ABSTRACT
BACKGROUND: Drug targets with genetic evidence are expected to increase clinical success by at least twofold. Yet, translating disease-associated genetic variants into functional knowledge remains a fundamental challenge of drug discovery. A key issue is that the vast majority of complex disease associations cannot be cleanly mapped to a gene. Immune disease-associated variants are enriched within regulatory elements found in T-cell-specific open chromatin regions. RESULTS: To identify genes and molecular programs modulated by these regulatory elements, we develop a CRISPRi-based single-cell functional screening approach in primary human T cells. Our pipeline enables the interrogation of transcriptomic changes induced by the perturbation of regulatory elements at scale. We first optimize an efficient CRISPRi protocol in primary CD4+ T cells via CROPseq vectors. Subsequently, we perform a screen targeting 45 non-coding regulatory elements and 35 transcription start sites and profile approximately 250,000 T -cell single-cell transcriptomes. We develop a bespoke analytical pipeline for element-to-gene (E2G) mapping and demonstrate that our method can identify both previously annotated and novel E2G links. Lastly, we integrate genetic association data for immune-related traits and demonstrate how our platform can aid in the identification of effector genes for GWAS loci. CONCLUSIONS: We describe "primary T cell crisprQTL" - a scalable, single-cell functional genomics approach for mapping regulatory elements to genes in primary human T cells. We show how this framework can facilitate the interrogation of immune disease GWAS hits and propose that the combination of experimental and QTL-based techniques is likely to address the variant-to-function problem.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Immune System Diseases , Humans , T-Lymphocytes , Regulatory Sequences, Nucleic Acid , Chromatin/genetics , Immune System Diseases/genetics , Genome-Wide Association Study , Polymorphism, Single NucleotideABSTRACT
The trapping of Poly-ADP-ribose polymerase (PARP) on DNA caused by PARP inhibitors (PARPi) triggers acute DNA replication stress and synthetic lethality (SL) in BRCA2-deficient cells. Hence, DNA damage is accepted as a prerequisite for SL in BRCA2-deficient cells. In contrast, here we show that inhibiting ROCK in BRCA2-deficient cells triggers SL independently from acute replication stress. Such SL is preceded by polyploidy and binucleation resulting from cytokinesis failure. Such initial mitosis abnormalities are followed by other M phase defects, including anaphase bridges and abnormal mitotic figures associated with multipolar spindles, supernumerary centrosomes and multinucleation. SL was also triggered by inhibiting Citron Rho-interacting kinase, another enzyme that, similarly to ROCK, regulates cytokinesis. Together, these observations demonstrate that cytokinesis failure triggers mitotic abnormalities and SL in BRCA2-deficient cells. Furthermore, the prevention of mitotic entry by depletion of Early mitotic inhibitor 1 (EMI1) augmented the survival of BRCA2-deficient cells treated with ROCK inhibitors, thus reinforcing the association between M phase and cell death in BRCA2-deficient cells. This novel SL differs from the one triggered by PARPi and uncovers mitosis as an Achilles heel of BRCA2-deficient cells.
Subject(s)
DNA Damage , Synthetic Lethal Mutations , Anaphase , Mitosis , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , rho-Associated Kinases/antagonists & inhibitors , BRCA2 Protein/genetics , HumansABSTRACT
BRCA2 is a well-established cancer driver in several human malignancies. While the remarkable success of PARP inhibitors proved the clinical potential of targeting BRCA deficiencies, the emergence of resistance mechanisms underscores the importance of seeking novel Synthetic Lethal (SL) targets for future drug development efforts. In this work, we performed a BRCA2-centric SL screen with a collection of plant-derived compounds from South America. We identified the steroidal alkaloid Solanocapsine as a selective SL inducer, and we were able to substantially increase its potency by deriving multiple analogs. The use of two complementary chemoproteomic approaches led to the identification of the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) as Solanocapsine's target responsible for its BRCA2-linked SL induction. Additional confirmatory evidence was obtained by using the highly specific dCK inhibitor (DI-87), which induces SL in multiple BRCA2-deficient and KO contexts. Interestingly, dCK-induced SL is mechanistically different from the one induced by PARP inhibitors. dCK inhibition generates substantially lower levels of DNA damage, and cytotoxic phenotypes are associated exclusively with mitosis, thus suggesting that the fine-tuning of nucleotide supply in mitosis is critical for the survival of BRCA2-deficient cells. Moreover, by using a xenograft model of contralateral tumors, we show that dCK impairment suffices to trigger SL in-vivo. Taken together, our findings unveil dCK as a promising new target for BRCA2-deficient cancers, thus setting the ground for future therapeutic alternatives to PARP inhibitors.
