Structure of CC Chemokine Receptor 5 with a Potent Chemokine Antagonist Reveals Mechanisms of Chemokine Recognition and Molecular Mimicry by HIV.

CCR5 is the primary chemokine receptor utilized by HIV to infect leukocytes, whereas CCR5 ligands inhibit infection by blocking CCR5 engagement with HIV gp120. To guide the design of improved therapeutics, we solved the structure of CCR5 in complex with chemokine antagonist [5P7]CCL5. Several structural features appeared to contribute to the anti-HIV potency of [5P7]CCL5, including the distinct chemokine orientation relative to the receptor, the near-complete occupancy of the receptor binding pocket, the dense network of intermolecular hydrogen bonds, and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc. Molecular modeling indicated that HIV gp120 mimicked the chemokine interaction with CCR5, providing an explanation for the ability of CCR5 to recognize diverse ligands and gp120 variants. Our findings reveal that structural plasticity facilitates receptor-chemokine specificity and enables exploitation by HIV, and provide insight into the design of small molecule and protein inhibitors for HIV and other CCR5-mediated diseases.

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity.

Chemokines and their receptors are orchestrators of cell migration in humans. Because dysregulation of the receptor-chemokine system leads to inflammation and cancer, both chemokines and receptors are highly sought therapeutic targets. Yet one of the barriers for their therapeutic targeting is the limited understanding of the structural principles behind receptor-chemokine recognition and selectivity. The existing structures do not include CXC subfamily complexes and lack information about the receptor distal N-termini, despite the importance of the latter in signaling, regulation, and bias. Here, we report the discovery of the geometry of the complex between full-length CXCR4, a prototypical CXC receptor and driver of cancer metastasis, and its endogenous ligand CXCL12. By comprehensive disulfide cross-linking, we establish the existence and the structure of a novel interface between the CXCR4 distal N-terminus and CXCL12 ß1-strand, while also recapitulating earlier findings from nuclear magnetic resonance, modeling and crystallography of homologous receptors. A cross-linking-informed high-resolution model of the CXCR4-CXCL12 complex pinpoints the interaction determinants and reveals the occupancy of the receptor major subpocket by the CXCL12 proximal N terminus. This newly found positioning of the chemokine proximal N-terminus provides a structural explanation of CXC receptor-chemokine selectivity against other subfamilies. Our findings challenge the traditional two-site understanding of receptor-chemokine recognition, suggest the possibility of new affinity and signaling determinants, and fill a critical void on the structural map of an important class of therapeutic targets. These results will aid the rational design of selective chemokine-receptor targeting small molecules and biologics with novel pharmacology.

Mapping the gene network landscape of Alzheimer's disease through integrating genomics and transcriptomics.

Integration of multi-omics data with molecular interaction networks enables elucidation of the pathophysiology of Alzheimer's disease (AD). Using the latest genome-wide association studies (GWAS) including proxy cases and the STRING interactome, we identified an AD network of 142 risk genes and 646 network-proximal genes, many of which were linked to synaptic functions annotated by mouse knockout data. The proximal genes were confirmed to be enriched in a replication GWAS of autopsy-documented cases. By integrating the AD gene network with transcriptomic data of AD and healthy temporal cortices, we identified 17 gene clusters of pathways, such as up-regulated complement activation and lipid metabolism, down-regulated cholinergic activity, and dysregulated RNA metabolism and proteostasis. The relationships among these pathways were further organized by a hierarchy of the AD network pinpointing major parent nodes in graph structure including endocytosis and immune reaction. Control analyses were performed using transcriptomics from cerebellum and a brain-specific interactome. Further integration with cell-specific RNA sequencing data demonstrated genes in our clusters of immunoregulation and complement activation were highly expressed in microglia.

P16INK4a Upregulation Mediated by SIX6 Defines Retinal Ganglion Cell Pathogenesis in Glaucoma.

Glaucoma, a blinding neurodegenerative disease, whose risk factors include elevated intraocular pressure (IOP), age, and genetics, is characterized by accelerated and progressive retinal ganglion cell (RGC) death. Despite decades of research, the mechanism of RGC death in glaucoma is still unknown. Here, we demonstrate that the genetic effect of the SIX6 risk variant (rs33912345, His141Asn) is enhanced by another major POAG risk gene, p16INK4a (cyclin-dependent kinase inhibitor 2A, isoform INK4a). We further show that the upregulation of homozygous SIX6 risk alleles (CC) leads to an increase in p16INK4a expression, with subsequent cellular senescence, as evidenced in a mouse model of elevated IOP and in human POAG eyes. Our data indicate that SIX6 and/or IOP promotes POAG by directly increasing p16INK4a expression, leading to RGC senescence in adult human retinas. Our study provides important insights linking genetic susceptibility to the underlying mechanism of RGC death and provides a unified theory of glaucoma pathogenesis.

