Synergistic Inhibition Guided Fragment-Linking Strategy and Quantitative Structure-Property Relationship Modeling To Design Inhalable Therapeutics for Asthma Targeting CSF1R.

The colony-stimulating factor-1 receptor (CSF1R) is a tyrosine-protein kinase that is a potential target for asthma therapeutics. We have applied a fragment-lead combination approach to identify small fragments that act synergistically with GW2580, a known inhibitor of CSF1R. Two fragment libraries were screened in combination with GW2580 by surface plasmon resonance (SPR). Binding affinity measurements confirmed that thirteen fragments bind specifically to the CSF1R, and a kinase activity assay further validated the inhibitory effect of these fragments. Several fragment compounds enhanced the inhibitory activity of the lead inhibitor. Computational solvent mapping, molecular docking, and modeling studies suggest that some of these fragments bind adjacent to the binding site of the lead inhibitor and further stabilize the inhibitor-bound state. Modeling results guided the computational fragment-linking approach to design potential next-generation compounds. The inhalability of these proposed compounds was predicted using quantitative structure-property relationships (QSPR) modeling based on an analysis of 71 drugs currently on the market. This work provides new insights into the development of inhalable small molecule therapeutics for asthma.

Gene Expression Signatures in Inflammatory and Sclerotic Morphea Skin and Sera Distinguish Morphea from Systemic Sclerosis.

Morphea is an inflammatory fibrotic disorder of the skin that has been likened to systemic sclerosis (SSc). We sought to examine the molecular landscape of morphea by examining lesional skin gene expression and blood biomarkers and comparing the gene expression profiles with those from site-matched nonlesional and SSc lesional skin. We found the morphea transcriptome is dominated by IFN-γ-mediated T helper 1 immune dysregulation, with a relative paucity of fibrosis pathways. Specifically, expression profiles of morphea skin clustered with the SSc inflammatory subset and were distinct from the those of SSc fibroproliferative subset. Unaffected morphea skin also differed from unaffected SSc skin because it did not exhibit pathological gene expression signatures. Examination of downstream IFN-γ-mediated chemokines, CXCL9 and CXCL10, revealed increased transcription in the skin but not in circulation. In contrast to transcriptional activity, CXCL9 was elevated in serum and was associated with active, widespread cutaneous involvement. Taken together, these results indicate that morphea is a skin-directed process characterized by T helper 1 immune-mediated dysregulation, which contrasts with fibrotic signatures and systemic transcriptional changes associated with SSc. The similarity between morphea and the inflammatory subset of SSc on transcriptional profiling indicates that therapies under development for this subset of SSc are also promising for treatment of morphea.

Mapping the energy landscape of PROTAC-mediated protein-protein interactions.

A principal challenge in computational modeling of macromolecules is the vast conformational space that arises out of large numbers of atomic degrees of freedom. Recently, growing interest in building predictive models of complexes mediated by Proteolysis Targeting Chimeras (PROTACs) has led to the application of state-of-the-art computational techniques to tackle this problem. However, repurposing existing tools to carry out protein-protein docking and linker conformer generation independently results in extensive sampling of structures incompatible with PROTAC-mediated complex formation. Here we show that it is possible to restrict the search to the space of protein-protein conformations that can be bridged by a PROTAC molecule with a given linker composition by using a cyclic coordinate descent algorithm to position PROTACs into complex-bound configurations. We use this methodology to construct potential energy and solvation energy landscapes of PROTAC-mediated interactions. Our results suggest that desolvation of amino acids at interfaces could play a dominant role in PROTAC-mediated complex formation.

Evaluation of interactions between the hepatitis C virus NS3/4A and sulfonamidobenzamide based molecules using molecular docking, molecular dynamics simulations and binding free energy calculations.

The Hepatitis C Virus (HCV) NS3/4A is an attractive target for the treatment of Hepatitis C infection. Herein, we present an investigation of HCV NS3/4A inhibitors based on a sulfonamidobenzamide scaffold. Inhibitor interactions with HCV NS3/4A were explored by molecular docking, molecular dynamics simulations, and MM/PBSA binding free energy calculations. All of the inhibitors adopt similar molecular docking poses in the catalytic site of the protease that are stabilized by hydrogen bond interactions with G137 and the catalytic S139, which are known to be important for potency and binding stability. The quantitative assessments of binding free energies from MM/PBSA correlate well with the experimental results, with a high coefficient of determination, R2 of 0.92. Binding free energy decomposition analyses elucidate the different contributions of Q41, F43, H57, R109, K136, G137, S138, S139, A156, M485, and Q526 in binding different inhibitors. The importance of these sidechain contributions was further confirmed by computational alanine scanning mutagenesis. In addition, the sidechains of K136 and S139 show crucial but distinct contributions to inhibitor binding with HCV NS3/4A. The structural basis of the potency has been elucidated, demonstrating the importance of the R155 sidechain conformation. This extensive exploration of binding energies and interactions between these compounds and HCV NS3/4A at the atomic level should benefit future antiviral drug design.

