ABSTRACT
Toll-like receptor (TLR) 7 and TLR8 are endosomal sensors of the innate immune system that are activated by GU-rich single stranded RNA (ssRNA). Multiple genetic and functional lines of evidence link chronic activation of TLR7/8 to the pathogenesis of systemic autoimmune diseases (sAID) such as Sjögren's syndrome (SjS) and systemic lupus erythematosus (SLE). This makes targeting TLR7/8-induced inflammation with small-molecule inhibitors an attractive approach for the treatment of patients suffering from systemic autoimmune diseases. Here, we describe how structure-based optimization of compound 2 resulted in the discovery of 34 (MHV370, (S)-N-(4-((5-(1,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)methyl)bicyclo[2.2.2]octan-1-yl)morpholine-3-carboxamide). Its in vivo activity allows for further profiling toward clinical trials in patients with autoimmune disorders, and a Phase 2 proof of concept study of MHV370 has been initiated, testing its safety and efficacy in patients with Sjögren's syndrome and mixed connective tissue disease.
ABSTRACT
Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM.
Subject(s)
Antineoplastic Agents , Leukemia, Myeloid, Acute , Multiple Myeloma , Transcription Factors , Antineoplastic Agents/pharmacology , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , RNA Interference , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Inappropriate activation of TLR7 and TLR8 is linked to several autoimmune diseases, such as lupus erythematosus. Here we report on the efficient structure-based optimization of the inhibition of TLR8, starting from a co-crystal structure of a small screening hit. Further optimization of the physicochemical properties for cellular potency and expansion of the structure-activity relationship for dual potency finally resulted in a highly potent TLR7/8 antagonist with demonstrated in vivo efficacy after oral dosing.
ABSTRACT
Bees are exposed to various stressors, including pesticides and lack of flowering resources. Despite potential interactions between these stressors, the impacts of pesticides on bees are generally assumed to be consistent across bee-attractive crops, and regulatory risk assessments of pesticides neglect interactions with flowering resources. Furthermore, impacts of fungicides on bees are rarely examined in peer-reviewed studies, although these are often the pesticides that bees are most exposed to. In a full-factorial semi-field experiment with 39 large flight cages, we assessed the single and combined impacts of the globally used azoxystrobin-based fungicide Amistar® and three types of flowering resources (Phacelia, buckwheat, and a floral mix) on Bombus terrestris colonies. Although Amistar is classified as bee-safe, Amistar exposure through Phacelia monocultures reduced adult worker body mass and colony growth (including a 55% decline in workers and an 88% decline in males), while the fungicide had no impact on colonies in buckwheat or the floral mix cages. Furthermore, buckwheat monocultures hampered survival and fecundity irrespective of fungicide exposure. This shows that bumblebees require access to complementary flowering species to gain both fitness and fungicide tolerance and that Amistar impacts are flowering resource-dependent. Our findings call for further research on how different flowering plants affect bees and their pesticide tolerance to improve guidelines for regulatory pesticide risk assessments and inform the choice of plants that are cultivated to safeguard pollinators.
Subject(s)
Fungicides, Industrial , Insecticides , Pesticides , Animals , Bees , Crops, Agricultural , Fungicides, Industrial/toxicity , Male , ReproductionABSTRACT
Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.
Subject(s)
Cell Cycle Proteins/genetics , Epidermis/drug effects , Re-Epithelialization/drug effects , Skin Ulcer/drug therapy , Small Molecule Libraries/pharmacology , Transcription Factors/genetics , Wounds, Nonpenetrating/drug therapy , Animals , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Disease Models, Animal , Epidermis/metabolism , Epidermis/pathology , Fluorescence Resonance Energy Transfer , Gene Expression Regulation , High-Throughput Screening Assays , Humans , Keratinocytes/drug effects , Keratinocytes/metabolism , Keratinocytes/pathology , Male , Mice , Mice, Inbred C57BL , Primary Cell Culture , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Precursors/antagonists & inhibitors , Protein Precursors/genetics , Protein Precursors/metabolism , Re-Epithelialization/genetics , Skin Ulcer/genetics , Skin Ulcer/metabolism , Skin Ulcer/pathology , Small Molecule Libraries/chemistry , Structure-Activity Relationship , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Transcription, Genetic , Wounds, Nonpenetrating/genetics , Wounds, Nonpenetrating/metabolism , Wounds, Nonpenetrating/pathologyABSTRACT
A series of inhibitors of Autotaxin (ATX) have been developed from a high throughput screening hit, 1a, which shows an alternative binding mode to known catalytic site inhibitors. Selectivity over the hERG channel and microsomal clearance were dependent on the lipophilicity of the compounds, and this was optimised by reduction of clogD whilst maintaining high affinity ATX inhibition. Compound 15a shows good oral exposure, and concentration dependent inhibition of formation of LPA in vivo, as shown in pharmacokinetic-pharmacodynamic (PK/PD) experiments.