Subject(s)
Antineoplastic Agents , Deoxycytidine Kinase , Humans , Deoxycytidine Kinase/genetics , Deoxycytidine Kinase/metabolism , Poly(ADP-ribose) Polymerase Inhibitors , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Nucleotides/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , BRCA2 Protein/geneticsABSTRACT
Alternative mode-of-inhibition of clinically validated targets is an effective strategy for circumventing existing clinical drug resistance. Herein, we report 1,3-diarylpyrazolyl-acylsulfonamides as potent inhibitors of HadAB/BC, a 3-hydroxyl-ACP dehydratase complex required to iteratively elongate the meromycolate chain of mycolic acids in Mycobacterium tuberculosis (Mtb). Mutations in compound 1-resistant Mtb mutants mapped to HadC (Rv0637; K157R), while chemoproteomics confirmed the compound's binding to HadA (Rv0635), HadB (Rv0636), and HadC. The compounds effectively inhibited the HadAB and HadBC enzyme activities and affected mycolic acid biosynthesis in Mtb, in a concentration-dependent manner. Unlike known 3-hydroxyl-ACP dehydratase complex inhibitors of clinical significance, isoxyl and thioacetazone, 1,3-diarylpyrazolyl-acylsulfonamides did not require activation by EthA and thus are not liable to EthA-mediated resistance. Further, the crystal structure of a key compound in a complex with Mtb HadAB revealed unique binding interactions within the active site of HadAB, providing a useful tool for further structure-based optimization of the series.
Subject(s)
Mycobacterium tuberculosis , Thioacetazone , Bacterial Proteins/metabolism , Mycolic Acids/chemistry , Thioacetazone/metabolism , Thioacetazone/pharmacology , Hydro-Lyases/chemistry , Hydro-Lyases/metabolism , Hydro-Lyases/pharmacologyABSTRACT
Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human "mammalian target of rapamycin" (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kß) and cyclic guanosine monophosphate-dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kß in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kß. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kß and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.
Subject(s)
Antimalarials , Plasmodium , Animals , Humans , Antimalarials/pharmacology , Antimalarials/therapeutic use , Plasmodium falciparum , MTOR Inhibitors , 1-Phosphatidylinositol 4-Kinase , Guanosine Monophosphate , Life Cycle Stages , TOR Serine-Threonine Kinases , Sirolimus , MammalsABSTRACT
The sensitivity of Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis, which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.
Subject(s)
Mycobacterium tuberculosis , Prodrugs , Tuberculosis , Animals , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Ethionamide/chemistry , Ethionamide/pharmacology , Ethionamide/therapeutic use , Mice , Prodrugs/pharmacology , Prodrugs/therapeutic use , Tuberculosis/drug therapyABSTRACT
N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing four different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared with parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.
