ABSTRACT
Seasonal and pandemic-associated influenza strains cause highly contagious viral respiratory infections that can lead to severe illness and excess mortality. Here, we report on the optimization of our small-molecule inhibitor F0045(S) targeting the influenza hemagglutinin (HA) stem with our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. A combination of SuFEx- and amide-based lead molecule diversification and structure-guided design led to identification and validation of ultrapotent influenza fusion inhibitors with subnanomolar EC50 cellular antiviral activity against several influenza A group 1 strains. X-ray structures of six of these compounds with HA indicate that the appended moieties occupy additional pockets on the HA surface and increase the binding interaction, where the accumulation of several polar interactions also contributes to the improved affinity. The compounds here represent the most potent HA small-molecule inhibitors to date. Our divergent HTMC platform is therefore a powerful, rapid, and cost-effective approach to develop bioactive chemical probes and drug-like candidates against viral targets.
Subject(s)
Antiviral Agents , Hemagglutinin Glycoproteins, Influenza Virus , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Humans , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Chemistry, Pharmaceutical/methods , High-Throughput Screening Assays/methods , Influenza, Human/drug therapy , Influenza, Human/virology , Crystallography, X-Ray/methods , Click Chemistry/methods , Animals , Influenza A virus/drug effects , Madin Darby Canine Kidney Cells , Viral Fusion Protein Inhibitors/pharmacology , Viral Fusion Protein Inhibitors/chemistry , DogsABSTRACT
Diversity Oriented Clicking (DOC) is a discovery method geared toward the rapid synthesis of functional libraries. It combines the best attributes of both classical and modern click chemistries. DOC strategies center upon the chemical diversification of core "SuFExable" hubs-exemplified by 2-Substituted-Alkynyl-1-Sulfonyl Fluorides (SASFs)-enabling the modular assembly of compounds through multiple reaction pathways. We report here a range of stereoselective Michael-type addition pathways from SASF hubs including reactions with secondary amines, carboxylates, 1H-1,2,3-triazole, and halides. These high yielding conjugate addition pathways deliver unprecedented ß-substituted alkenyl sulfonyl fluorides as single isomers with minimal purification, greatly enriching the repertoire of DOC and holding true to the fundamentals of modular click chemistry. Further, we demonstrate the potential for biological function - a key objective of click chemistry - of this family of SASF-derived molecules as covalent inhibitors of human neutrophil elastase.
Subject(s)
Click Chemistry , Fluorides , Leukocyte Elastase , Proteinase Inhibitory Proteins, Secretory , Sulfinic Acids , Click Chemistry/methods , Fluorides/chemical synthesis , Fluorides/chemistry , Fluorides/pharmacology , Humans , Leukocyte Elastase/antagonists & inhibitors , Proteinase Inhibitory Proteins, Secretory/chemical synthesis , Proteinase Inhibitory Proteins, Secretory/chemistry , Proteinase Inhibitory Proteins, Secretory/pharmacology , Sulfinic Acids/chemical synthesis , Sulfinic Acids/chemistry , Sulfinic Acids/pharmacologyABSTRACT
The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H-15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed ß7-ß8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (Kd = 3-8 µM). Inhibitor 6c displayed tighter binding (Kd â¼ 0.3 µM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.
ABSTRACT
Molecular glues are small molecules that stabilize protein-protein interactions, enabling new molecular pharmacologies, such as targeted protein degradation. They offer advantages over proteolysis targeting chimeras (PROTACs), which present challenges associated with the size and properties of heterobifunctional constructions, but glues lack the rational design principles analogous to PROTACs. One notable exception is the ability to alter the structure of Cereblon (CRBN)-based molecular glues and redirect their activity toward new neo-substrate proteins. We took a focused approach toward modifying the CRBN ligand, 5'-amino lenalidomide, to alter its neo-substrate specificity using high-throughput chemical diversification by parallelized sulfur(VI)-fluoride exchange (SuFEx) transformations. We synthesized over 3,000 analogs of 5'-amino lenalidomide using this approach and screened the crude products using a phenotypic screen for cell viability, identifying dozens of analogs with differentiated activity. We characterized four compounds that degrade G-to-S phase transition 1 (GSPT1) protein, providing a proof-of-concept model for SuFEx-based discovery of CRBN molecular glues.
Subject(s)
Ubiquitin-Protein Ligases , Ubiquitin-Protein Ligases/metabolism , Proteolysis , LenalidomideABSTRACT
Influenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs), and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low micromolar to nanomolar affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 (H1/PR8) HA trimer against â¼72,000 compounds. The crystal structure of H1/PR8 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small-molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities.
Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , Fluorescence Polarization/methods , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H5N1 Subtype/drug effects , Influenza, Human/virology , Small Molecule Libraries/pharmacology , Antiviral Agents/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/metabolism , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/metabolism , Small Molecule Libraries/chemistryABSTRACT
Activity-based protein profiling (ABPP) has been used extensively to discover and optimize selective inhibitors of enzymes. Here, we show that ABPP can also be implemented to identify the converse-small-molecule enzyme activators. Using a kinetically controlled, fluorescence polarization-ABPP assay, we identify compounds that stimulate the activity of LYPLAL1-a poorly characterized serine hydrolase with complex genetic links to human metabolic traits. We apply ABPP-guided medicinal chemistry to advance a lead into a selective LYPLAL1 activator suitable for use in vivo. Structural simulations coupled to mutational, biochemical and biophysical analyses indicate that this compound increases LYPLAL1's catalytic activity likely by enhancing the efficiency of the catalytic triad charge-relay system. Treatment with this LYPLAL1 activator confers beneficial effects in a mouse model of diet-induced obesity. These findings reveal a new mode of pharmacological regulation for this large enzyme family and suggest that ABPP may aid discovery of activators for additional enzyme classes.
Subject(s)
Enzyme Activators/chemistry , Enzyme Activators/pharmacology , Lysophospholipase/metabolism , Small Molecule Libraries/pharmacology , Animals , Drug Discovery , Enzyme Activators/pharmacokinetics , Fluorescence Polarization , HEK293 Cells , High-Throughput Screening Assays/methods , Humans , Insulin Resistance , Lysophospholipase/chemistry , Lysophospholipase/genetics , Male , Metabolic Syndrome/drug therapy , Metabolic Syndrome/metabolism , Mice, Inbred C57BL , Mice, Obese , Molecular Dynamics Simulation , Molecular Structure , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacokinetics , Structure-Activity RelationshipABSTRACT
The bengamides comprise an interesting family of natural products isolated from sponges belonging to the prolific Jaspidae family. Their outstanding antitumor properties, coupled with their unique mechanism of action and unprecedented molecular structures, have prompted an intense research activity directed towards their total syntheses, analogue design, and biological evaluations for their development as new anticancer agents. Together with these biological studies in cancer research, in recent years, the bengamides have been identified as potential antibiotics by their impressive biological activities against various drug-resistant bacteria such as Mycobacterium tuberculosis and Staphylococcus aureus. This review reports on the new advances in the chemistry and biology of the bengamides during the last years, paying special attention to their development as promising new antibiotics. Thus, the evolution of the bengamides from their initial exploration as antitumor agents up to their current status as antibiotics is described in detail, highlighting the manifold value of these marine natural products as valid hits in medicinal chemistry.
Subject(s)
Antineoplastic Agents , Biological Products , Mycobacterium tuberculosis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biological Products/chemistry , Biological Products/pharmacology , Molecular StructureABSTRACT
Sulfur fluoride exchange (SuFEx) has emerged as the new generation of click chemistry. We report here a SuFEx-enabled, agnostic approach for the discovery and optimization of covalent inhibitors of human neutrophil elastase (hNE). Evaluation of our ever-growing collection of SuFExable compounds toward various biological assays unexpectedly revealed a selective and covalent hNE inhibitor: benzene-1,2-disulfonyl fluoride. Synthetic derivatization of the initial hit led to a more potent agent, 2-(fluorosulfonyl)phenyl fluorosulfate with IC50 0.24 µM and greater than 833-fold selectivity over the homologous neutrophil serine protease, cathepsin G. The optimized, yet simple benzenoid probe only modified active hNE and not its denatured form.
Subject(s)
Fluorides/chemistry , Leukocyte Elastase/antagonists & inhibitors , Serine Proteinase Inhibitors/chemistry , Sulfur Compounds/chemistry , Click Chemistry , Enzyme Activation/drug effects , Humans , Inhibitory Concentration 50 , Leukocyte Elastase/chemistry , Leukocyte Elastase/metabolism , Molecular Structure , Protein Binding , Protein Folding , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/pharmacology , Sulfinic Acids/chemical synthesis , Sulfinic Acids/chemistry , Sulfinic Acids/pharmacologyABSTRACT
Folate and related derivatives are essential small molecules required for survival. Of significant interest is the biological role and necessity of folate in the crosstalk between commensal organisms and their respective hosts, including the tremendously complex human distal gut microbiome. Here, we designed a folate-based probe consisting of a photo-crosslinker to detect and quantitate folate-binding proteins from proteomic samples. We demonstrate the selectivity of our probe for the well-established human folate-binding protein dihydrofolate reductase and show no promiscuous labeling occurs with human caspase-3 or bovine serum albumin, which served as negative controls. Affinity-based enrichment of folate-binding proteins from an E. coli lysate in combination with mass spectrometry proteomics verified the ability of our probe to isolate low-abundance folate-dependent proteins. We envision that our probe will serve as a tool to elucidate the roles of commensal microbial folate-binding proteins in health and microbiome-related diseases.