A receptor-like protein mediates plant immune responses to herbivore-associated molecular patterns.

Herbivory is fundamental to the regulation of both global food webs and the extent of agricultural crop losses. Induced plant responses to herbivores promote resistance and often involve the perception of specific herbivore-associated molecular patterns (HAMPs); however, precisely defined receptors and elicitors associated with herbivore recognition remain elusive. Here, we show that a receptor confers signaling and defense outputs in response to a defined HAMP common in caterpillar oral secretions (OS). Staple food crops, including cowpea (Vigna unguiculata) and common bean (Phaseolus vulgaris), specifically respond to OS via recognition of proteolytic fragments of chloroplastic ATP synthase, termed inceptins. Using forward-genetic mapping of inceptin-induced plant responses, we identified a corresponding leucine-rich repeat receptor, termed INR, specific to select legume species and sufficient to confer inceptin-induced responses and enhanced defense against armyworms (Spodoptera exigua) in tobacco. Our results support the role of plant immune receptors in the perception of chewing herbivores and defense.

Control of Unsaturation in De Novo Fatty Acid Biosynthesis by FabA.

Carrier protein-dependent biosynthesis provides a thiotemplated format for the production of natural products. Within these pathways, many reactions display exquisite substrate selectivity, a regulatory framework proposed to be controlled by protein-protein interactions (PPIs). In Escherichia coli, unsaturated fatty acids are generated within the de novo fatty acid synthase by a chain length-specific interaction between the acyl carrier protein AcpP and the isomerizing dehydratase FabA. To evaluate PPI-based control of reactivity, interactions of FabA with AcpP bearing multiple sequestered substrates were analyzed through NMR titration and guided high-resolution docking. Through a combination of quantitative binding constants, residue-specific perturbation analysis, and high-resolution docking, a model for substrate control via PPIs has been developed. The in silico results illuminate the mechanism of FabA substrate selectivity and provide a structural rationale with atomic detail. Helix III positioning in AcpP communicates sequestered chain length identity recognized by FabA, demonstrating a powerful strategy to regulate activity by allosteric control. These studies broadly illuminate carrier protein-dependent pathways and offer an important consideration for future inhibitor design and pathway engineering.

Crowdsourced identification of multi-target kinase inhibitors for RET- and TAU- based disease: The Multi-Targeting Drug DREAM Challenge.

A continuing challenge in modern medicine is the identification of safer and more efficacious drugs. Precision therapeutics, which have one molecular target, have been long promised to be safer and more effective than traditional therapies. This approach has proven to be challenging for multiple reasons including lack of efficacy, rapidly acquired drug resistance, and narrow patient eligibility criteria. An alternative approach is the development of drugs that address the overall disease network by targeting multiple biological targets ('polypharmacology'). Rational development of these molecules will require improved methods for predicting single chemical structures that target multiple drug targets. To address this need, we developed the Multi-Targeting Drug DREAM Challenge, in which we challenged participants to predict single chemical entities that target pro-targets but avoid anti-targets for two unrelated diseases: RET-based tumors and a common form of inherited Tauopathy. Here, we report the results of this DREAM Challenge and the development of two neural network-based machine learning approaches that were applied to the challenge of rational polypharmacology. Together, these platforms provide a potentially useful first step towards developing lead therapeutic compounds that address disease complexity through rational polypharmacology.

Graph-Convolutional Neural Net Model of the Statistical Torsion Profiles for Small Organic Molecules.

We present a graph-convolutional neural network (GCNN)-based method for learning and prediction of statistical torsional profiles (STP) in small organic molecules based on the experimental X-ray structure data. A specialized GCNN torsion profile model is trained using the structures in the Crystallography Open Database (COD). The GCNN-STP model captures torsional preferences over a wide range of torsion rotor chemotypes and correctly predicts a variety of effects from the vicinal atoms and moieties. GCNN-STP statistical profiles also show good agreement with quantum chemically (DFT) calculated torsion energy profiles. Furthermore, we demonstrate the application of the GCNN-STP statistical profiles for conformer generation. A web server that allows interactive profile prediction and viewing is made freely available at https://www.molsoft.com/tortool.html.

Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists.

CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer. These disease associations have motivated numerous preclinical studies and clinical trials (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2-chemokine axis. To aid drug discovery efforts, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein-protein interactions, receptor-chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

Unique metabolite preferences of the drug transporters OAT1 and OAT3 analyzed by machine learning.