Identification of Small Molecule Inhibitors against Staphylococcus aureus Dihydroorotase via HTS.

Drug-resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)-an integral step in the de novo pyrimidine biosynthesis pathway. This study sets forth a high-throughput screening (HTS) of 3000 fragment compounds by a colorimetry-based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca-asp determined the best compound to be a competitive inhibitor with a KD value of 11 µM, which is 10-fold tighter than Ca-asp. Preliminary structure-activity relationship (SAR) provides the foundation for further structure-based antimicrobial inhibitor design against S. aureus.

Baicalein Is a Phytohormone that Signals Through the Progesterone and Glucocorticoid Receptors.

While flavonoids have been studied extensively for estrogen receptor activity, they have not been well studied for their ability to modify progesterone receptor (PR) and glucocorticoid receptor (GR) signaling. Three flavonoid compounds, tangeretin, wogonin, and baicalein, were selected for testing for PR and GR activity based on their structural similarity to known phytoprogesterone-like compounds. Each compound was docked in the binding pocket of PR and GR. Of these compounds, baicalein was predicted to be most likely to bind to both receptors. A fluorescence polarization competitive binding assay for PR and GR confirmed that baicalein binds to both the PR and GR with IC50 values of 15.30 µM and 19.26 µM, respectively. In Ishikawa PR-B and T47D cells, baicalein acted as a PR antagonist in a hormone response element (HRE) luciferase (Luc) assay. In OVCAR5 cells, which only express GR, baicalein was a GR agonist via an HRE/Luc assay and induced GR target genes, FKBP5 and GILZ. RU486, a PR and GR antagonist, abrogated baicalein's activity in OVCAR5 cells, confirming baicalein's activity is mediated through the GR. In vivo, baicalein administered intraperitoneally to female mice twice a week for 4 weeks at a dose of 25 mg/kg induced the GR target gene GILZ in the reproductive tract, which was blocked by RU486. In summary, baicalein has PR antagonist and GR agonist activity in vitro and demonstrates GR agonist activity in the uterus in vivo.

Structural approaches to pathway-specific antimicrobial agents.

This perspective provides an overview of the evolution of antibiotic discovery from a largely phenotypic-based effort, through an intensive structure-based design focus, to a more holistic approach today. The current focus on antibiotic development incorporates assay and discovery conditions that replicate the host environment as much as feasible. They also incorporate several strategies, including target identification and validation within the whole cell environment, a variety of target deconvolution methods, and continued refinement of structure-based design approaches.

Na+/K+-ATPase-Targeted Cytotoxicity of (+)-Digoxin and Several Semisynthetic Derivatives.

(+)-Digoxin (1) is a well-known cardiac glycoside long used to treat congestive heart failure and found more recently to show anticancer activity. Several known cardenolides (2-5) and two new analogues, (+)-8(9)-ß-anhydrodigoxigenin (6) and (+)-17-epi-20,22-dihydro-21α-hydroxydigoxin (7), were synthesized from 1 and evaluated for their cytotoxicity toward a small panel of human cancer cell lines. A preliminary structure-activity relationship investigation conducted indicated that the C-12 and C-14 hydroxy groups and the C-17 unsaturated lactone unit are important for 1 to mediate its cytotoxicity toward human cancer cells, but the C-3 glycosyl residue seems to be less critical for such an effect. Molecular docking profiles showed that the cytotoxic 1 and the noncytotoxic derivative 7 bind differentially to Na+/K+-ATPase. The HO-12ß, HO-14ß, and HO-3'aα hydroxy groups of (+)-digoxin (1) may form hydrogen bonds with the side-chains of Asp121 and Asn122, Thr797, and Arg880 of Na+/K+-ATPase, respectively, but the altered lactone unit of 7 results in a rotation of its steroid core, which depotentiates the binding between this compound and Na+/K+-ATPase. Thus, 1 was found to inhibit Na+/K+-ATPase, but 7 did not. In addition, the cytotoxic 1 did not affect glucose uptake in human cancer cells, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na+/K+-ATPase but not by interacting with glucose transporters.

KxWO is a Novel Ferroelectric Nanomaterial for Application as a Room Temperature Acetone Sensor.

