Scholarly Works, Virginia Tech Center for Drug Discovery

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Activation of PAD4 in NET formation.
Rohrbach, Amanda S.; Slade, Daniel J.; Thompson, Paul R.; Mowen, Kerri A. (2012)
Peptidylarginine deiminases, or PADs, convert arginine residues to the non-ribosomally encoded amino acid citrulline in a variety of protein substrates. PAD4 is expressed in granulocytes and is essential for the formation of neutrophil extracellular traps (NETs) via PAD4-mediated histone citrullination. Citrullination of histones is thought to promote NET formation by inducing chromatin decondensation and facilitating the expulsion of chromosomal DNA that is coated with antimicrobial molecules. Numerous stimuli have been reported to lead to PAD4 activation and NET formation. However, how this signaling process proceeds and how PAD4 becomes activated in cells is largely unknown. Herein, we describe the various stimuli and signaling pathways that have been implicated in PAD4 activation and NET formation, including the role of reactive oxygen species generation. To provide a foundation for the above discussion, we first describe PAD4 structure and function, and how these studies led to the development of PAD-specific inhibitors. A comprehensive survey of the receptors and signaling pathways that regulate PAD4 activation will be important for our understanding of innate immunity, and the identification of signaling intermediates in PAD4 activation may also lead to the generation of pharmaceuticals to target NET-related pathogenesis.
Antimalarial 5,6-Dihydro-alpha-pyrones from Cryptocarya rigidifolia: Related Bicyclic Tetrahydro-alpha-Pyrones Are Artifacts
Liu, Yixi; Rakotondraibe, L. Harinantenaina; Brodie, Peggy J.; Wiley, Jessica D.; Cassera, Maria B.; Miller, James S.; Ratovoson, F.; Rakotobe, Etienne; Rasamison, Vincent E.; Kingston, David G. I. (American Chemical Society, 2015-06-01)
Antiproliferative Compounds from Cleistanthus boivinianus from the Madagascar Dry Forest
Liu, Yixi; Young, Kelly; Rakotondraibe, L. Harinantenaina; Brodie, Peggy J.; Wiley, Jessica D.; Cassera, Maria B.; Callmander, Martin W.; Rakotondrajaona, R.; Rakotobe, Etienne; Rasamison, Vincent E.; TenDyke, Karen; Shen, Yongchun; Kingston, David G. I. (American Chemical Society, 2015-07-01)
Antiproliferative triterpenoid saponins from Leptaulus citroides Baill. from the Madagascar rain forest
Su, Qingxi; Brodie, Peggy J.; Liu, Yixi; Miller, James S.; Andrianjafy, Naina M.; Antsiferana, Rabodo; Rasamison, Vincent E.; Kingston, David G. I. (Springer, 2016)
Bioassay-guided fractionation of EtOH extracts obtained from the roots and wood of the Madagascan plant Leptaulus citroides Baill. (Cardiopteridaceae) led to the isolation of ethyl esters of three new triterpenoid saponins (1–3) and the known sesquiterpenoid cinnamosmolide (4). The structures of 1–3 were elucidated by extensive 1D and 2D NMR experiments and mass spectrometry. Compounds 1, 2, and 4 showed moderate cytotoxicity against the A2780 human ovarian cancer cell line with IC50 values of 2.8, 10.2 and 2.0 lM, respectively.