The multispecific organic anion transporters, OAT1 (SLC22A6) and OAT3 (SLC22A8), the main kidney elimination pathways for many common drugs, are often considered to have largely-redundant roles. However, whereas examination of metabolomics data from Oat-knockout mice (Oat1 and Oat3KO) revealed considerable overlap, over a hundred metabolites were increased in the plasma of one or the other of these knockout mice. Many of these relatively unique metabolites are components of distinct biochemical and signaling pathways, including those involving amino acids, lipids, bile acids, and uremic toxins. Cheminformatics, together with a "logical" statistical and machine learning-based approach, identified a number of molecular features distinguishing these unique endogenous substrates. Compared with OAT1, OAT3 tends to interact with more complex substrates possessing more rings and chiral centers. An independent "brute force" approach, analyzing all possible combinations of molecular features, supported the logical approach. Together, the results suggest the potential molecular basis by which OAT1 and OAT3 modulate distinct metabolic and signaling pathways in vivo As suggested by the Remote Sensing and Signaling Theory, the analysis provides a potential mechanism by which "multispecific" kidney proximal tubule transporters exert distinct physiological effects. Furthermore, a strong metabolite-based machine-learning classifier was able to successfully predict unique OAT1 versus OAT3 drugs; this suggests the feasibility of drug design based on knockout metabolomics of drug transporters. The approach can be applied to other SLC and ATP-binding cassette drug transporters to define their nonredundant physiological roles and for analyzing the potential impact of drug-metabolite interactions.

Differential activities of maize plant elicitor peptides as mediators of immune signaling and herbivore resistance.

Plant elicitor peptides (Peps) are conserved regulators of defense responses and models for the study of damage-associated molecular pattern-induced immunity. Although present as multigene families in most species, the functional relevance of these multigene families remains largely undefined. While Arabidopsis Peps appear largely redundant in function, previous work examining Pep-induced responses in maize (Zm) implied specificity of function. To better define the function of individual ZmPeps and their cognate receptors (ZmPEPRs), activities were examined by assessing changes in defense-associated phytohormones, specialized metabolites and global gene expression patterns, in combination with heterologous expression assays and analyses of CRISPR/Cas9-generated knockout plants. Beyond simply delineating individual ZmPep and ZmPEPR activities, these experiments led to a number of new insights into Pep signaling mechanisms. ZmPROPEP and other poaceous precursors were found to contain multiple active Peps, a phenomenon not previously observed for this family. In all, seven new ZmPeps were identified and the peptides were found to have specific activities defined by the relative magnitude of their response output rather than by uniqueness. A striking correlation was observed between individual ZmPep-elicited changes in levels of jasmonic acid and ethylene and the magnitude of induced defense responses, indicating that ZmPeps may collectively regulate immune output through rheostat-like tuning of phytohormone levels. Peptide structure-function studies and ligand-receptor modeling revealed structural features critical to the function of ZmPeps and led to the identification of ZmPep5a as a potential antagonist peptide able to competitively inhibit the activity of other ZmPeps, a regulatory mechanism not previously observed for this family.

Mtor inhibitors associated with higher cardiovascular adverse events-A large population database analysis.

There are limited real-world data available regarding adverse events (AEs) of immunosuppressants. We utilized the FDA Adverse Event Reporting System (FAERS) database from 2004 to 2018 to perform a retrospective database analysis. We analyzed AE reports due to the individual agents tacrolimus, sirolimus, or everolimus and compared reporting odds ratios of the mTOR inhibitors to tacrolimus. The mTOR inhibitors arm had 1282 reports with 4176 AEs, while the tacrolimus arm had a total of 7587 reports with 20 940 individual AEs. mTOR inhibitors had significantly higher incidences of cardiovascular (ROR 1.95, 95% CI 1.70, 2.23), dermatologic (ROR 1.34, 95% CI 1.04, 1.73), endocrine (ROR 1.52, 95% CI 1.26, 1.82), gastrointestinal (ROR 1.15, 95% CI 1.01, 1.30), infectious disease (ROR 1.35, 95% 1.20, 1.52), musculoskeletal (ROR 1.39, 95% CI 1.13, 1.70), pulmonary (ROR 3.46, 95% 2.97, 4.03), renal (ROR 1.27, 95% CI 1.10, 1.46), and vascular AEs (ROR 3.10, 95% CI 2.14, 4.49). Across every organ type, mTOR inhibitors had greater cardiovascular AEs compared to tacrolimus, specifically in arteriosclerosis, heart failure, hypotension, tachycardia, chest pain, edema, and pericardial disorders. mTOR inhibitors may be associated with higher cardiovascular AEs. Further investigation is required to determine the potential mechanism of this effect.