Subject(s)
Amides/pharmacology , Cinnamates/pharmacology , Drug Development , Enzyme Inhibitors/pharmacology , Phosphoric Diester Hydrolases/metabolism , Tetrazoles/pharmacology , Amides/chemical synthesis , Amides/chemistry , Animals , Cinnamates/chemical synthesis , Cinnamates/chemistry , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Mice , Models, Molecular , Molecular Structure , Rats , Structure-Activity Relationship , Tetrazoles/chemical synthesis , Tetrazoles/chemistryABSTRACT
Inappropriate activation of endosomal TLR7 and TLR8 occurs in several autoimmune diseases, in particular systemic lupus erythematosus (SLE). Herein, the development of a TLR8 antagonist competition assay and its application for hit generation of dual TLR7/8 antagonists are reported. The structure-guided optimization of the pyridone hit 3 using this biochemical assay in combination with cellular and TLR8 cocrystal structural data resulted in the identification of a highly potent and selective TLR7/8 antagonist (27) with in vivo efficacy. The two key steps for optimization were (i) a core morph guided by a TLR7 sequence alignment to achieve a dual TLR7/8 antagonism profile and (ii) introduction of a fluorine in the piperidine ring to reduce its basicity, resulting in attractive oral pharmacokinetic (PK) properties and improved TLR8 binding affinity.
Subject(s)
Lupus Erythematosus, Systemic/drug therapy , Pyridones/chemistry , Pyridones/pharmacology , Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 8/antagonists & inhibitors , Animals , Cells, Cultured , Drug Discovery , Humans , Indazoles/chemistry , Indazoles/pharmacokinetics , Indazoles/pharmacology , Lupus Erythematosus, Systemic/metabolism , Male , Mice, Inbred C57BL , Models, Molecular , Pyridones/pharmacokinetics , Rats, Sprague-Dawley , Toll-Like Receptor 7/chemistry , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/chemistry , Toll-Like Receptor 8/metabolismABSTRACT
Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12.
Subject(s)
ELAV Proteins/chemistry , ELAV-Like Protein 1/chemistry , RNA Recognition Motif/genetics , RNA-Binding Proteins/chemistry , 3' Untranslated Regions , AU Rich Elements/genetics , Crystallography, X-Ray , Dimerization , ELAV-Like Protein 1/genetics , Humans , Magnetic Resonance Spectroscopy , RNA-Binding Proteins/genetics , Ribonucleoside Diphosphate Reductase/chemistry , Tumor Suppressor Proteins/chemistryABSTRACT
A series of inhibitors of Autotaxin (ATX) has been developed using the binding mode of known inhibitor, PF-8380, as a template. Replacement of the benzoxazolone with a triazole zinc-binding motif reduced crystallinity and improved solubility relative to PF-8380. Modification of the linker region removed hERG activity and led to compound 12 - a selective, high affinity, orally-bioavailable inhibitor of ATX. Compound 12 concentration-dependently inhibits autotaxin and formation of LPA in vivo, as shown in pharmacokinetic-pharmacodynamic experiments.