The cardiolipin-binding peptide elamipretide mitigates fragmentation of cristae networks following cardiac ischemia reperfusion in rats
Allen, Mitchell E.; Pennington, Edward Ross; Perry, Justin B.; Dadoo, Sahil; Makrecka-Kuka, Marina; Dambrova, Maija; Moukdar, Fatiha; Patel, Hetal D.; Han, Xianlin; Kidd, Grahame K.; Benson, Emily K.; Raisch, Tristan B.; Poelzing, Steven; Brown, David A.; Shaikh, Saame Raza (2020-07-17)
Allen and Pennington et al. show that the cardiolipin-binding peptide elamipretide mitigates disease-induced fragmentation of cristae networks following cardiac ischemia reperfusion in rats. This study suggests that elamipretide targets mitochondrial membranes to sustain cristae networks, improving their bioenergetic function. Mitochondrial dysfunction contributes to cardiac pathologies. Barriers to new therapies include an incomplete understanding of underlying molecular culprits and a lack of effective mitochondria-targeted medicines. Here, we test the hypothesis that the cardiolipin-binding peptide elamipretide, a clinical-stage compound under investigation for diseases of mitochondrial dysfunction, mitigates impairments in mitochondrial structure-function observed after rat cardiac ischemia-reperfusion. Respirometry with permeabilized ventricular fibers indicates that ischemia-reperfusion induced decrements in the activity of complexes I, II, and IV are alleviated with elamipretide. Serial block face scanning electron microscopy used to create 3D reconstructions of cristae ultrastructure reveals that disease-induced fragmentation of cristae networks are improved with elamipretide. Mass spectrometry shows elamipretide did not protect against the reduction of cardiolipin concentration after ischemia-reperfusion. Finally, elamipretide improves biophysical properties of biomimetic membranes by aggregating cardiolipin. The data suggest mitochondrial structure-function are interdependent and demonstrate elamipretide targets mitochondrial membranes to sustain cristae networks and improve bioenergetic function.
Cation competition and recruitment around the c-kit1 G-quadruplex using polarizable simulations
Salsbury, Alexa M.; Lemkul, Justin A. (2021-06-01)
Nucleic acid-ion interactions are fundamentally important to the physical, energetic, and conformational properties of DNA and RNA. These interactions help fold and stabilize highly ordered secondary and tertiary structures, such as G-quadruplexes (GQs), which are functionally relevant in telomeres, replication initiation sites, and promoter sequences. The c-kit protooncogene encodes for a receptor tyrosine kinase and is linked to gastrointestinal stromal tumors, mast cell disease, and leukemia. This gene contains three unique GQ-forming sequences that have proposed antagonistic effects on gene expression. The dominant GQ, denoted c-kit1, has been shown to decrease expression of c-kit transcripts, making the c-kit1GQa promising drug target. Toward disease intervention, more information is needed regarding its conformational dynamics and ion binding properties. Therefore, we performed molecular dynamics simulations of the c-kit1 GQ with K+, Na+, Li+, and mixed salt solutions using the Drude-2017 polarizable force field. We evaluated GQ structure, ion sampling, core energetics, ion dehydration and binding, and ion competition and found that each analysis supported the known GQ-ion specificity trend (K+ > Na+ > Li+). We also found that K+ ions coordinate in the tetrad core antiprismatically, whereas Na+ and Li+ align coplanar to guanine tetrads, partially because of their attraction to surrounding water. Further, we showed that K+ occupancy is higher around the c-kit1 GQ and its nucleobases than Na+ and Li+, which tend to interact with backbone and sugar moieties. Finally, we showed that K+ binding to the c-kit1GQ is faster and more frequent than Na+ and Li+. Such descriptions of GQ-ion dynamics suggest the rate of dehydration as the dominant factor for preference of K+ by DNA GQs and provide insight into noncanonical nucleic acids for which little experimental data exist.
Characterization of the Ornithine Hydroxylation Step in Albachelin Biosynthesis
Bufkin, Kendra; Sobrado, Pablo (MDPI, 2017-10-01)
N-Hydroxylating monooxygenases (NMOs) are involved in siderophore biosynthesis. Siderophores are high affinity iron chelators composed of catechol and hydroxamate functional groups that are synthesized and secreted by microorganisms and plants. Recently, a new siderophore named albachelin was isolated from a culture of Amycolatopsis alba growing under iron-limiting conditions. This work focuses on the expression, purification, and characterization of the NMO, abachelin monooxygenase (AMO) from A. alba. This enzyme was purified and characterized in its holo (FAD-bound) and apo (FAD-free) forms. The apo-AMO could be reconstituted by addition of free FAD. The two forms of AMO hydroxylate ornithine, while lysine increases oxidase activity but is not hydroxylated and display low affinity for NADPH.