Subject(s)
Cross-Linking Reagents/chemistry , Folic Acid Transporters/analysis , Folic Acid/chemistry , Molecular Probes/chemistry , Caspase 3/chemistry , Chromatography, High Pressure Liquid , Escherichia coli/chemistry , Humans , Microbiota/physiology , Photochemical Processes , Proteomics , Serum Albumin, Bovine/metabolism , Tandem Mass Spectrometry , Tetrahydrofolate Dehydrogenase/chemistryABSTRACT
Optimization of small-molecule probes or drugs is a synthetically lengthy, challenging, and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible sulfur(VI) fluoride exchange (SuFEx) click chemistry. A high-throughput screening hit benzyl (cyanomethyl)carbamate (Ki = 8 µM) against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RNâS(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products were directly screened to yield drug-like inhibitors with 480-fold higher potency (Ki = 18 nM). We showed that the improved molecule is active in a bacteria-host coculture. Since this SuFEx linkage reaction succeeds on picomole scale for direct screening, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Exotoxins/antagonists & inhibitors , Sulfur Compounds/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Catalytic Domain , Click Chemistry , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/metabolism , Cysteine Proteinase Inhibitors/toxicity , Drug Discovery , Exotoxins/chemistry , Exotoxins/metabolism , High-Throughput Screening Assays , Humans , Jurkat Cells , Microsomes, Liver/metabolism , Proof of Concept Study , Protein BindingABSTRACT
Diversity Oriented Clicking (DOC) is a unified click-approach for the modular synthesis of lead-like structures through application of the wide family of click transformations. DOC evolved from the concept of achieving "diversity with ease", by combining classic C-C π-bond click chemistry with recent developments in connective SuFEx-technologies. We showcase 2-Substituted-Alkynyl-1-Sulfonyl Fluorides (SASFs) as a new class of connective hub in concert with a diverse selection of click-cycloaddition processes. Through the selective DOC of SASFs with a range of dipoles and cyclic dienes, we report a diverse click-library of 173 unique functional molecules in minimal synthetic steps. The SuFExable library comprises 10 discrete heterocyclic core structures derived from 1,3- and 1,5-dipoles; while reaction with cyclic dienes yields several three-dimensional bicyclic Diels-Alder adducts. Growing the library to 278 discrete compounds through late-stage modification was made possible through SuFEx click derivatization of the pendant sulfonyl fluoride group in 96 well-plates-demonstrating the versatility of the DOC approach for the rapid synthesis of diverse functional structures. Screening for function against MRSA (USA300) revealed several lead hits with improved activity over methicillin.
Subject(s)
Click Chemistry , Sulfinic Acids/chemistry , Cycloaddition Reaction , Molecular StructureABSTRACT
Tetrahydroquinoline (THQ)-type compounds are a class of potential larvicides against mosquitoes. The structure-activity relationships (SAR) of these compounds were previously investigated (Smith et al., Bioorg. Med. Chem. Lett. 2003, 13, 1943-1946), and one of cis-forms (with respect to the configurations of 2-methyl and 4-anilino substitutions on the THQ basic structure) was stereoselectively synthesized. However, the absolute configurations of C2 and C4 were not determined. In this study, four THQ-type compounds with cis configurations were synthesized, and two were submitted for X-ray crystal structure analysis. This analysis demonstrated that two enantiomers are packed into the crystal form. We synthesized the cis-form of the fluorinated THQ compound, according to the published method, and the enantiomers were separated via chiral HPLC. The absolute configurations of the enantiomers were determined by X-ray crystallography. Each of the enantiomers was tested for activity against mosquito larvae in vivo and competitive binding to the ecdysone receptor in vitro. Compared to the (2S,4R) enantiomer, the (2R,4S) enantiomer showed 55 times higher activity in the mosquito larvicidal assay, and 36 times higher activity in the competitive receptor binding assay.