Structure based design and synthesis of novel Toll-like Receptor 2 (TLR 2) lipid antagonists.

Toll-like receptors (TLRs) play key role in innate immune response to Damage Associated Molecular Patterns (DAMPs) and Pathogen Associated Molecular Patterns (PAMPs). DAMP/PAMP-mediated activation of TLRs triggers NFκB signaling resulting in pro-inflammatory cytokine release. Using TLR2-Pam2CSK4 agonist co-crystal structure information, we designed and synthesized a novel series of Toll-like Receptor 2 (TLR2) lipid antagonists and identified compounds 14, 15 and 17 with sub-micromolar potency. TLR2 antagonists that we identified are stable for > 1.0 h in both gastric juice and PBS buffer and could be used as research tools.

Lanosterol reverses protein aggregation in cataracts.

The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people, and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for cataract prevention and treatment.

Biomimetic microbioreactor-supramolecular nanovesicles improve enzyme therapy of hepatic cancer.

A novel biomimetic nanovesicle-loaded supramolecular enzyme-based therapeutics has been developed. Here, using a biomimetic lipid-D-α-tocopherol polyethylene glycol succinate (TPGS) hybrid semi-permeable membrane, cyclodextrin supramolecular docking, metal-ion-aided coordination complexing, we combined multiple functional motifs into a single biomimetic microbioreactor-supramolecular nanovesicle (MiSuNv) that allowed effective transport of arginine deiminase (ADI) to hepatic tumor cells to enhance arginine depletion. We compared two intercalated enzyme-carrying supermolecular motifs mainly comprising of 2-hydroxypropyl-ß-cyclodextrin and sulfobutyl-ether-ß-cyclodextrin, the only two cyclodextrin derivatives approved for injection by the United States Food and Drug Administration. The ADI-specific antitumor effects were enhanced by TPGS (one constituent of MiSuNv, having synergistic antitumor effects), as ADI was separated from adverse external environment by a semi-permeable membrane and sequestered in a favorable internal microenvironment with an optimal pH and metal-ion combination. ADI@MiSuNv contributed to cell cycle arrest, apoptosis and autophagy through the enhanced efficacy of enzyme treatment against Hep3B xenograft tumors in rats.

Transcriptome and binding data indicate that citral inhibits single strand DNA-binding proteins.

The mechanism of phytotoxicity of citral was probed in Arabidopsis thaliana using RNA-Seq and in silico binding analyses. Inhibition of growth by 50% by citral downregulated transcription of 9156 and 5541 genes in roots and shoots, respectively, after 1 h. Only 56 and 62 genes in roots and shoots, respectively, were upregulated. In the shoots, the downregulation increased at 3 h (6239 genes downregulated, vs 66 upregulated). Of all genes affected in roots at 1 h (time of greatest effect), 7.69% of affected genes were for nucleic acid binding functions. Genes for single strand DNA binding proteins (SSBP) WHY1, WHY 2 and WHY3 were strongly downregulated in the shoot up until 12 h after citral exposure. Effects were strong in the root at just 1 h after the treatment and then at 12 and 24 h. Similar effects occurred with the transcription factors MYC-2, ANAC and SCR-SHR, which were also significantly downregulated for the first hour of treatment, and downregulation occurred again after 12 and 24 h treatment. Downregulation of ANAC in the first hour of treatment was significantly (P < 0.0001) decreased more than eight times compared to the control. In silico molecular docking analysis suggests binding of citral isomers to the SSBPs WHY1, WHY2, and WHY3, as well as with other transcription factors such as MYC-2, ANAC and SCR-SHR. Such effects could account for the profound and unusual effects of citral on downregulation of gene transcription.

Druggable exosites of the human kino-pocketome.

Small molecules binding at any of the multiple regulatory sites on the molecular surface of a protein kinase may stabilize or disrupt the corresponding interaction, leading to consequent modulation of the kinase cellular activity. As such, each of these sites represents a potential drug target. Even targeting sites outside the immediate ATP site, the so-called exosites, may cause desirable biological effects through an allosteric mechanism. Targeting exosites can alleviate adverse effects and toxicity that is common when ATP-site compounds bind promiscuously to many other types of kinases. In this study we have identified, catalogued, and annotated all potentially druggable exosites on the protein kinase domains within the existing structural human kinome. We then priority-ranked these exosites by those most amenable to drug design. In order to identify pockets that are either consistent across the kinome, or unique and specific to a particular structure, we have also implemented a normalized representation of all pockets, and displayed these graphically. Finally, we have built a database and designed a web-based interface for users interested in accessing the 3-dimensional representations of these pockets. We envision this information will assist drug discovery efforts searching for untargeted binding pockets in the human kinome.