Chemical and Biological Methods to Detect Post-Translational Modifications of Arginine
Slade, Daniel J.; Subramanian, Venkataraman; Fuhrmann, Jakob; Thompson, Paul R. (2014-02)
Posttranslational modifications (PTMs) of protein embedded arginines are increasingly being recognized as playing an important role in both prokaryotic and eukaryotic biology, and it is now clear that these PTMs modulate a number of cellular processes including DNA binding, gene transcription, protein-protein interactions, immune system activation, and proteolysis. There are currently four known enzymatic PTMs of arginine ( i.e., citrullination, methylation, phosphorylation, ADP-ribosylation), and two non-enzymatic PTMs (i.e., carbonylation, advanced glycation end-products (AGEs)). Enzymatic modification of arginine is tightly controlled during normal cellular function, and can be drastically altered in response to various second messengers and in different disease states. Non-enzymatic arginine modifications are associated with a loss of metabolite regulation during normal human aging. This abnormally large number of modifications to a single amino acid creates a diverse set of structural perturbations that can lead to altered biological responses. While the biological role of methylation has been the most extensively characterized of the arginine PTMs, recent advances have shown that the once obscure modification known as citrullination is involved in the onset and progression of inflammatory diseases and cancer. This review will highlight the reported arginine PTMs and their methods of detection, with a focus on new chemical methods to detect protein citrullination.
Chemical Proteomic Platform To Identify Citrullinated Proteins
Lewallen, Daniel M.; Bicker, Kevin L.; Subramanian, Venkataraman; Clancy, Kathleen W.; Slade, Daniel J.; Martell, Julianne; Dreyton, Christina J.; Sokolove, Jeremy; Weerapana, Eranthie; Thompson, Paul R. (2015-11-20)
Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and are routinely used for disease diagnosis. Protein citrullination is also increased in cancer and other autoimmune disorders, suggesting that citrullinated proteins may serve as biomarkers for diseases beyond RA. To identify these citrullinated proteins, we developed biotin-conjugated phenylglyoxal (biotin-PG). Using this probe and our platform technology, we identified >50 intracellular citrullinated proteins. More than 20 of these are involved in RNA splicing, suggesting, for the first time, that citrullination modulates RNA biology. Overall, this chemical proteomic platform will play a key role in furthering ourunderstanding of protein citrullination in rheumatoid arthritis and potentially a wider spectrum of inflammatory diseases.
Citrulination unravels stem cells
Slade, Daniel J.; Subramanian, Venkataraman; Thompson, Paul R. (2014-05)
Maintenance of the pluripotent stem cell state is regulated by the post-translational modification of histones. The discovery that citrullination of the linker histone H1 is critical to this
Crystal structure of the MACPF domain of human complement protein C8 alpha in complex with the C8 gamma subunit
Slade, Daniel J.; Lovelace, Leslie L.; Chruszcz, Maksymilian; Minor, Wladek; Lebioda, Lukasz; Sodetz, James M. (Academic Press – Elsevier, 2008-05-29)
Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that assemble on bacterial membranes to form a pore-like structure referred to as the "membrane attack complex" (MAC). C8 contains three genetically distinct subunits (C8α, C8β, Cγ.) arranged as a disulfide-linked C8α-γ dimer that is noncovalently associated with C8β. C6, C7 C8α, C8β and C9 are homologous. All contain N- and C-terminal modules and an intervening 40-kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. The C8γ subunit is unrelated and belongs to the lipocalin family of proteins that display a β-barrel fold and generally bind small, hydrophobic ligands. Several hundred proteins with MACPF domains have been identified based on sequence similarity; however, the structure and function of most are unknown. Crystal structures of the secreted bacterial protein Plu-MACPF and the human C8α MACPF domain were recently reported and both display a fold similar to the bacterial pore-forming cholesterol-dependent cytolysins (CDC). In the present study, we determined the crystal structure of the human C8α MACPF domain disulfide-linked to C8γ (αMACPF-γ) at 2.15 Å resolution. The αMACPF portion has the predicted CDC-like fold and shows two regions of interaction with C8γ. One is in a previously characterized 19-residue insertion (indel) in C8α and fills the entrance to the putative C8γ ligand binding site. The second is a hydrophobic pocket that makes contact with residues on the side of the C8γ β-barrel. The latter interaction induces conformational changes in αMACPF that are likely important for C8 function. Also observed is structural conservation of the MACPF signature motif Y/W-G-T/S-H-F/Y-X6-G-G in αMACPF and Plu-MACPF, and conservation of several key glycine residues known to be important for refolding and pore formation by CDCs.
