ABSTRACT
PARP14 is an interferon-stimulated gene that is overexpressed in multiple tumor types, influencing pro-tumor macrophage polarization as well as suppressing the antitumor inflammation response by modulating IFN-γ and IL-4 signaling. PARP14 is a 203â kDa protein that possesses a catalytic domain responsible for the transfer of mono-ADP-ribose to its substrates. PARP14 also contains three macrodomains and a WWE domain which are binding modules for mono-ADP-ribose and poly-ADP-ribose, respectively, in addition to two RNA recognition motifs. Catalytic inhibitors of PARP14 have been shown to reverse IL-4 driven pro-tumor gene expression in macrophages, however it is not clear what roles the non-enzymatic biomolecular recognition motifs play in PARP14-driven immunology and inflammation. To further understand this, we have discovered a heterobifunctional small molecule designed based on a catalytic inhibitor of PARP14 that binds in the enzyme's NAD+ -binding site and recruits cereblon to ubiquitinate it and selectively target it for degradation.
Subject(s)
Poly(ADP-ribose) Polymerases/metabolism , Small Molecule Libraries/pharmacology , Humans , Macrophages/drug effects , Macrophages/metabolism , Molecular Structure , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistryABSTRACT
We have identified a class of azabenzimidazoles as potent and selective JAK1 inhibitors. Investigations into the SAR are presented along with the structural features required to achieve selectivity for JAK1 versus other JAK family members. An example from the series demonstrated highly selective inhibition of JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it to serve as a JAK1 selective tool compound to further probe the biology of JAK1 selective inhibitors.
Subject(s)
Imidazoles/pharmacology , Janus Kinase 1/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , STAT3 Transcription Factor/antagonists & inhibitors , Animals , Cell Cycle/drug effects , Cell Death/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Female , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Janus Kinase 1/metabolism , Mice , Mice, Nude , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , STAT3 Transcription Factor/metabolism , Structure-Activity RelationshipABSTRACT
PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.003 µM, exhibits >300-fold selectivity over all PARP family members, and its profile enables further study of PARP14 biology and disease association both in vitro and in vivo. Inhibition of PARP14 with RBN012759 reverses IL-4-driven protumor gene expression in macrophages and induces an inflammatory mRNA signature similar to that induced by immune checkpoint inhibitor therapy in primary human tumor explants. These data support an immune suppressive role of PARP14 in tumors and suggest potential utility of PARP14 inhibitors in the treatment of cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Inflammation/drug therapy , Interleukin-4/antagonists & inhibitors , Kidney Neoplasms/drug therapy , Macrophages/drug effects , Poly(ADP-ribose) Polymerases/metabolism , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Dose-Response Relationship, Drug , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , HEK293 Cells , Humans , Inflammation/genetics , Inflammation/metabolism , Interleukin-4/genetics , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Structure , Poly(ADP-ribose) Polymerases/genetics , RAW 264.7 Cells , RNA, Messenger/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.
Subject(s)
Drug Resistance, Neoplasm/drug effects , Interferon Type I/metabolism , Neoplasms/drug therapy , Nucleoside Transport Proteins/genetics , Nucleoside Transport Proteins/metabolism , Small Molecule Libraries/administration & dosage , Adaptive Immunity/drug effects , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , HEK293 Cells , HeLa Cells , Humans , Mice , Neoplasms/genetics , Neoplasms/metabolism , Signal Transduction/drug effects , Small Molecule Libraries/pharmacology , Tumor Escape/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Optimization of a series of azabenzimidazoles identified from screening hit 2 and the information gained from a co-crystal structure of the azabenzimidazole-based lead 6 bound to CDK9 led to the discovery of azaindoles as highly potent and selective CDK9 inhibitors. With the goal of discovering a highly selective and potent CDK9 inhibitor administrated intravenously that would enable transient target engagement of CDK9 for the treatment of hematological malignancies, further optimization focusing on physicochemical and pharmacokinetic properties led to azaindoles 38 and 39. These compounds are highly potent and selective CDK9 inhibitors having short half-lives in rodents, suitable physical properties for intravenous administration, and the potential to achieve profound but transient inhibition of CDK9 in vivo.
