Discovery of Inhibitors for Proliferating Cell Nuclear Antigen Using a Computational-Based Linked-Multiple-Fragment Screen

Proliferating
cell nuclear antigen (PCNA) is a central factor in DNA replication
and repair pathways that plays an essential role in genome stability.
The functional roles of PCNA are mediated through an extensive list
of protein–protein interactions, each of which transmits specific
information in protein assemblies. The flexibility at the PCNA–protein
interaction interfaces offers opportunities for the discovery of functionally
selective inhibitors of DNA repair pathways. Current fragment-based
drug design methodologies can be limited by the flexibility of protein
interfaces. These factors motivated an approach to defining compounds
that could leverage previously identified subpockets on PCNA that
are suitable for fragment-binding sites. Methodologies for screening
multiple connected fragment-binding events in distinct subpockets
are deployed to improve the selection of fragment combinations. A
flexible backbone based on N-alkyl-glycine amides
offers a scaffold to combinatorically link multiple fragments for
in silico screening libraries that explore the diversity of subpockets
at protein interfaces. This approach was applied to discover new potential
inhibitors of DNA replication and repair that target PCNA in a multiprotein
recognition site. The screens of the libraries were designed to computationally
filter ligands based upon the fragments and positions to <1%, which
were synthesized and tested for direct binding to PCNA. Molecular
dynamics simulations also revealed distinct features of these novel
molecules that block key PCNA–protein interactions. Furthermore,
a Bayesian classifier predicted 15 of the 16 new inhibitors to be
modulators of protein–protein interactions, demonstrating the
method’s utility as an effective screening filter. The cellular
activities of example ligands with similar affinity for PCNA demonstrate
unique properties for novel selective synergy with therapeutic DNA-damaging
agents in drug-resistant contexts.