Differences in Conformational Sampling and Intrinsic Electric Fields Drive Ion Binding in Telomeric and TERRA G-Quadruplexes
Poleto, Marcelo D.; Lemkul, Justin A. (American Chemical Society, 2023-10-17)
The formation of G-quadruplexes (GQs) occurs in guanine-rich sequences of DNA and RNA, producing highly stable and structurally diverse noncanonical nucleic acid structures. GQs play crucial roles in regulating transcription, translation, and replication and maintaining the genome, among others; thus, changes to their structures can lead to diseases such as cancer. Previous studies using polarizable molecular dynamics simulations have shown differences in ion binding properties between telomeric and telomeric repeat-containing RNA GQs despite architectural similarities. Here, we used volume-based metadynamics and repulsive potential simulations in conjunction with polarizable force fields to quantify the impact of ion binding on the GQ dynamics and ion binding free energies. Furthermore, we describe how GQs exert electric fields on their surroundings to link dynamics with variations in the electronic structure. Our findings provide new insights into the energetic, physical, and conformational properties of GQs and expose subtle but important differences between DNA and RNA GQs with the same fold.
Discovery of Two Inhibitors of the Type IV Pilus Assembly ATPase PilB as Potential Antivirulence Compounds
Dye, Keane J.; Vogelaar, Nancy J.; O'Hara, Megan; Sobrado, Pablo; Santos, Webster; Carlier, Paul R.; Yang, Zhaomin (American Society for Microbiology, 2022-12)
Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. With the pressing antibiotic resistance pandemic, antivirulence has been increasingly explored as an alternative strategy against bacterial infections. The bacterial type IV pilus (T4P) is a well-documented virulence factor and an attractive target for small molecules for antivirulence purposes. The PilB ATPase is essential for T4P biogenesis because it catalyzes the assembly of monomeric pilins into the polymeric pilus filament. Here, we describe the identification of two PilB inhibitors by a high-throughput screen (HTS) in vitro and their validation as effective inhibitors of T4P assembly in vivo. We used Chloracidobacterium thermophilum PilB as a model enzyme to optimize an ATPase assay for the HTS. From a library of 2,320 compounds, benserazide and levodopa, two approved drugs for Parkinson's disease, were identified and confirmed biochemically to be PilB inhibitors. We demonstrate that both compounds inhibited the T4P-dependent motility of the bacteria Myxoccocus xanthus and Acinetobacter nosocomialis. Additionally, benserazide and levodopa were shown to inhibit A. nosocomialis biofilm formation, a T4P-dependent process. Using M. xanthus as a model, we showed that both compounds inhibited T4P assembly in a dose-dependent manner. These results suggest that these two compounds are effective against the PilB protein in vivo. The potency of benserazide and levodopa as PilB inhibitors both in vitro and in vivo demonstrate potentials of the HTS and its two hits here for the development of anti-T4P chemotherapeutics.IMPORTANCE Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. Here, we report the development and implementation of a method to identify anti-T4P chemicals from compound libraries by high-throughput screen. This led to the identification and validation of two T4P inhibitors both in the test tubes and in bacteria. The discovery and validation pipeline reported here as well as the confirmation of two anti-T4P inhibitors provide new venues and leads for the development of chemotherapeutics against antibiotic-resistant infections.