Subject(s)
Cyclin-Dependent Kinase 9/antagonists & inhibitors , Drug Discovery , Indoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Cyclin-Dependent Kinase 9/metabolism , Dose-Response Relationship, Drug , Humans , Indoles/chemical synthesis , Indoles/chemistry , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are posttranslational modifications found on multiple amino acids. There are 12 enzymatically active mono(ADP-ribose) polymerase (monoPARP) enzymes and 4 enzymatically active poly(ADP-ribose) polymerase (polyPARP) enzymes that use nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate to generate these modifications. While there are approved drugs and clinical trials ongoing for the enzymes that perform PARylation, MARylation is gaining recognition for its role in immune function, inflammation, and cancer. However, there is a lack of chemical probes to study the function of monoPARPs in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop biochemical assays to enable hit finding, and determination of the potency and selectivity of inhibitors. Complicating the development of enzymatic assays is that it is poorly understood how monoPARPs engage their substrates. To overcome this, we have developed a family-wide approach to developing robust high-throughput monoPARP assays where the enzymes are immobilized and forced to self-modify using biotinylated-NAD+, which is detected using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) readout. Herein we describe the development of assays for 12 monoPARPs and 3 polyPARPs and apply them to understand the potency and selectivity of a focused library of inhibitors across this family.
Subject(s)
ADP Ribose Transferases/antagonists & inhibitors , Enzyme Inhibitors/isolation & purification , High-Throughput Screening Assays , Poly(ADP-ribose) Polymerase Inhibitors/isolation & purification , Protein Processing, Post-Translational/genetics , ADP Ribose Transferases/chemistry , ADP Ribose Transferases/genetics , ADP-Ribosylation/genetics , Adenosine Diphosphate Ribose/genetics , Enzyme Inhibitors/pharmacology , Humans , NAD/chemistry , Poly ADP Ribosylation/genetics , Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Poly(ADP-ribose) Polymerases/drug effects , Poly(ADP-ribose) Polymerases/genetics , Substrate SpecificityABSTRACT
Poly(ADP-ribose) polymerase (PARP) enzymes use nicotinamide adenine dinucleotide (NAD+) to modify up to seven different amino acids with a single mono(ADP-ribose) unit (MARylation deposited by PARP monoenzymes) or branched poly(ADP-ribose) polymers (PARylation deposited by PARP polyenzymes). To enable the development of tool compounds for PARP monoenzymes and polyenzymes, we have developed active site probes for use in in vitro and cellular biophysical assays to characterize active site-directed inhibitors that compete for NAD+ binding. These assays are agnostic of the protein substrate for each PARP, overcoming a general lack of knowledge around the substrates for these enzymes. The in vitro assays use less enzyme than previously described activity assays, enabling discrimination of inhibitor potencies in the single-digit nanomolar range, and the cell-based assays can differentiate compounds with sub-nanomolar potencies and measure inhibitor residence time in live cells.
Subject(s)
Fluorescent Dyes/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Binding, Competitive , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/metabolism , HEK293 Cells , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , NAD/chemistry , NAD/metabolism , Nanoparticles/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/chemical synthesis , Poly(ADP-ribose) Polymerase Inhibitors/metabolism , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/genetics , Protein Binding , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Surface Plasmon ResonanceABSTRACT
JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of diseases. Particularly, pSTAT3 is observed in response to the treatment with inhibitors of oncogenic signaling pathways such as EGFR, MAPK, and AKT and is associated with resistance or poorer response to agents targeting these pathways. Among the JAK family kinases, JAK1 has been shown to be the primary driver of STAT3 phosphorylation and signaling; therefore, selective JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein, an account of the medicinal chemistry optimization from the promiscuous kinase screening hit 3 to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor, is reported. Compound 21 has good preclinical pharmacokinetics. Compound 21 displayed an enhanced antitumor activity in combination with an approved EGFR inhibitor, osimertinib, in a preclinical non-small-cell lung cancer (NSCLC) xenograft NCI-H1975 model.
Subject(s)
Indoles/therapeutic use , Janus Kinase 1/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Acrylamides/pharmacology , Aniline Compounds/pharmacology , Animals , Cell Line, Tumor , Drug Design , Drug Discovery , Drug Screening Assays, Antitumor , Drug Synergism , ErbB Receptors/antagonists & inhibitors , Female , Humans , Indoles/chemical synthesis , Indoles/pharmacokinetics , Mice, Nude , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
A CDK9 inhibitor having short target engagement would enable a reduction of Mcl-1 activity, resulting in apoptosis in cancer cells dependent on Mcl-1 for survival. We report the optimization of a series of amidopyridines (from compound 2), focusing on properties suitable for achieving short target engagement after intravenous administration. By increasing potency and human metabolic clearance, we identified compound 24, a potent and selective CDK9 inhibitor with suitable predicted human pharmacokinetic properties to deliver transient inhibition of CDK9. Furthermore, the solubility of 24 was considered adequate to allow i.v. formulation at the anticipated effective dose. Short-term treatment with compound 24 led to a rapid dose- and time-dependent decrease of pSer2-RNAP2 and Mcl-1, resulting in cell apoptosis in multiple hematological cancer cell lines. Intermittent dosing of compound 24 demonstrated efficacy in xenograft models derived from multiple hematological tumors. Compound 24 is currently in clinical trials for the treatment of hematological malignancies.