Systems Biology Analysis Reveals Eight SLC22 Transporter Subgroups, Including OATs, OCTs, and OCTNs.

The SLC22 family of OATs, OCTs, and OCTNs is emerging as a central hub of endogenous physiology. Despite often being referred to as "drug" transporters, they facilitate the movement of metabolites and key signaling molecules. An in-depth reanalysis supports a reassignment of these proteins into eight functional subgroups, with four new subgroups arising from the previously defined OAT subclade: OATS1 (SLC22A6, SLC22A8, and SLC22A20), OATS2 (SLC22A7), OATS3 (SLC22A11, SLC22A12, and Slc22a22), and OATS4 (SLC22A9, SLC22A10, SLC22A24, and SLC22A25). We propose merging the OCTN (SLC22A4, SLC22A5, and Slc22a21) and OCT-related (SLC22A15 and SLC22A16) subclades into the OCTN/OCTN-related subgroup. Using data from GWAS, in vivo models, and in vitro assays, we developed an SLC22 transporter-metabolite network and similar subgroup networks, which suggest how multiple SLC22 transporters with mono-, oligo-, and multi-specific substrate specificity interact to regulate metabolites. Subgroup associations include: OATS1 with signaling molecules, uremic toxins, and odorants, OATS2 with cyclic nucleotides, OATS3 with uric acid, OATS4 with conjugated sex hormones, particularly etiocholanolone glucuronide, OCT with neurotransmitters, and OCTN/OCTN-related with ergothioneine and carnitine derivatives. Our data suggest that the SLC22 family can work among itself, as well as with other ADME genes, to optimize levels of numerous metabolites and signaling molecules, involved in organ crosstalk and inter-organismal communication, as proposed by the remote sensing and signaling theory.

Molecular Basis of Action of a Small-Molecule Positive Allosteric Modulator Agonist at the Type 1 Cholecystokinin Holoreceptor.

Allosteric modulation of receptors provides mechanistic safety while effectively achieving biologic endpoints otherwise difficult or impossible to obtain by other means. The theoretical case has been made for the development of a positive allosteric modulator (PAM) of the type 1 cholecystokinin receptor (CCK1R) having minimal intrinsic agonist activity to enhance meal-induced satiety for the treatment of obesity, while reducing the risk of side effects and/or toxicity. Unfortunately, such a drug does not currently exist. In this work, we have identified a PAM agonist of the CCK1R, SR146131, and determined its putative binding mode and receptor activation mechanism by combining molecular modeling, chimeric CCK1R/CCK2R constructs, and site-directed mutagenesis. We probed the structure-activity relationship of analogs of SR146131 for impact on agonism versus cooperativity of the analogs. This identified structural features that might be responsible for binding affinity and potency while retaining PAM activity. SR146131 and several of its analogs were docked into the receptor structure, which had the natural endogenous peptide agonist, cholecystokinin, already in the bound state (by docking), providing a refined structural model of the intact CCK1R holoreceptor. Both SR146131 and its analogs exhibited unique probe-dependent cooperativity with orthosteric peptide agonists and were simultaneously accommodated in this model, consistent with the derived structure-activity relationships. This provides improved understanding of the molecular basis for CCK1R-directed drug development.

In silico discovery of small molecules that inhibit RfaH recruitment to RNA polymerase.

RfaH is required for virulence in several Gram-negative pathogens including Escherichia coli and Klebsiella pneumoniae. Through direct interactions with RNA polymerase (RNAP) and ribosome, RfaH activates the expression of capsule, cell wall and pilus biosynthesis operons by reducing transcription termination and activating translation. While E. coli RfaH has been extensively studied using structural and biochemical approaches, limited data are available for other RfaH homologs. Here we set out to identify small molecule inhibitors of E. coli and K. pneumoniae RfaHs. Results of biochemical and functional assays show that these proteins act similarly, with a notable difference between their interactions with the RNAP ß subunit gate loop. We focused on high-affinity RfaH interactions with the RNAP ß' subunit clamp helices as a shared target for inhibition. Among the top 10 leads identified by in silico docking using ZINC database, 3 ligands were able to inhibit E. coli RfaH recruitment in vitro. The most potent lead was active against both E. coli and K. pneumoniae RfaHs in vitro. Our results demonstrate the feasibility of identifying RfaH inhibitors using in silico docking and pave the way for rational design of antivirulence therapeutics against antibiotic-resistant pathogens.