Effects of Familial Alzheimer's Disease Mutations on the Folding Free Energy and Dipole-Dipole Interactions of the Amyloid β-Peptide
Davidson, Darcy S.; Kraus, Joshua A.; Montgomery, Julia M.; Lemkul, Justin A. (American Chemical Society, 2022-10-06)
Familial Alzheimer's disease (FAD) mutations of the amyloid β-peptide (Aβ) are known to lead to early onset and more aggressive Alzheimer's disease. FAD mutations such as "Iowa" (D23N), "Arctic" (E22G), "Italian" (E22K), and "Dutch" (E22Q) have been shown to accelerate Aβ aggregation relative to the wild-type (WT). The mechanism by which these mutations facilitate increased aggregation is unknown, but each mutation results in a change in the net charge of the peptide. Previous studies have used nonpolarizable force fields to study Aβ, providing some insight into how this protein unfolds. However, nonpolarizable force fields have fixed charges that lack the ability to redistribute in response to changes in local electric fields. Here, we performed polarizable molecular dynamics simulations on the full-length Aβ42of WT and FAD mutations and calculated folding free energies of the Aβ15-27fragment via umbrella sampling. By studying both the full-length Aβ42and a fragment containing mutations and the central hydrophobic cluster (residues 17-21), we were able to systematically study how these FAD mutations impact secondary and tertiary structure and the thermodynamics of folding. Electrostatic interactions, including those between permanent and induced dipoles, affected side-chain properties, salt bridges, and solvent interactions. The FAD mutations resulted in shifts in the electronic structure and solvent accessibility at the central hydrophobic cluster and the hydrophobic C-terminal region. Using umbrella sampling, we found that the folding of the WT and E22 mutants is enthalpically driven, whereas the D23N mutant is entropically driven, arising from a different unfolding pathway and peptide-bond dipole response. Together, the unbiased, full-length, and umbrella sampling simulations of fragments reveal that the FAD mutations perturb nearby residues and others in hydrophobic regions to potentially alter solubility. These results highlight the role electronic polarizability plays in amyloid misfolding and the role of heterogeneous microenvironments that arise as conformational change takes place.
Electronic Polarization at the Interface between the p53 Transactivation Domain and Two Binding Partners
Corrigan, Alexsandra N.; Lemkul, Justin A. (American Chemical Society, 2022-07-07)
Intrinsically disordered proteins (IDPs) are an abundant class of highly charged proteins that participate in numerous crucial biological processes, often in regulatory roles. IDPs do not have one major free energy minimum with a dominant structure, instead existing as conformational ensembles of multiple semistable conformations. p53 is a prototypical protein with disordered regions and binds to many structurally diverse partners, making it a useful model for exploring the role of electrostatic interactions at IDP binding interfaces. In this study, we used the Drude-2019 force field to simulate the p53 transactivation domain with two protein partners to probe the role of electrostatic interactions in IDP protein-protein interactions. We found that the Drude-2019 polarizable force field reasonably reproduced experimental chemical shifts of the p53 transactivation domain (TAD) in one complex for which these data are available. We also found that the proteins in these complexes displayed dipole response at specific residues of each protein and that residues primarily involved in binding showed a large percent change in dipole moment between the unbound and complexed states. Probing the role of electrostatic interactions in IDP binding can allow us greater fundamental understanding of these interactions and may help with targeting p53 or its partners for drug design.
Enzyme-Triggered Chemodynamic Therapy via a Peptide-H2S Donor Conjugate with Complexed Fe2+
Zhu, Yumeng; Archer, William R.; Morales, Katlyn F.; Schulz, Michael D.; Wang, Yin; Matson, John B. (Wiley-V C H Verlag, 2023-04)
Inducing high levels of reactive oxygen species (ROS) inside tumor cells is a cancer therapy method termed chemodynamic therapy (CDT). Relying on delivery of Fenton reaction promoters such as Fe2+, CDT takes advantage of overproduced ROS in the tumor microenvironment. We developed a peptide-H2S donor conjugate, complexed with Fe2+, termed AAN-PTC-Fe2+. The AAN tripeptide was specifically cleaved by legumain, an enzyme overexpressed in glioma cells, to release carbonyl sulfide (COS). Hydrolysis of COS by carbonic anhydrase formed H2S, an inhibitor of catalase, an enzyme that detoxifies H2O2. Fe2+ and H2S together increased intracellular ROS levels and decreased viability in C6 glioma cells compared with controls lacking either Fe2+, the AAN sequence, or the ability to generate H2S. AAN-PTC-Fe2+ performed better than temezolimide while exhibiting no cytotoxicity toward H9C2 cardiomyocytes. This study provides an H2S-amplified, enzyme-responsive platform for synergistic cancer treatment.
