LC-MS-based metabolomics for detecting adulteration in Tribulus terrestris-derived dietary supplements

Folin-Ciocâlteu, RP-HPLC (reverse phase-high performance liquid chromatography), and LC-MS (liquid chromatography-mass spectrometry) provide complementary information for describing cider (Malus spp.) apple juice

When I think about what it takes to keep a high-performing MS laboratory running—especially those dealing with tight timelines, strict regulations, and ever-expanding datasets—I’m reminded that the software we rely on isn’t just a tool. It’s the backbone of our workflows, our decisions, and ultimately, our results.Whether you’re in pharma, environmental testing, or food safety, managing the complexity of targeted mass spectrometry workflows while maintaining accuracy, compliance, and efficiency requires a lot of work.That’s where a purpose-built solution makes a difference. I’ve worked closely with labs that are navigating these challenges—and from the many conversations and experiences, I put together five main capabilities you should look for in your software when it comes to targeted MS quantitation:<h3>1. Simplified data and workflow management</h3>Too many labs are still relying on disconnected systems or folder-based setups that can lead to inconsistent methods and reports. When utilizing modern software solutions, everything—methods, templates, data, reports—can be centrally managed, reducing errors and training time while ensuring everyone is working from the same validated workflows.It’s a big plus if the software also supports multi-consensus reporting, making it easy to compare results across sequences and analyses for greater confidence in the data. By packaging complete workflows and setting predefined views, labs can streamline routine operations and ensure method repeatability, so anyone can run the analysis reliably and consistently.<h3>2. Controlled access in and out of the lab</h3>Many labs are still tied to legacy setups that make remote work challenging. Researchers often rely on standalone workstations that store data locally, making it hard to access critical results when off-site. Transferring data via USB drives or external hard disks not only adds inefficiency, but it can also raise serious security concerns. These outdated practices slow down collaboration and increase the risk of data loss or breaches.Hybrid work is here to stay. With the ability to control and access instruments and data, users can securely set up runs, review results, and manage workflows from anywhere. You’re no longer tied to a specific bench, workstation, or even building. Productivity doesn’t have to stop when you step out of the lab.“Managing all our instruments and work with data remotely via a centralized server means we don’t need to be in the laboratory to access our data.” – Dr. Kayleigh Arthur, Owlstone Medical Ltd.<h3>3. It’s built for MS workflows</h3>Whether you’re running triple quads, high-res accurate mass, or both—life’s much easier when working with software solutions that give you native instrument control and deep integration for targeted workflows. That includes support for MSⁿ, intact mass deconvolution, spectral libraries, and high-throughput quantitation.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_5_Things_to_Consider_When_Selecting_Software_for_Targeted_MS_Quantitation_9e5e9dc842.jpg" alt="Thermo Fisher Scientific: 5 Things to Consider When Selecting Software for Targeted MS Quantitation." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_5_Things_to_Consider_When_Selecting_Software_for_Targeted_MS_Quantitation_9e5e9dc842.jpg 245w," sizes="100vw" width="1536" height="411">Just as important is what happens after data acquisition. Integrated, built-in reporting capabilities—paired with customizable reporting options—should make it easy to flag results and automate the necessary calculations, all within the software. You need a powerful combination that can streamline review and ensures consistency across analyses.<h3>4. Scalability</h3>From small bioanalytical teams to global enterprises, can it be scaled up or down without the need for reinstallation or manual data transfer? It should be built to handle complex, multi-site deployments with ease.Add to that network resilience, automated data protection, and centralized updates—and you’ll get a system designed to grow with your organization.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_5_Things_to_Consider_When_Selecting_Software_for_Targeted_MS_Quantitation_2_8ec325678b.jpg" alt="Thermo Fisher Scientific: 5 Things to Consider When Selecting Software for Targeted MS Quantitation." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_5_Things_to_Consider_When_Selecting_Software_for_Targeted_MS_Quantitation_2_8ec325678b.jpg 245w," sizes="100vw" width="1200" height="660"><article><div><h3>5. Stress-free compliance</h3>Let’s face it, if you have to do it, compliance is non-negotiable, but it can be time-consuming. The system should include built-in features that support GxP, 21 CFR Part 11, and audit readiness—like audit trails, version control, and detailed user management.The ability to query and interrogate data within the system would ensure that data integrity requirements are met, delivering full traceability, and verification of data throughout its lifecycle. Your QA team will be very pleased!<h3>Conclusion</h3>Managing targeted MS quantitation shouldn’t feel like assembling a puzzle with missing pieces. A modern software should bring together everything you need—scalable infrastructure, powerful MS data tools, and built-in compliance features—into one cohesive solution. It must support your science, your team, and your business goals.If you’re ready to rethink how your lab handles MS data, this is the right time to explore what’s possible.<blockquote>Join our <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/webinar/5232">upcoming webinar</a> to learn about networked software for MS labs.Visit us on&nbsp;<a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/showcase/72003132/admin/dashboard/">LinkedIn:</a>&nbsp;#targetedquantitation #compliance #massspectrometry #control</blockquote></div></article><section><blockquote><h4>Laercio Fernandes</h4>Laércio Fernandes is a Product Marketing Manager for software at Thermo Fisher Scientific. With a background rooted in analytical chemistry, Laércio has over 20 years of experience in the analytical field with a focus on chromatography instrumentation, enterprise software and compliance. His background in sales and support has enabled him to have a good overview and understanding of different processes and challenges that laboratories may face, which has allowed him to better support our customers in order to improve their workflows and productivity.</blockquote></section>