A newly synthesized nanomaterial known as KxW7O22 (KxWO) exhibits a stable room-temperature ferroelectric property. This unique ferroelectric property has revealed that KxWO is a promising material for application in a breath sensor, which can be used for patients to monitor their daily health condition and diagnose disease at every early stage with low cost, convenience, and non-invasion. In this study, we successfully synthesized nano-structured KxWO through a low cost but high yield hydrothermal method. The sensing response of KxWO to acetone is examined based on a chemiresistive effect. For the first time, we systematically studied how material structures and the component, potassium (K), can affect KxWO-based sensing performance. The results indicate that the low temperature ferroelectric property of KxWO causes an excellent response to acetone, which is the biomarker for diabetes. The lowest detection limit can be down to 0.1 ppm and the KxWO-based sensor can operate at room temperature. In addition, the Kx component KxWO and its crystal structure also play an important role in improving its sensing performance. Our results provide advanced research in (1) exploring the study of KxWO material properties by tailoring the concentration of the potassium in KxWO and introducing the surfactant Pluronic L-121 in the growing process, and (2) optimizing KxWO sensing performance by controlling its material properties.

Cytotoxic and non-cytotoxic cardiac glycosides isolated from the combined flowers, leaves, and twigs of Streblus asper.

A new non-cytotoxic [(+)-17ß-hydroxystrebloside (1)] and two known cytotoxic [(+)-3'-de-O-methylkamaloside (2) and (+)-strebloside (3)] cardiac glycosides were isolated and identified from the combined flowers, leaves, and twigs of Streblus asper collected in Vietnam, with the absolute configuration of 1 established from analysis of its ECD and NMR spectroscopic data and confirmed by computational ECD calculations. A new 14,21-epoxycardanolide (3a) was synthesized from 3 that was treated with base. A preliminary structure-activity relationship study indicated that the C-14 hydroxy group and the C-17 lactone unit and the established conformation are important for the mediation of the cytotoxicity of 3. Molecular docking profiles showed that the cytotoxic 3 and its non-cytotoxic analogue 1 bind differentially to Na+/K+-ATPase. Compound 3 docks deeply in the Na+/K+-ATPase pocket with a sole pose, and its C-10 formyl and C-5, C-14, and C-4' hydroxy groups may form hydrogen bonds with the side-chains of Glu111, Glu117, Thr797, and Arg880 of Na+/K+-ATPase, respectively. However, 1 fits the cation binding sites with at least three different poses, which all depotentiate the binding between 1 and Na+/K+-ATPase. Thus, 3 was found to inhibit Na+/K+-ATPase, but 1 did not. In addition, the cytotoxic and Na+/K+-ATPase inhibitory 3 did not affect glucose uptake in human lung cancer cells, against which it showed potent activity, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na+/K+-ATPase but not by interacting with glucose transporters.

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease.

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 310 -helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.

Identification of Small Molecules Exhibiting Oxacillin Synergy through a Novel Assay for Inhibition of vraTSR Expression in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) strains that are resistant to all forms of penicillin have become an increasingly common and urgent problem threatening human health. They are responsible for a wide variety of infectious diseases ranging from minor skin abscesses to life-threatening severe infections. The vra operon that is conserved among S. aureus strains encodes a three-component signal transduction system (vraTSR) that is responsible for sensing and responding to cell wall stress. We developed a novel and multifaceted assay to identify compounds that potentiate the activity of oxacillin, essentially restoring efficacy of oxacillin against MRSA, and performed high-throughput screening (HTS) to identify oxacillin potentiators. HTS of 13,840 small-molecule compounds from an antimicrobial-focused Life Chemicals library, using the MRSA cell-based assay, identified three different inhibitor scaffolds. Checkerboard assays for synergy with oxacillin, reverse transcriptase PCR (RT-PCR) assays against vraR expression, and direct confirmation of interaction with VraS by surface plasmon resonance (SPR) further verified them to be viable hit compounds. A subsequent structure-activity relationship (SAR) study of the best scaffold with diverse analogs was utilized to improve potency and provides a strong foundation for further development.

Exploring small molecules with pan-genotypic inhibitory activities against hepatitis C virus NS3/4A serine protease.