Subject(s)
Cyclin-Dependent Kinase 9/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Pyridines/chemistry , Animals , Apoptosis/drug effects , Binding Sites , Cell Line, Tumor , Cyclin-Dependent Kinase 9/metabolism , Dogs , Drug Evaluation, Preclinical , Half-Life , Hematologic Neoplasms/drug therapy , Hematologic Neoplasms/pathology , Humans , Mice , Molecular Docking Simulation , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyridines/metabolism , Pyridines/pharmacology , Pyridines/therapeutic use , Rats , Solubility , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Checkpoint kinase-1 (Chk1, CHEK1) is a Ser/Thr protein kinase that mediates the cellular response to DNA-damage. A novel class of 2-ureido thiophene carboxamide urea (TCU) Chk1 inhibitors is described. Inhibitors in this chemotype were optimized for cellular potency and selectivity over Cdk1.
Subject(s)
Chemistry, Pharmaceutical/methods , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Kinases/chemistry , Amides/chemistry , Apoptosis , Carbon/chemistry , Cell Cycle , Checkpoint Kinase 1 , DNA Damage , Drug Design , Humans , Inhibitory Concentration 50 , Models, Chemical , Phenyl Ethers/chemistry , Thiophenes/chemistry , Urea/chemistryABSTRACT
Checkpoint kinase 1 (CHK1) inhibitors are potential cancer therapeutics that can be utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule CHK1 inhibitors from different chemical scaffolds have been developed and evaluated in clinical trials in combination with chemotherapeutics and radiation treatment. Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and a related series of triazoloquinolines (TZQs), led to the identification of fused-ring bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs), and 7-carboxamide indoles. X-ray crystal structures reveal a key intramolecular noncovalent sulfur-oxygen interaction in aligning the hinge-binding carboxamide group to the thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an indole NH was also effective in locking the carboxamide in the preferred bound conformation to CHK1. Optimization on the 7-CTP series resulted in the identification of lead compound 44, which displayed respectable drug-like properties and good in vitro and in vivo potency.
Subject(s)
Checkpoint Kinase 1/antagonists & inhibitors , Drug Discovery , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Checkpoint Kinase 1/chemistry , DNA Damage , Humans , Indoles/chemistry , Models, Molecular , Protein Domains , Pyridines/chemistryABSTRACT
The intramolecular Morita-Baylis-Hillman (MBH) reaction has been achieved with unprecedented levels of enantioselectivity. Using a co-catalyst system involving pipecolinic acid and N-methylimidazole, cyclic MBH products have been obtained with enantiomer ratios of 92:8 (84% ee). In addition, reactions may be carried out with a "kinetic resolution quench" involving acetic anhydride and an asymmetric acylation catalyst such that ee enhancement occurs to deliver products with >98% ee with an isolated yield of 50%. [reaction: see text]
ABSTRACT
[structure: see text] Collagen is the most abundant protein in animals. Interstrand N-H...O=C hydrogen bonds between backbone amide groups form a ladder in the middle of the collagen triple helix. Isosteric replacement of the hydrogen-bond-donating amide with an ester or (E)-alkene markedly decreases the conformational stability of the triple helix. Thus, this recurring hydrogen bond is critical to the structural integrity of collagen. In this context, an ester isostere confers more stability than does an (E)-alkene.
Subject(s)
Alkenes/chemistry , Collagen/chemistry , Models, Molecular , Protein Conformation , Amino Acid Sequence , Esters , Hydrogen Bonding , Molecular Structure , Stereoisomerism , ThermodynamicsABSTRACT
INTRODUCTION: The family of inhibitor of apoptosis proteins (IAPs) plays a key role in the suppression of proapoptotic signaling; hence, a small molecule that disrupts the binding of IAPs with their functional partner should restore apoptotic response to proapoptotic stimuli in cells. The continued publication of new patent applications of IAP antagonists over the past 4 years is a testament to the continued interest surrounding the IAP family of proteins. AREAS COVERED: This review summarizes the IAP antagonist patent literature from 2010 to 2014. Monovalent and bivalent Smac mimetics will be covered as well as two new developments in the field: IAP antagonists coupled to or merged with other targeted agents and new BIR2 selective IAP antagonists. EXPERT OPINION: In addition to the well-explored scaffolds for monovalent and bivalent Smac-mimetics, some companies have taken more drastic approaches to explore new chemical space - for example, fragment-based approaches and macrocyclic inhibitors. Furthermore, other companies have designed compounds with alternative biological profiles - tethering to known kinase binding structures, trying to target to the mitochondria or introducing selective binding to the BIR2 domain. An overview of the status for the four small molecule IAP antagonists being evaluated in active human clinical trials is also provided.