Subject(s)
Ecdysone/agonists , Quinolines/pharmacology , Animals , Crystallography, X-Ray , Dose-Response Relationship, Drug , Insecta , Models, Molecular , Molecular Structure , Quinolines/chemistry , Structure-Activity RelationshipABSTRACT
Soluble epoxide hydrolase (sEH) is a bifunctional enzyme responsible for lipid metabolism and is a promising drug target. Here, we report the first-in-class PROTAC small-molecule degraders of sEH. Our optimized PROTAC selectively targets the degradation of cytosolic but not peroxisomal sEH, resulting in exquisite spatiotemporal control. Remarkably, our sEH PROTAC molecule has higher potency in cellular assays compared to the parent sEH inhibitor as measured by the significantly reduced ER stress. Interestingly, our mechanistic data indicate that our PROTAC directs the degradation of cytosolic sEH via the lysosome, not through the proteasome. The molecules presented here are useful chemical probes to study the biology of sEH with the potential for therapeutic development. Broadly, our results represent a proof of concept for the superior cellular potency of sEH degradation over sEH enzymatic inhibition, as well as subcellular compartment-selective modulation of a protein by PROTACs.
Subject(s)
Endoplasmic Reticulum Stress , Epoxide Hydrolases , Proteolysis Targeting Chimera , Cytosol/metabolism , Epoxide Hydrolases/chemistry , Epoxide Hydrolases/metabolism , Proteolysis Targeting Chimera/pharmacology , Endoplasmic Reticulum Stress/physiologyABSTRACT
Untargeted metabolomics of disease-associated intestinal microbiota can detect quantitative changes in metabolite profiles and complement other methodologies to reveal the full effect of intestinal dysbiosis. Here, we used the T cell transfer mouse model of colitis to identify small-molecule metabolites with altered abundance due to intestinal inflammation. We applied untargeted metabolomics to detect metabolite signatures in cecal, colonic, and fecal samples from healthy and colitic mice and to uncover differences that would aid in the identification of colitis-associated metabolic processes. We provided an unbiased spatial survey of the GI tract for small molecules, and we identified the likely source of metabolites and biotransformations. Several prioritized metabolites that we detected as being altered in colitis were evaluated for their ability to induce inflammatory signaling in cultured macrophages, such as NF-κB signaling and the expression of cytokines and chemokines upon LPS stimulation. Multiple previously uncharacterized anti-inflammatory and inflammation-augmenting metabolites were thus identified, with phytosphingosine showing the most effective anti-inflammatory activity in vitro. We further demonstrated that oral administration of phytosphingosine decreased inflammation in a mouse model of colitis induced by the compound TNBS. The collection of distinct metabolites we identified and characterized, many of which have not been previously associated with colitis, may offer new biological insight into IBD-associated inflammation and disease pathogenesis.
Subject(s)
Colitis , T-Lymphocytes , Anti-Inflammatory Agents , Humans , MetabolomicsABSTRACT
Transcriptional coregulators, which mediate chromatin-dependent transcriptional signaling, represent tractable targets to modulate tumorigenic gene expression programs with small molecules. Genetic loss-of-function studies have recently implicated the transcriptional coactivator, ENL, as a selective requirement for the survival of acute leukemia and highlighted an essential role for its chromatin reader YEATS domain. Motivated by these discoveries, we executed a screen of nearly 300,000 small molecules and identified an amido-imidazopyridine inhibitor of the ENL YEATS domain (IC50 = 7 µM). Improvements to the initial screening hit were enabled by adopting and expanding upon a SuFEx-based approach to high-throughput medicinal chemistry, ultimately demonstrating that it is compatible with cell-based drug discovery. Through these efforts, we discovered SR-0813, a potent and selective ENL/AF9 YEATS domain inhibitor (IC50 = 25 nM). Armed with this tool and a first-in-class ENL PROTAC, SR-1114, we detailed the biological response of AML cells to pharmacological ENL disruption for the first time. Most notably, we discovered that ENL YEATS inhibition is sufficient to selectively suppress ENL target genes, including HOXA9/10, MYB, MYC, and a number of other leukemia proto-oncogenes. Cumulatively, our study establishes YEATS domain inhibition as a viable approach to disrupt the pathogenic function of ENL in acute leukemia and provides the first thoroughly characterized chemical probe for the ENL YEATS domain.