Among the many Hepatitis C virus (HCV) genotypes and subtypes, genotypes 1b and 3a are most prevalent in United States and Asia, respectively. A total of 132 commercially available analogs of a previous lead compound were initially investigated against wild-type HCV genotype 1b NS3/4A protease. Ten compounds showed inhibitory activities (IC50 values) below 10 µM with comparable direct binding affinities (KD values) determined by surface plasmon resonance (SPR). To identify pan-genotypic inhibitors, these ten selected compounds were tested against four additional genotypes (1a, 2a, 3a, and 4) and three drug-resistant mutants (A156S, R155K, and V36M). Four new analogs have been identified with better activities against all five tested genotypes than the prior lead compound. Further, the original lead compound did not show activity against genotype 3a NS3/4A, whereas four newly identified compounds exhibited IC50 values below 33 µM against genotype 3a NS3/4A. Encouragingly, the best new compound F1813-0710 possessed promising activity toward genotype 3a, which is a huge improvement over the previous lead compound that had no effect on genotype 3a. This intriguing observation was further analyzed by molecular docking and molecular dynamics (MD) simulations to understand their different binding interactions, which should benefit future pan-genotypic inhibitor design and drug discovery.

Identification and design of novel small molecule inhibitors against MERS-CoV papain-like protease via high-throughput screening and molecular modeling.

The development of new therapeutic agents against the coronavirus causing Middle East Respiratory Syndrome (MERS) is a continuing imperative. The initial MERS-CoV epidemic was contained entirely through public health measures, but episodic cases continue, as there are currently no therapeutic agents effective in the treatment of MERS-CoV, although multiple strategies have been proposed. In this study, we screened 30,000 compounds from three different compound libraries against one of the essential proteases, the papain-like protease (PLpro), using a fluorescence-based enzymatic assay followed by surface plasmon resonance (SPR) direct binding analysis for hit confirmation. Mode of inhibition assays and competition SPR studies revealed two compounds to be competitive inhibitors. To improve upon the inhibitory activity of the best hit compounds, a small fragment library consisting of 352 fragments was screened in the presence of each hit compound, resulting in one fragment that enhanced the IC50 value of the best hit compound by 3-fold. Molecular docking and MM/PBSA binding energy calculations were used to predict potential binding sites, providing insight for design and synthesis of next-generation compounds.

Microbiome dysbiosis is associated with disease duration and increased inflammatory gene expression in systemic sclerosis skin.

BACKGROUND: Infectious agents have long been postulated to be disease triggers for systemic sclerosis (SSc), but a definitive link has not been found. Metagenomic analyses of high-throughput data allows for the unbiased identification of potential microbiome pathogens in skin biopsies of SSc patients and allows insight into the relationship with host gene expression. METHODS: We examined skin biopsies from a diverse cohort of 23 SSc patients (including lesional forearm and non-lesional back samples) by RNA-seq. Metagenomic filtering and annotation was performed using the Integrated Metagenomic Sequencing Analysis (IMSA). Associations between microbiome composition and gene expression were analyzed using single-sample gene set enrichment analysis (ssGSEA). RESULTS: We find the skin of SSc patients exhibits substantial changes in microbial composition relative to controls, characterized by sharp decreases in lipophilic taxa, such as Propionibacterium, combined with increases in a wide range of gram-negative taxa, including Burkholderia, Citrobacter, and Vibrio. CONCLUSIONS: Microbiome dysbiosis is associated with disease duration and increased inflammatory gene expression. These data provide a comprehensive portrait of the SSc skin microbiome and its association with local gene expression, which mirrors the molecular changes in lesional skin.

Hit-to-Lead: Hit Validation and Assessment.

High-throughput screening assays have become nearly ubiquitous in the search for small compounds or peptides that can modulate biological processes for therapeutic purposes. While many assays have become quite robust, with well-established protocols, the subsequent steps of validating the hits and choosing the best ones to take forward into leads for further chemical development are less established. In this chapter, we describe a variety of approaches, including chemical assessment, the use of various computational approaches, a variety of counter-screens, and "orthogonal" biophysical assays using nuclear magnetic resonance, surface plasmon resonance, isothermal titration calorimetry or thermal shift assays as methods for validating and assessing the quality of hits.

Discovery of small molecule inhibitors of adenovirus by disrupting E3-19K/HLA-A2 interactions.

The binding of the adenovirus (Ad) protein E3-19K with the human leukocyte antigen (HLA) plays an important role in Ad infections, which is the causative agent of a series of gastrointestinal, respiratory and ocular diseases. The objective of this research is to evaluate the essential interactions between E3-19K and HLA-A2 using the X-ray crystal structure of the E3-19K/HLA-A2 complex, and to identify small molecules that could potentially disrupt their binding. Computational methods, including molecular dynamic simulations, MM/GBSA calculations, and computational solvent mapping, were implemented to determine potential binding site(s) for small molecules. The previous experimentally determined hot spot residues, Q54 and E177 in HLA-A2, were also predicted to be the dominant residues for binding to E3-19K by our theoretical calculations. Several other residues were also found to play pivotal roles for the binding of E3-19K with HLA-A2. Residues adjacent to E177, including Q54 and several other residues theoretically predicted to be crucial in HLA-A2 were selected as a potential binding pocket to perform virtual screening with 1200 compounds from the Prestwick library. Seven hits were validated by surface plasmon resonance (SPR) as binders to HLA-A2 as a first step in identifying molecules that can perturb its association with the Ad E3-19K protein.