Flavin oxidation in flavin dependent N-monooxygenase
Sobrado, Pablo; Robinson, Reeder; Klancher, Catherine; Rodriguez, Pedro (2019-01-02)
Fluorescence Polarization Binding Assay for Aspergillus fumigatus Virulence Factor UDP-Galactopyranose Mutase
Qi, Jun; Oppenheimer, Michelle; Sobrado, Pablo (Hindawi, 2011-08-21)
Aspergillus fumigatus is an opportunistic human pathogenic fungus responsible for deadly lung infections in immunocompromised individuals. Galactofuranose (Galf) residues are essential components of the cell wall and play an important role in A. fumigatus virulence. The flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the isomerization of UDP-galactopyranose to UDP-galactofuranose, the biosynthetic precursor of Galf. Thus, inhibitors of UGM that block the biosynthesis of Galf can lead to novel chemotherapeutics for treating A. fumigatus-related diseases. Here, we describe the synthesis of fluorescently labeled UDP analogs and the development of a fluorescence polarization (FP) binding assay for A. fumigatus UGM (AfUGM). High-affinity binding to AfUGM was only obtained with the chromophore TAMRA, linked to UDP by either 2 or 6 carbons with Kd values of 2.6 ± 0.2 μM and 3.0 ± 0.7 μM, respectively. These values were ~6 times lower than when UDP was linked to fluorescein. The FP assay was validated against several known ligands and displayed an excellent Z′ factor (0.79 ± 0.02) and good tolerance to dimethyl sulfoxide.
Genetic Reporter System for Positioning of Proteins at the Bacterial Pole
Fixen, Kathryn R.; Janakiraman, Anuradha; Garrity, Sean; Slade, Daniel J.; Gray, Andrew N.; Karahan, Nilay; Hochschild, Ann; Goldberg, Marcia B. (2012)
Spatial organization within bacteria is fundamental to many cellular processes, although the basic mechanisms underlying localization of proteins to specific sites within bacteria are poorly understood. The study of protein positioning has been limited by a paucity of methods that allow rapid large-scale screening for mutants in which protein positioning is altered. We developed a genetic reporter system for protein localization to the pole within the bacterial cytoplasm that allows saturation screening for mutants in Escherichia coli in which protein localization is altered. Utilizing this system, we identify proteins required for proper positioning of the Shigella autotransporter IcsA. Autotransporters, widely distributed bacterial virulence proteins, are secreted at the bacterial pole. We show that the conserved cell division protein FtsQ is required for localization of IcsA and other autotransporters to the pole. We demonstrate further that this system can be applied to the study of proteins other than autotransporters that display polar positioning within bacterial cells.
High-Throughput Screen for Inhibitors of the Type IV Pilus Assembly ATPase PilB
Dye, Keane J.; Vogelaar, Nancy J.; Sobrado, Pablo; Yang, Zhaomin (2021-03)
The bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant. Antivirulence chemotherapeutics are considered a promising alternative to antibiotics because they target the disease process instead of bacterial viability. However, a roadblock to the discovery of anti-T4P compounds is the lack of a high-throughput screen (HTS) that can be implemented relatively easily and economically. Here, we describe the first HTS for the identification of inhibitors specifically against the T4P assembly ATPase PilB in vitro. Chloracidobacterium thermophilum PilB (CtPilB) had been demonstrated to have robust ATPase activity and the ability to bind its expected ligands in vitro. We utilized CtPilB and MANT-ATP, a fluorescent ATP analog, to develop a binding assay and adapted it for an HTS. As a proof of principle, we performed a pilot screen with a small compound library of kinase inhibitors and identified quercetin as a PilB inhibitor in vitro. Using Myxococcus xanthus as a model bacterium, we found quercetin to reduce its T4P-dependent motility and T4P assembly in vivo. These results validated our HTS as effective in identifying PilB inhibitors. This assay may prove valuable in seeking leads for the development of antivirulence chemotherapeutics against PilB, an essential and universal component of all bacterial T4P systems. IMPORTANCE Many bacterial pathogens use their type IV pili (T4P) to facilitate and maintain infection of a human host. Small chemical compounds that inhibit the production or assembly of T4P hold promise in the treatment and prevention of infections, especially in the era of increasing threats from antibiotic-resistant bacteria. However, few chemicals are known to have inhibitory or anti-T4P activity. Their identification has not been easy due to the lack of a method for the screening of compound collections or libraries on a large scale. Here, we report the development of an assay that can be scaled up to screen compound libraries for inhibitors of a critical T4P assembly protein. We further demonstrate that it is feasible to use whole cells to examine potential inhibitors for their activity against T4P assembly in a bacterium.

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