ABSTRACT
Members of the CA class of cysteine proteases have multifaceted roles in physiology and virulence for many bacteria. Streptococcal pyrogenic exotoxin B (SpeB) is secreted by Streptococcus pyogenes and implicated in the pathogenesis of the bacterium through degradation of key human immune effector proteins. Here, we developed and characterized a clickable inhibitor, 2S-alkyne, based on X-ray crystallographic analysis and structure-activity relationships. Our SpeB probe showed irreversible enzyme inhibition in biochemical assays and labeled endogenous SpeB in cultured S. pyogenes supernatants. Importantly, application of 2S-alkyne decreased S. pyogenes survival in the presence of human neutrophils and supports the role of SpeB-mediated proteolysis as a mechanism to limit complement-mediated host defense. We posit that our SpeB inhibitor will be a useful chemical tool to regulate, label, and quantitate secreted cysteine proteases with SpeB-like activity in complex biological samples and a lead candidate for new therapeutics designed to sensitize S. pyogenes to host immune clearance.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Cysteine Proteases/drug effects , Cysteine Proteinase Inhibitors/pharmacology , Streptococcus pyogenes/enzymology , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemistry , Drug Design , Protein Conformation , Streptococcus pyogenes/pathogenicity , Structure-Activity Relationship , VirulenceABSTRACT
Controlled site-specific bioconjugation through chemical methods to native DNA remains an unanswered challenge. Herein, we report a simple solution to achieve this conjugation through the tactical combination of two recently developed technologies: one for the manipulation of DNA in organic media and another for the chemoselective labeling of alcohols. Reversible adsorption of solid support (RASS) is employed to immobilize DNA and facilitate its transfer into dry acetonitrile. Subsequent reaction with P(V)-based Ψ reagents takes place in high yield with exquisite selectivity for the exposed 3' or 5' alcohols on DNA. This two-stage process, dubbed SENDR for Synthetic Elaboration of Native DNA by RASS, can be applied to a multitude of DNA conformations and sequences with a variety of functionalized Ψ reagents to generate useful constructs.
ABSTRACT
[This corrects the article DOI: 10.1021/acscentsci.0c00680.].
ABSTRACT
Plant-derived lignans, consumed daily by most individuals, are thought to protect against cancer and other diseases1; however, their bioactivity requires gut bacterial conversion to enterolignans2. Here, we dissect a four-species bacterial consortium sufficient for all five reactions in this pathway. A single enzyme (benzyl ether reductase, encoded by the gene ber) was sufficient for the first two biotransformations, variable between strains of Eggerthella lenta, critical for enterolignan production in gnotobiotic mice and unique to Coriobacteriia. Transcriptional profiling (RNA sequencing) independently identified ber and genomic loci upregulated by each of the remaining substrates. Despite their low abundance in gut microbiomes and restricted phylogenetic range, all of the identified genes were detectable in the distal gut microbiomes of most individuals living in northern California. Together, these results emphasize the importance of considering strain-level variations and bacterial co-occurrence to gain a mechanistic understanding of the bioactivation of plant secondary metabolites by the human gut microbiome.
Subject(s)
Actinobacteria/genetics , Gastrointestinal Microbiome/genetics , Gene Expression Profiling , Lignans/metabolism , Actinobacteria/classification , Actinobacteria/metabolism , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biotransformation , Genome, Bacterial/genetics , Humans , Lignans/chemistry , Metabolic Networks and Pathways/genetics , Mice , Microbial Consortia/genetics , Phylogeny , Species SpecificityABSTRACT
Lead identification and optimization are essential steps in the development of a new drug. It requires cost-effective, selective and sensitive chemical tools. Here, we report a novel method using nanobodies that allows the efficient screening for potent ligands. The method is illustrated with the cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), a virulence factor secreted by the opportunistic pathogen Pseudomonas aeruginosa. 18 nanobodies selective to Cif were isolated by bio-panning from nanobody-phage library constructed from immunized llama. 8 out of 18 nanobodies were identified as potent inhibitors of Cif enzymatic activity with IC50s in the range of 0.3-6.4⯵M. A nanobody VHH219 showed high affinity (KDâ¯=â¯0.08â¯nM) to Cif and the highest inhibitory potency, IC50â¯=â¯0.3⯵M. A displacement sandwich ELISA (dsELISA) with VHH219 was then developed for classification of synthetic small molecule inhibitors according their inhibitory potency. The developed assay allowed identification of new inhibitor with highest potency reported so far (0.16⯱â¯0.02⯵M). The results from dsELISA assay correlates strongly with a conventional fluorogenic assay (Râ¯=â¯0.9998) in predicting the inhibitory potency of the tested compounds. However, the novel dsELISA is an order of magnitude more sensitive and allows the identification and ranking of potent inhibitors missed by the classic fluorogenic assay method. These data were supported with Octet biolayer interferometry measurements. The novel method described herein relies solely on the binding properties of the specific neutralizing nanobody, and thus is applicable to any pharmacological target for which such a nanobody can be found, independent of any requirement for catalytic activity.