Subject(s)
Biomimetic Materials/pharmacology , Drug Design , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Animals , Apoptosis/drug effects , Humans , Molecular Targeted Therapy , Oligopeptides/chemistry , Patents as Topic , Signal Transduction/drug effectsABSTRACT
[reaction: see text] Nucleophile-loaded peptides and proline have been found to function synergistically as cocatalysts for the asymmetric ketone-based Baylis-Hillman reaction. Although neither compound is effective independently in terms of rate or enantioselectivity, their combination leads to catalysis where enantioselectivities up to 81% have been observed.
Subject(s)
Aldehydes/chemistry , Peptides/chemistry , Proline/chemistry , Butanones/chemistry , Catalysis , StereoisomerismABSTRACT
Ethyl diazoacetate undergoes dirhodium(II)-catalyzed reactions with aryl aldehydes to form 1,3-dioxolanes as mixtures of diastereoisomers in good yields. Carbonyl ylides are reaction intermediates. Catalyst dependent diastereocontrol is observed for reactions with p-nitrobenzaldehyde, but not for those with p-anisaldehyde or benzaldehyde, so that at least with transformations involving p-nitrobenzaldehyde a metal-stabilized ylide is responsible for product formation. Higher yields are obtained with catalysis by dirhodium(II) carboxamidates than with the carboxylates. Diastereoselectivity in 1,3-dioxolane formation that occurs through the "free" ylide can be effectively controlled so that only one diastereomer is produced through the use of 2,6-di-tert-butyl-4-methylphenyl diazoacetate (BDA) or dicyclohexylmethyl diazoacetate (DCM). The thermodynamically least stable all-cis trisubstituted 1,3-dioxolane is the primary product from p-nitrobenzaldehyde "cycloaddition" to the metal-stabilized ylide. Reactions that take place in the presence of p-anisaldehyde and dimethyl acetylenedicarboxylate (DMAD) result in the formation of one 2,5-dihydrofuran-2-carboxylate stereoisomer in good yield. In contrast, with p-nitrobenzaldehyde and DMAD both dihydrofuran stereoisomers are produced along with, mainly, the dioxolane derived from the metal-stabilized ylide; there is in this case competition between addition reactions of the "free" ylide and the metal-associated ylide.
ABSTRACT
Olefinic analogues of effective peptide-based catalysts for the kinetic resolution of functionalized racemic secondary alcohols have been synthesized. The isosteric replacement of the peptide amide bond in 1 with an (E)-alkene to form 2 has enabled the evaluation of the kinetic role of particular amides within these catalysts.
ABSTRACT
A series of dimeric compounds based on the AVPI motif of Smac were designed and prepared as antagonists of the inhibitor of apoptosis proteins (IAPs). Optimization of cellular potency, physical properties, and pharmacokinetic parameters led to the identification of compound 14 (AZD5582), which binds potently to the BIR3 domains of cIAP1, cIAP2, and XIAP (IC50 = 15, 21, and 15 nM, respectively). This compound causes cIAP1 degradation and induces apoptosis in the MDA-MB-231 breast cancer cell line at subnanomolar concentrations in vitro. When administered intravenously to MDA-MB-231 xenograft-bearing mice, 14 results in cIAP1 degradation and caspase-3 cleavage within tumor cells and causes substantial tumor regressions following two weekly doses of 3.0 mg/kg. Antiproliferative effects are observed with 14 in only a small subset of the over 200 cancer cell lines examined, consistent with other published IAP inhibitors. As a result of its in vitro and in vivo profile, 14 was nominated as a candidate for clinical development.
Subject(s)
Alkynes/pharmacology , Antineoplastic Agents/pharmacology , Biomimetic Materials/pharmacology , Drug Discovery , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Neoplasms/drug therapy , Oligopeptides/pharmacology , Alkynes/chemical synthesis , Alkynes/chemistry , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Biomimetic Materials/chemical synthesis , Biomimetic Materials/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dimerization , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Inhibitor of Apoptosis Proteins/metabolism , Mice , Molecular Conformation , Neoplasms/pathology , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
B-Raf represents an attractive target for anticancer therapy and the development of small molecule B-Raf inhibitors has delivered new therapies for metastatic melanoma patients. We have discovered a novel class of small molecules that inhibit mutant B-Raf(V600E) kinase activity both in vitro and in vivo. Investigations into the structure-activity relationships of the series are presented along with efforts to improve upon the cellular potency, solubility, and pharmacokinetic profile. Compounds selectively inhibited B-Raf(V600E) in vitro and showed preferential antiproliferative activity in mutant B-Raf(V600E) cell lines and exhibited selectivity in a kinase panel against other kinases. Examples from this series inhibit growth of a B-Raf(V600E) A375 xenograft in vivo at a well-tolerated dose. In addition, aminoquinazolines described herein were shown to display pERK elevation in nonmutant B-Raf cell lines in vitro.