of surface IgMdim B cells in the peripheral circulation (0.08% compared with 5–20% in healthy controls). Using expression vectors in 293T cells or Jurkat T cells, we show that the mutant Igβ can form disulfide-linked complexes and bring the μ H chain to the cell surface as part of the BCR but is inefficient at both tasks. The results show that minor changes in the ability of the Igα/Igβ complex to bring the BCR to the cell surface have profound effects on B cell development.

Here we describe three new small-molecule activators of BMP signaling found by high throughput screening of a library of ~600,000 small molecules. Using a cell-based luciferase assay in the BMP4-responsive human cervical carcinoma clonal cell line, C33A-2D2, we identified three compounds with similar chemotypes that each ventralize zebrafish embryos and stimulate increased expression of the BMP target genes, bmp2b and szl. Because these compounds ventralize zebrafish embryos, we have termed them “ventromorphins.” As expected for BMP pathway activators, they induce the differentiation of C2C12 myoblasts to osteoblasts. Affymetrix RNA analysis confirmed the differentiation results and showed that ventromorphins treatment elicits a response similar to BMP4 treatment. Unlike Isoliquiritigenin (SJ000286237), a flavone that maximally activates the pathway after 24 hours of treatment, all three ventromorphins induced SMAD1/5/8 phosphorylation within 30 minutes of treatment and achieved peak activity within 1 hour, indicating that their responses are consistent with directly activated BMP signaling.