The Flavonoid Apigenin Is a Progesterone Receptor Modulator with In Vivo Activity in the Uterus.

Apigenin is a flavonoid with well-documented anti-cancer properties; however, its mechanisms of action are still unclear. We previously identified apigenin as a potential phytoprogestin, a natural product with a chemical scaffold that interacts with the progesterone receptor (PR). Our objective was to characterize the ability of apigenin to interact with PR through molecular docking studies, in vitro activity assays, and the ability of apigenin to elicit progestin-like effects in vivo. Molecular docking confirmed that apigenin could interact with PR, though with lower affinity than progesterone due to fewer van der Waals interactions. In Ishikawa cells stably expressing PR-B, apigenin significantly increased progesterone response element/luciferase (PRE/Luc) activity at 5 and 10 µM, but not in the parental Ishikawa cells that lack PR expression. In the presence of 100 nM of progesterone, 10 µM apigenin reduced PRE/Luc activity, indicative of mixed agonist activity. Apigenin also triggered degradation of PR in Ishikawa PR-B cells as measured by western blot. Apigenin reduced proliferation of Ishikawa cells, but through a PR-independent mechanism. In contrast, apigenin and progesterone both stimulated proliferation of T47D cells, an effect blocked by RU486. Apigenin activated other nuclear receptors evidenced by increased luciferase activity in MDA-MB-231 cells, which are PR negative. In vivo, apigenin blocked the genistein-stimulated increase in uterine epithelial cell height; stimulated endometrial expression of Hand2, a transcription factor stimulated by PR, and significantly reduced genistein-induced proliferation. In summary, apigenin is a phytoprogestin, with mixed agonist activity that demonstrates activity in vivo by hindering estrogen receptor-mediated uterine proliferation.

Determination of absolute configuration and binding efficacy of benzimidazole-based FabI inhibitors through the support of electronic circular dichroism and MM-GBSA techniques.

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis (FtFabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward FtFabI. The IC50 of benzimidazole (-)-1 is ∼100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (-)/(+)-1 and (-)/(+)-2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (-)-rotation for both compounds 1 and 2, while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with FtFabI show much tighter binding MM-GBSA free energies for S-1 and S-2 than for their enantiomers, R-1 and R-2, consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (-) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (-) to (S)- and (+) to (R)- for compounds 1 and 2, and to further evaluate structural changes to improve efficacy.

Structural characterization of Porphyromonas gingivalis enoyl-ACP reductase II (FabK).

Enoyl-acyl carrier protein (ACP) reductase II (FabK) is a critical rate-limiting enzyme in the bacterial type II fatty-acid synthesis (FAS II) pathway. FAS II pathway enzymes are markedly disparate from their mammalian analogs in the FAS I pathway in both structure and mechanism. Enzymes involved in bacterial fatty-acid synthesis represent viable drug targets for Gram-negative pathogens, and historical precedent exists for targeting them in the treatment of diseases of the oral cavity. The Gram-negative organism Porphyromonas gingivalis represents a key causative agent of the costly and highly prevalent disease known as chronic periodontitis, and exclusively expresses FabK as its enoyl reductase enzyme in the FAS-II pathway. Together, these characteristics distinguish P. gingivalis FabK (PgFabK) as an attractive and novel narrow-spectrum antibacterial target candidate. PgFabK is a flavoenzyme that is dependent on FMN and NADPH as cofactors for the enzymatic reaction, which reduces the enoyl substrate via a ping-pong mechanism. Here, the structure of the PgFabK enzyme as determined using X-ray crystallography is reported to 1.9 Šresolution with endogenous FMN fully resolved and the NADPH cofactor partially resolved. PgFabK possesses a TIM-barrel motif, and all flexible loops are visible. The determined structure has allowed insight into the structural basis for the NADPH dependence observed in PgFabK and the role of a monovalent cation that has been observed in previous studies to be stringently required for FabK activity. The PgFabK structure and the insights gleaned from its analysis will facilitate structure-based drug-discovery efforts towards the prevention and treatment of P. gingivalis infection.