Designer TGFβ Superfamily Ligands with Diversified Functionality

Abstract

Transforming Growth Factor – beta (TGFβ) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.

Regulation of FSHβ induction in LβT2 cells by BMP2 and an Activin A/BMP2 chimera, AB215

“…1A). Consistent with our previous findings, using C2C12 cells (Allendorph et al 2011), AB215 has a higher potency than BMP2 in activating a Smad1/5/8-responsive ID1-luciferase reporter in HEK293T cells (Fig. 1B).…”

“…Activin A was purchased from R&D systems (http://www.rndsystems.com). AB204 and AB215 were prepared as described previously (Allendorph et al 2011). In brief, Activin A/BMP2 (AB2) chimera library is a synthetic assortment of six sequence segments originating from two parental molecules, and AB215 is a member of AB2 chimera library.…”

“…We have previously created a library of Activin A/BMP2 (AB2) chimeras denoted by the code (BXXXXX), where X is either A (Activin A) or B (BMP2) (Allendorph et al 2011). AB215 (BABBBA) was designed to have the majority of the type 2 receptor-binding epitope of Activin A (segments 2 and 6) and the majority of type 1 receptor-binding epitope of BMP2 (segments 1, 3, 4, and 5) (Fig.…”

“…Our prediction was that such a swap would yield a chimeric ligand with enhanced type 2 receptor affinity and signaling potency that retains BMP2-like type 1 receptor utilization and SMAD signaling specificity. Indeed, one such ligand, AB215, utilizes the same signaling receptors and Smads as BMP2 but possesses signaling activity and potency that are enhanced relative to BMP2 as demonstrated in in vitro signaling assays (Allendorph et al 2011). In this study, we sought to determine whether AB215 functions synergistically with Activin A in the regulation of the FSHb production in a manner resembling BMP2.…”

Compound screening platform using human induced pluripotent stem cells to identify small molecules that promote chondrogenesis

“…These results show that we can differentiate KiPSCs-SL into chondrocytes in 96-well plate format, enabling a streamlined differentiation protocol that could be performed on an automated platform. AB235 and NB61, two recently described novel compounds, are chimeric ligands of Activin/BMP2 (Allendorph et al, 2011). Since Activin and BMP2 can promote chondrogenesis in vitro and in vivo (Jiang et al, 1993;Majumdar et al, 2001), these chimeras should have chondrogenic potential.…”