Subject(s)
Drug Design , Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/metabolism , Triazoles/pharmacology , Administration, Oral , Animals , Benzoxazoles/pharmacology , Drug Stability , Humans , Male , Microsomes/metabolism , Phosphodiesterase Inhibitors/administration & dosage , Phosphodiesterase Inhibitors/chemical synthesis , Phosphodiesterase Inhibitors/pharmacokinetics , Piperazines/pharmacology , Rats, Sprague-Dawley , Solubility , Triazoles/administration & dosage , Triazoles/chemical synthesis , Triazoles/pharmacokineticsABSTRACT
DGCR8 (DiGeorge Critical Region 8) is an essential microRNA (miRNA) processing protein that recognizes primary transcripts of miRNAs (pri-miRNAs) and triggers their cleavage by the Drosha nuclease. We previously found that Fe(III) heme binds and activates DGCR8. Here we report that in HeLa cells, DGCR8 undergoes two proteolytic events that produce two C-terminal fragments called DGCR8(C1) and DGCR8(C2) , respectively. DGCR8(C2) accumulates during apoptosis and is generated through cleavage by a caspase. The caspase cleavage site is located in the central loop of the heme-binding domain. Cleavage of DGCR8 by caspase-3 in vitro results in loss of the otherwise tightly bound Fe(III) heme cofactor, dissociation of the N- and C-terminal proteolytic fragments, and inhibition of the pri-miRNA processing activity. These results reveal an intrinsic mechanism in the DGCR8 protein that seems to have evolved for regulating miRNA processing via association with Fe(III) heme and proteolytic cleavage by caspases. Decreased expression of miRNAs has been observed in apoptotic cells, and this change was attributed to caspase-mediated cleavage of a down-stream miRNA processing nuclease Dicer. We suggest that both the Drosha and Dicer cleavage steps of the miRNA maturation pathway may be inhibited in apoptosis and other biological processes where caspases are activated.
Subject(s)
Caspases/metabolism , Proteins/metabolism , Amino Acid Sequence , Apoptosis , HeLa Cells , Heme/metabolism , Humans , MicroRNAs/metabolism , Molecular Sequence Data , Proteins/chemistry , Proteolysis , RNA-Binding Proteins , Sequence AlignmentABSTRACT
DiGeorge critical region 8 (DGCR8) is essential for maturation of microRNAs (miRNAs) in animals. In the cleavage of primary transcripts of miRNAs (pri-miRNAs) by the Drosha nuclease, the DGCR8 protein directly binds and recognizes pri-miRNAs through a mechanism currently controversial. Our previous data suggest that DGCR8 trimerizes upon cooperative binding to pri-mir-30a. However, a separate study proposed a model in which a DGCR8 molecule contacts one or two pri-miRNA molecules using its two double-stranded RNA binding domains. Here, we extensively characterized the interaction between DGCR8 and pri-miRNAs using biochemical and structural methods. First, a strong correlation was observed between the association of DGCR8 with pri-mir-30a and the rate of pri-miRNA processing in vitro. Second, we show that the high binding cooperativity allows DGCR8 to distinguish pri-miRNAs from a nonspecific competitor with subtle differences in dissociation constants. The highly cooperative binding of DGCR8 to a pri-miRNA is mediated by the formation of higher-order structures, most likely a trimer of DGCR8 dimers, on the pri-miRNA. These properties are not limited to its interaction with pri-mir-30a. Furthermore, the amphipathic C-terminal helix of DGCR8 is important both for trimerization of DGCR8 on pri-miRNAs and for the cleavage of pri-miRNAs by Drosha. Finally, our three-dimensional model from electron tomography analysis of the negatively stained DGCR8-pri-mir-30a complex directly supports the trimerization model. Our study provides a molecular basis for recognition of pri-miRNAs by DGCR8. We further propose that the higher-order structures of the DGCR8-pri-miRNA complexes trigger the cleavage of pri-miRNAs by Drosha.
Subject(s)
MicroRNAs/metabolism , Ribonuclease III/metabolism , Animals , DEAD-box RNA Helicases , DiGeorge Syndrome/genetics , DiGeorge Syndrome/metabolism , MicroRNAs/chemistry , MicroRNAs/genetics , Protein Structure, Tertiary/genetics , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , Ribonuclease III/chemistry , Ribonuclease III/geneticsABSTRACT
Maturation of microRNAs (miRNAs, approximately 22nt) from long primary transcripts [primary miRNAs (pri-miRNAs)] is regulated during development and is altered in diseases such as cancer. The first processing step is a cleavage mediated by the Microprocessor complex containing the Drosha nuclease and the RNA-binding protein DiGeorge critical region 8 (DGCR8). We previously reported that dimeric DGCR8 binds heme and that the heme-bound DGCR8 is more active than the heme-free form. Here, we identified a conserved dimerization domain in DGCR8. Our crystal structure of this domain (residues 298-352) at 1.7 A resolution demonstrates a previously unknown use of a WW motif as a platform for extensive dimerization interactions. The dimerization domain of DGCR8 is embedded in an independently folded heme-binding domain and directly contributes to association with heme. Heme-binding-deficient DGCR8 mutants have reduced pri-miRNA processing activity in vitro. Our study provides structural and biochemical bases for understanding how dimerization and heme binding of DGCR8 may contribute to regulation of miRNA biogenesis.
