ABSTRACT
Dysregulation of IL17A drives numerous inflammatory and autoimmune disorders with inhibition of IL17A using antibodies proven as an effective treatment. Oral anti-IL17 therapies are an attractive alternative option, and several preclinical small molecule IL17 inhibitors have previously been described. Herein, we report the discovery of a novel class of small molecule IL17A inhibitors, identified via a DNA-encoded chemical library screen, and their subsequent optimization to provide in vivo efficacious inhibitors. These new protein-protein interaction (PPI) inhibitors bind in a previously undescribed mode in the IL17A protein with two copies binding symmetrically to the central cavities of the IL17A homodimer.
Subject(s)
DNA , Drug Discovery , Interleukin-17 , Small Molecule Libraries , Interleukin-17/metabolism , Interleukin-17/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , DNA/metabolism , DNA/chemistry , Humans , Animals , Structure-Activity Relationship , Protein Binding , MiceABSTRACT
DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from Mycobacterium tuberculosis (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.
Subject(s)
Antitubercular Agents , Enzyme Inhibitors , Mycobacterium tuberculosis , Polyketide Synthases , Small Molecule Libraries , Thiolester Hydrolases , Animals , Humans , Mice , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Crystallography, X-Ray , Disease Models, Animal , Drug Discovery , Drug Evaluation, Preclinical , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/drug effects , Polyketide Synthases/metabolism , Polyketide Synthases/chemistry , Polyketide Synthases/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Thiolester Hydrolases/antagonists & inhibitors , Thiolester Hydrolases/metabolism , Thiolester Hydrolases/chemistry , Thiolester Hydrolases/genetics , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
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
The c-MET receptor tyrosine kinase has received considerable attention as a cancer drug target yet there remains a need for inhibitors which are selective for c-MET and able to target emerging drug-resistant mutants. We report here the discovery, by screening a DNA-encoded chemical library, of a highly selective c-MET inhibitor which was shown by X-ray crystallography to bind to the kinase in an unprecedented manner. These results represent a novel mode of inhibiting c-MET with a small molecule and may provide a route to targeting drug-resistant forms of the kinase whilst avoiding potential toxicity issues associated with broad kinome inhibition.
Subject(s)
Antineoplastic Agents , Proto-Oncogene Proteins c-met , Antineoplastic Agents/pharmacology , Cell Line, Tumor , DNA , Protein Kinase Inhibitors/chemistry , Small Molecule Libraries/chemistryABSTRACT
Mer is a member of the TAM (Tyro3, Axl, Mer) kinase family that has been associated with cancer progression, metastasis, and drug resistance. Their essential function in immune homeostasis has prompted an interest in their role as modulators of antitumor immune response in the tumor microenvironment. Here we illustrate the outcomes of an extensive lead-generation campaign for identification of Mer inhibitors, focusing on the results from concurrent, orthogonal high-throughput screening approaches. Data mining, HT (high-throughput), and DECL (DNA-encoded chemical library) screens offered means to evaluate large numbers of compounds. We discuss campaign strategy and screening outcomes, and exemplify series resulting from prioritization of hits that were identified. Concurrent execution of HT and DECL screening successfully yielded a large number of potent, selective, and novel starting points, covering a range of selectivity profiles across the TAM family members and modes of kinase binding, and offered excellent start points for lead development.
Subject(s)
Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , c-Mer Tyrosine Kinase/antagonists & inhibitors , Animals , Crystallography, X-Ray , Data Mining , Drug Discovery , Humans , Models, Molecular , c-Mer Tyrosine Kinase/chemistry , c-Mer Tyrosine Kinase/metabolismABSTRACT
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.
Subject(s)
DNA Probes/genetics , DNA/genetics , Farnesyltranstransferase/metabolism , Peptide Hydrolases/metabolism , Protein Kinases/metabolism , Base Sequence , DNA/chemistry , DNA Probes/chemistry , DNA Probes/metabolism , Enzyme Activation , Farnesyltranstransferase/analysis , Humans , Peptide Hydrolases/analysis , Polymerase Chain Reaction , Protein Kinases/analysisABSTRACT
Retinoic acid receptor alpha (RARα) selective compounds may guide the design of drugs that can be used in conjunction with hormonal adjuvant therapy in the treatment of breast cancer. Herein we report a modified synthesis of a known RARα antagonist, 2-fluoro-4-[[[8-bromo-2,2-dimethyl-4-(4-methylphenyl)chroman-6-yl]carbonyl]amino]benzoic acid and a synthesis of its unknown, desfluoro analog, 4-[[[8-bromo-2,2-dimethyl-4-(4-methylphenyl)chroman-6-yl]carbonyl]amino]benzoic acid. The modified route allows for facile reaction workups, increased yields, lower cost and incorporates a green alternative step. Structure-activity relationship studies determined through functional cell-based assays, demonstrated antagonism to RARα for both compounds. Molecular modeling within the RARα binding pocket was used to compare binding interactions of the desfluoro analog to a known RAR antagonist.