Subject(s)
Congenital Disorders of Glycosylation , Congenital Disorders of Glycosylation/genetics , Congenital Disorders of Glycosylation/metabolism , Gene Expression Regulation , Humans , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/deficiency , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/genetics , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Proteasome Endopeptidase Complex/metabolismABSTRACT
PSENEN/PEN2 is the smallest subunit of the γ-secretase complex, an intramembrane protease that cleaves proteins within their transmembrane domains. Mutations in components of the γ-secretase underlie familial Alzheimer disease. In addition to its proteolytic activity, supplementary, γ-secretase independent, functions in the macroautophagy/autophagy-lysosome system have been proposed. Here, we screened for PSENEN-interacting proteins and identified CLN3. Mutations in CLN3 are causative for juvenile neuronal ceroid lipofuscinosis, a rare lysosomal storage disorder considered the most common neurodegenerative disease in children. As mutations in the PSENEN and CLN3 genes cause different neurodegenerative diseases, understanding shared cellular functions of both proteins might be pertinent for understanding general cellular mechanisms underlying neurodegeneration. We hypothesized that CLN3 modulates γ-secretase activity and that PSENEN and CLN3 play associated roles in the autophagy-lysosome system. We applied CRISPR gene-editing and obtained independent isogenic HeLa knockout cell lines for PSENEN and CLN3. Following previous studies, we demonstrate that PSENEN is essential for forming a functional γ-secretase complex and is indispensable for γ-secretase activity. In contrast, CLN3 does not modulate γ-secretase activity to a significant degree. We observed in PSENEN- and CLN3-knockout cells corresponding alterations in the autophagy-lysosome system. These include reduced activity of lysosomal enzymes and lysosome number, an increased number of autophagosomes, increased lysosome-autophagosome fusion, and elevated levels of TFEB (transcription factor EB). Our study strongly suggests converging roles of PSENEN and CLN3 in the autophagy-lysosome system in a γ-secretase activity-independent manner, supporting the idea of common cytopathological processes underlying different neurodegenerative diseases.Abbreviations: Aß, amyloid-beta; AD, Alzheimer disease; APP, amyloid precursor protein; ATP5MC, ATP synthase membrane subunit c; DQ-BSA, dye-quenched bovine serum albumin; ER, endoplasmic reticulum; GFP, green fluorescent protein; ICC, immunocytochemistry; ICD, intracellular domain; JNCL, juvenile neuronal ceroid lipofuscinosis; KO, knockout; LC3, microtubule associated protein 1 light chain 3; NCL, neuronal ceroid lipofuscinoses; PSEN, presenilin; PSENEN/PEN2: presenilin enhancer, gamma-secretase subunit; TAP, tandem affinity purification; TEV, tobacco etch virus; TF, transferrin; WB, Western blot; WT, wild type.
Subject(s)
Alzheimer Disease , Neuronal Ceroid-Lipofuscinoses , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Autophagy/genetics , Child , Humans , Lysosomes/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Molecular Chaperones/metabolism , Neuronal Ceroid-Lipofuscinoses/genetics , Neuronal Ceroid-Lipofuscinoses/metabolism , Presenilins/genetics , Presenilins/metabolism , Transcription Factors/metabolismABSTRACT
OATP2B1, a member of the solute carrier (SLC) transporter family, is an important mechanism of substrate drug uptake in the intestine and liver and therefore a determinant of clinical pharmacokinetics and site of drug-drug interactions. Other SLC transporters have emerged as pharmacology targets. Studies of SLC transporter uptake to-date relied on radioisotope- or fluorescence-labeled reagents or low-throughput quantification of unlabeled compounds in cell lysate. In this study, we developed a cell-based MALDI MS workflow for investigation of OATP2B1 cellular uptake by optimizing the substrate, matrix, matrix-analyte ratio, and matrix application and normalization method. This workflow was automated and applied to characterize substrate transport kinetics and to test 294 top-marketed drugs for OATP2B1 inhibition and quantify inhibitory potencies necessary for extrapolation of clinical drug-drug interaction potential. Intra-assay reproducibility of this MALDI MS method was high (CV < 10%), and results agreed well (83% overlap) with previously published radioisotope assay data. Our results indicate that fast and robust MALDI MS cellular assays could emerge as a high-throughput label-free alternative for direct assessment of drug transporter function in DDIs and toxicities as well as enable drug discovery for transporters as pharmacology targets.
Subject(s)
Organic Anion Transporters/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Biological Transport , HumansABSTRACT
Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients' cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.
Subject(s)
Myotonic Dystrophy , Animals , Cyclin-Dependent Kinases , Disease Models, Animal , Humans , Mice , Myotonic Dystrophy/drug therapy , Myotonic Dystrophy/genetics , RNA , RNA Splicing/genetics , Trinucleotide Repeat Expansion/geneticsABSTRACT
CRISPR/Cas9-based gene knockouts (KOs) enable precise perturbation of target gene function in human cells, which is ideally assessed in an unbiased fashion by molecular omics readouts. Typically, this requires the lengthy process of isolating KO subclones. We show here that KO subclones are phenotypically heterogenous, regardless of the guide RNA used. We present an experimental strategy that avoids subcloning and achieves fast and efficient gene silencing on cell pools, based on the synergistic combination of two guide RNAs mapping at close (40-300 bp) genomic proximity. Our strategy results in better predictable indel generation with a low allelic heterogeneity, concomitant with low or undetectable residual target protein expression, as determined by MS3 mass spectrometry proteomics. Our method is compatible with nondividing primary cells and can also be used to study essential genes. It enables the generation of high confidence omics data which solely reflect the phenotype of the target ablation.
Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Gene Knockout Techniques/methods , Animals , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , DNA/genetics , Gene Silencing/physiology , Hep G2 Cells , Humans , INDEL Mutation/genetics , RNA, Guide, Kinetoplastida/geneticsABSTRACT
N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.
Subject(s)
Endoplasmic Reticulum , Gene Expression Regulation , Endoplasmic Reticulum/metabolism , Humans , K562 Cells , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/genetics , Proteasome Endopeptidase Complex/metabolismABSTRACT
The two tandem bromodomains of the BET (bromodomain and extraterminal domain) proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1, whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models, whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET-targeted therapies.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antineoplastic Agents/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Histone Acetyltransferases/antagonists & inhibitors , Immunologic Factors/pharmacology , Molecular Targeted Therapy , Transcription Factors/antagonists & inhibitors , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antineoplastic Agents/therapeutic use , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Drug Discovery , Gene Expression Regulation, Neoplastic/drug effects , HEK293 Cells , Histone Acetyltransferases/chemistry , Histone Acetyltransferases/genetics , Humans , Immune System Diseases/drug therapy , Immunologic Factors/chemistry , Immunologic Factors/therapeutic use , Inflammation/drug therapy , Neoplasms/drug therapy , Protein Domains/drug effects , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
OATP2B1 is an intestinal and hepatic drug uptake transporter. Intestinal OATP2B1 has been elucidated as the mechanism of unexpected clinical drug-drug interactions (DDIs), where drug exposure was unexpectedly decreased with unchanged half-life. Hepatic OATP2B1 may be an understudied clinical DDI mechanism. The aim of the present work was to understand the prevalence of clinically relevant intestinal and hepatic OATP2B1 inhibitors in marketed drug space. HEK293 cells stably overexpressing human OATP2B1 or vector control were generated and cultured for 72 h in a 96-well format. OATP2B1-mediated uptake of dibromofluorescein (DBF) was found to be optimal at 10 µM concentration and 30 min incubation time. A total of 294 drugs (top 300 marketed drugs, excluding biologics and restricted drugs, supplemented with â¼100 small-molecule drugs) were screened for OATP2B1 inhibition at 10 µM. Drugs demonstrating ≥50% inhibition in this screen were advanced for IC50 determination, which was extrapolated to clinical intestinal and hepatic OATP2B1 inhibition as per 2017 FDA DDI guidance. Of the 294 drugs screened, 67 elicited ≥50% inhibition of OATP2B1-mediated DBF uptake at 10 µM screening concentration. For the 67 drugs flagged in the single-concentration inhibition screen, upon evaluation of a full concentration range, IC50 values could be determined for 58 drugs. OATP2B1 IC50 values established for these 58 drugs were extrapolated as potentially clinically relevant at the intestinal level for 38 orally administered drugs (Igut/IC50 ≥ 10), and 17 were flagged as potential clinical inhibitors of hepatic OATP2B1 uptake (1 + Iin,max,u/IC50 ≥ 1.1). This analysis of 294 drugs demonstrated prevalence of clinically relevant intestinal and hepatic OATP2B1 inhibitors to be 13 and 6%, respectively. As OATP2B1-inhibitor drugs are not exceedingly rare, these results suggest that clinical OATP2B1 DDIs have been rarely observed because OATP2B1 is uncommonly the predominant determinant of drug disposition.
Subject(s)
Drug Evaluation, Preclinical/methods , Organic Anion Transporters/antagonists & inhibitors , Biological Transport , Cell Survival/drug effects , Drug Interactions , Erlotinib Hydrochloride/pharmacology , Fluoresceins/metabolism , HEK293 Cells , Half-Life , Humans , Inhibitory Concentration 50 , Intestinal Mucosa/metabolism , Liver/metabolism , Organic Anion Transporters/genetics , Organic Anion Transporters/metabolism , TransfectionABSTRACT
Epigenetic regulatory mechanisms are central to the development and survival of all eukaryotic organisms. These mechanisms critically depend on the marking of chromatin domains with distinctive histone tail modifications (PTMs) and their recognition by effector protein complexes. Here we used quantitative proteomic approaches to unveil interactions between PTMs and associated reader protein complexes of Plasmodium falciparum, a unicellular parasite causing malaria. Histone peptide pull-downs with the most prominent and/or parasite-specific PTMs revealed the binding preference for 14 putative and novel reader proteins. Amongst others, they highlighted the acetylation-level-dependent recruitment of the BDP1/BDP2 complex and identified an PhD-finger protein (PHD 1, PF3D7_1008100) that could mediate a cross-talk between H3K4me2/3 and H3K9ac marks. Tagging and interaction proteomics of 12 identified proteins unveiled the composition of 5 major epigenetic complexes, including the elusive TBP-associated-factor complex as well as two distinct GCN5/ADA2 complexes. Furthermore, it has highlighted a remarkable degree of interaction between these five (sub)complexes. Collectively, this study provides an extensive inventory of PTM-reader interactions and composition of epigenetic complexes. It will not only fuel further explorations of gene regulation amongst ancient eukaryotes, but also provides a stepping stone for exploration of PTM-reader interactions for antimalarial drug development.