Subject(s)
Proteins/chemistry , Proteins/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Proteins/genetics , RNA-Binding Proteins , Spectrometry, Mass, Electrospray IonizationABSTRACT
In prokaryotes, cognate toxin-antitoxin pairs have long been known, but no three-dimensional structure has been available for any given complex from Mycobacterium tuberculosis. Here we report the crystal structure and activity of a member of the VapBC family of complexes from M. tuberculosis. The toxin VapC-5 is a compact, 150 residues, two domain alpha/beta protein. Bent around the toxin is the VapB-5 antitoxin, a 33-residue alpha-helix. Assays suggest that the toxin is an Mg-enabled endoribonuclease, inhibited by the antitoxin. The lack of DNase activity is consistent with earlier suggestions that the complex represses its own operon. Furthermore, analysis of the interactions in the binding of the antitoxin to the toxin suggest that exquisite control is required to protect the bacteria cell from toxic VapC-5.
Subject(s)
Bacterial Proteins/chemistry , DNA-Binding Proteins/chemistry , Membrane Glycoproteins/chemistry , Mycobacterium tuberculosis/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Protein Structure, Quaternary/physiology , Protein Structure, Secondary/physiology , Protein Structure, Tertiary/physiologyABSTRACT
microRNAs (miRNAs) are extensively involved in developmental programming. Some miRNAs are highly conserved, while others are lineage specific. All miRNAs maturate through a series of processing steps. Here we review recent progresses in the studies of early steps in miRNA biogenesis, focusing on animal systems. The miRNA maturation pathways are surprisingly diverse, involving transcription by RNA polymerase II or III, cleavage by the Drosha nuclease or the spliceosome, and sometimes modifications by the adenosine deaminase ADAR. The relationship between the diversity in miRNA biogenesis and the apparently rapid evolution of miRNA genes and functions is discussed.
Subject(s)
MicroRNAs/biosynthesis , Animals , DNA-Directed RNA Polymerases/metabolism , Evolution, Molecular , Humans , RNA Precursors/metabolism , Spliceosomes/metabolism , Transcription, GeneticABSTRACT
MicroRNAs (miRNAs) regulate the expression of a large number of protein-coding genes. Their primary transcripts (pri-miRNAs) have to undergo multiple processing steps to reach the functional form. Little is known about how the processing of miRNAs is modulated. Here we show that the RNA-binding protein DiGeorge critical region-8 (DGCR8), which is essential for the first processing step, is a heme-binding protein. The association with heme promotes dimerization of DGCR8. The heme-bound DGCR8 dimer seems to trimerize upon binding pri-miRNAs and is active in triggering pri-miRNA cleavage, whereas the heme-free monomer is much less active. A heme-binding region of DGCR8 inhibits the pri-miRNA-processing activity of the monomer. This putative autoinhibition is overcome by heme. Our finding that heme is involved in pri-miRNA processing suggests that the gene-regulation network of miRNAs and signal-transduction pathways involving heme might be connected.
Subject(s)
Heme/metabolism , MicroRNAs/metabolism , Proteins/metabolism , RNA Processing, Post-Transcriptional , Amino Acid Sequence , Dimerization , Heme/chemistry , Hemeproteins/chemistry , Hemeproteins/metabolism , Humans , Molecular Sequence Data , Protein Structure, Tertiary , Proteins/chemistry , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolismABSTRACT
Mycobacteria have low-permeability outer membranes that render them resistant to most antibiotics. Hydrophilic nutrients can enter by way of transmembrane-channel proteins called porins. An x-ray analysis of the main porin from Mycobacterium smegmatis, MspA, revealed a homooctameric goblet-like conformation with a single central channel. This is the first structure of a mycobacterial outer-membrane protein. No structure-related protein was found in the Protein Data Bank. MspA contains two consecutive beta barrels with nonpolar outer surfaces that form a ribbon around the porin, which is too narrow to fit the thickness of the mycobacterial outer membrane in contemporary models.