ABSTRACT
Inhibition of poly (ADP-ribose) polymerase-1 (PARP1), a DNA repair enzyme, has proven to be a successful strategy for the treatment of various cancers. With the appropriate selection conditions and protein design, DNA-encoded library (DEL) technology provides a powerful avenue to identify small molecules with the desired mechanism of action towards a target of interest. However, DNA-binding proteins, such as PARP1, can be challenging targets for DEL screening due to non-specific protein-DNA interactions. To overcome this, we designed and screened a PARP1 catalytic domain construct without the autoinhibitory helical domain. This allowed us to interrogate an active, functionally-relevant form of the protein resulting in the discovery of novel isoindolinone PARP1 inhibitors with single-digit nanomolar potency. These inhibitors also demonstrated little to no PARP1-DNA trapping, a property that could be advantageous in the clinic.
Subject(s)
DNA , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase Inhibitors , Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors , Poly (ADP-Ribose) Polymerase-1/metabolism , Humans , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/chemical synthesis , DNA/chemistry , DNA/metabolism , Structure-Activity Relationship , Drug Discovery , Molecular Structure , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/chemical synthesis , Dose-Response Relationship, Drug , Isoindoles/chemistry , Isoindoles/pharmacology , Isoindoles/chemical synthesis , Catalytic DomainABSTRACT
Here, we describe an immunoassay approach for the detection of enzyme activity by quantitative PCR (qPCR) or parallel DNA sequencing which relies on activity-based probes linked to barcoding DNAs. We demonstrate this technique in the detection of serine hydrolase activities using a fluorophosphonate-oligonucleotide conjugate.
Subject(s)
DNA Probes/chemistry , DNA/chemistry , Hydrolases/genetics , Hydrolases/metabolism , Immunoassay , Base Sequence , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Phosphates/chemistry , Phosphates/metabolism , Polymerase Chain Reaction , Serine/genetics , Serine/metabolismABSTRACT
A sensing approach is applied to encode quantitative enzymatic activity information into DNA sequence populations. The method utilizes DNA-linked peptide substrates as activity probes. Signal detection involves chemical manipulation of a probe population downstream of sample exposure and application of purifying, selective pressure for enzyme products. Selection-induced changes in DNA abundance indicate sample activity. The detection of protein kinase, protease, and farnesyltransferase activities is demonstrated. The assays were employed to measure enzyme inhibition by small molecules and activity in cell lysates using parallel DNA sequencing or quantitative PCR. This strategy will allow the extensive infrastructure for genetic analysis to be applied to proteomic assays, which has a number of advantages in throughput, sensitivity, and sample multiplexing.