Subject(s)
Epigenesis, Genetic/genetics , Gene Expression Regulation/genetics , Histones/metabolism , Plasmodium falciparum/genetics , Protein Processing, Post-Translational/genetics , Chromatin/metabolism , Humans , Malaria, Falciparum/genetics , Malaria, Falciparum/parasitology , MethylationABSTRACT
Kinobeads are a set of promiscuous kinase inhibitors immobilized on sepharose beads for the comprehensive enrichment of endogenously expressed protein kinases from cell lines and tissues. These beads enable chemoproteomics profiling of kinase inhibitors of interest in dose-dependent competition studies in combination with quantitative mass spectrometry. We present improved bead matrices that capture more than 350 protein kinases and 15 lipid kinases from human cell lysates, respectively. A multiplexing strategy is suggested that enables determination of apparent dissociation constants in a single mass spectrometry experiment. Miniaturization of the procedure enabled determining the target selectivity of the clinical BCR-ABL inhibitor dasatinib in peripheral blood mononuclear cell (PBMC) lysates from individual donors. Profiling of a set of Jak kinase inhibitors revealed kinase off-targets from nearly all kinase families underpinning the need to profile kinase inhibitors against the kinome. Potently bound off-targets of clinical inhibitors suggest polypharmacology, e.g. through MRCK alpha and beta, which bind to decernotinib with nanomolar affinity.
Subject(s)
Dasatinib/pharmacology , Janus Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Proteome/metabolism , Proteomics/methods , Animals , Chromatography, Affinity/methods , Dogs , HEK293 Cells , Humans , Janus Kinases/metabolism , K562 Cells , Mice , Microspheres , Monocytes/metabolism , Protein Binding , Proteome/chemistry , Sepharose/analogs & derivatives , Substrate SpecificityABSTRACT
One of the attractive properties of artemisinins is their extremely fast-killing capability, quickly relieving malaria symptoms. Nevertheless, the unique benefits of these medicines are now compromised by the prolonged parasite clearance times and the increasing frequency of treatment failures, attributed to the increased tolerance of Plasmodium falciparum to artemisinin. This emerging artemisinin resistance threatens to undermine the effectiveness of antimalarial combination therapies. Herein, we describe the medicinal chemistry efforts focused on a cGMP-dependent protein kinase (PKG) inhibitor scaffold, leading to the identification of novel chemical entities with very potent, similar to artemisinins, fast-killing potency against asexual blood stages that cause disease, and activity against gametocyte activation that is required for transmission. Furthermore, we confirm that selective PKG inhibitors have a slow speed of kill, while chemoproteomic analysis suggests for the first time serine/arginine protein kinase 2 (SRPK2) targeting as a novel strategy for developing antimalarial compounds with extremely fast-killing properties.
Subject(s)
Antimalarials/pharmacology , Artemisinins/chemistry , Plasmodium falciparum/drug effects , Antimalarials/chemistry , Antimalarials/metabolism , Artemisinins/metabolism , Artemisinins/pharmacology , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic GMP-Dependent Protein Kinases/genetics , Cyclic GMP-Dependent Protein Kinases/metabolism , ERG1 Potassium Channel/antagonists & inhibitors , ERG1 Potassium Channel/metabolism , Humans , Inhibitory Concentration 50 , Mutagenesis, Site-Directed , Plasmodium falciparum/growth & development , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Solubility , Structure-Activity Relationship , Thiazoles/chemistryABSTRACT
Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.