Directed Evolution of the BpsA Carrier Protein Domain for Enhanced Activation by Non-Cognate 4'-Phosphopantetheinyl Transferases Implications for ASD Drug Discovery

Autism Spectrum Disorder (ASD) is a complex neurodevelopmen-
tal condition characterized by challenges in social interaction, commu-
nication, and repetitive behaviors. Despite significant research efforts,
effective treatments for ASD remain limited, largely due to the disor-
der’s heterogeneous nature and multifactorial etiology.

Often drug discovery approaches have fallen short in addressing the diverse
symptoms and underlying biological mechanisms of ASD. In this context,
the BpsA carrier protein and 4’-phosphopantetheinyl transferases (PPTases)
present a promising opportunity for novel therapeutic development. BpsA is
a multifunctional enzyme involved in the biosynthesis of secondary metabo-
lites, such as antibiotics, through the activation by PPTases.
These enzymes catalyze the transfer of the 4’-phosphopantetheine moi-
ety from coenzyme A to specific serine residues on carrier proteins, con-
verting them from their inactive apo-form to their active holo-form. While

through directing evolution we can engineer protein, mimicing natural selec-
tion demands for sophisticated screening in order to evolve proteins or nucleic
acids.Applying directed evolution to the BpsA carrier protein domain, we aim
to enhance its activation by non-cognate PPTases, potentially leading to the
discovery of new bioactive compounds with therapeutic relevance to ASD.
The chemical changes resulting from directed evolution can extend be-
yond the primary structure of the synthesized compounds. The introduction
of new functional groups or the modification of existing ones can lead to the
generation of structurally diverse derivatives. The evolved BpsA variants
may interact with tailoring enzymes, such as oxidoreductases, methyltrans-
ferases, or glycosyltransferases, in novel ways. Enzyme modifications are
practical. This method can result in the production of compounds with
modified functional groups, altered oxidation states, or the addition of sugar
moieties.
While semisynthetic derivatization often proves key in order to develop
our new drug, the novel compounds produced through the action of evolved
BpsA can serve as starting points for extensive chemical modifications. These
derivatization techniques, such as selective protection, deprotection, or func-
tional group interconversions, can generate libraries of structurally diverse
analogs.
In turn, evolved BpsA variants could enable the production of new in-
termediates that can be again transformed by biocatalytic enzymes. These
enzymes, such as oxidases, reductases, or hydrolases, can introduce additional
chemical modifications, expanding the structural diversity of the final prod-
ucts. Exploring new chemical spaces we leverage the full power of directed
evolution and maybe discover novel bioactive compounds with therapeutic
relevance for ASD and , perhaps,other conditions. The chemical changes and
new chemistry arising from the engineered BpsA variants can provide a rich
source of lead compounds for drug discovery efforts, offering new opportuni-
ties for the development of sophisticated treatments.

In this article, we will explore the options we have, engineering such a new
drug, we will focus mostly around molecular action and directed evolution
technique, while delivering proper analysis regarding our BpsA variants.