5 Things to Consider When Selecting Software for Targeted MS Quantitation

In a recent blog post we discussed how <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/article/5073">centralized data management</a> enhances security and control in analytical laboratories. But beyond security, networked environments offer other architectural advantages that fundamentally change how data flows through the lab.One key benefit is the separation of data acquisition from processing and review—a structural shift that brings measurable improvements in performance, uptime, and compliance.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Decoupling_Acquisition_From_Data_Review_Unlocking_Flexibility_and_Efficiency_in_Chromatography_and_Mass_Spectrometry_Workflows_a982d7ba0d.jpeg" alt="Thermo Fisher Scientific Decoupling Acquisition From Data Review Unlocking Flexibility and Efficiency in Chromatography and Mass Spectrometry Workflows" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Decoupling_Acquisition_From_Data_Review_Unlocking_Flexibility_and_Efficiency_in_Chromatography_and_Mass_Spectrometry_Workflows_a982d7ba0d.jpeg 245w," sizes="100vw" width="1024" height="512"><h3>Maximizing instrument uptime</h3>In many labs, data acquisition and review occur on the same computer, which tends to be placed next to the instrument. This ties up the acquisition workstation long after a run is complete, especially when processing tasks—such as baseline correction, peak integration, or spectral deconvolution—are resource-intensive.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Thermo_Fisher_Scientific_Decoupling_Acquisition_From_Data_Review_Unlocking_Flexibility_and_Efficiency_in_Chromatography_and_Mass_Spectrometry_Workflows_304df97f03.jpg" alt="Thermo Fisher Scientific Decoupling Acquisition From Data Review Unlocking Flexibility and Efficiency in Chromatography and Mass Spectrometry Workflows" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Thermo_Fisher_Scientific_Decoupling_Acquisition_From_Data_Review_Unlocking_Flexibility_and_Efficiency_in_Chromatography_and_Mass_Spectrometry_Workflows_304df97f03.jpg 245w," sizes="100vw" width="1024" height="428"><article><div>Separating these tasks means the acquisition PC can be dedicated solely to capturing raw data, while automated transfer and processing occur on a separate system. &nbsp;If processing is the biggest bottleneck in your workflow, this has clear advantages.<h3>Enabling remote review and flexible access</h3>When data is stored centrally and accessed over the network, analysts can log in from remote locations or satellite labs to perform review and approval workflows.Central data storage also moves away from flat file sharing into a secure, user-controlled, archived storage location, which offers enhanced security and controlled access to archived dataDecoupling supports hybrid work environments and multi-user labs while supporting traceability through system audit trails and role-based access control.<h3>Scaling processing power independently</h3>Mass spectrometry data analysis—especially when using techniques like data-independent acquisition (DIA), MSⁿ workflows, or top-down proteomics—demands significant computational resources. These tasks benefit from multithreading, GPU acceleration, or distributed computing environments.By shifting processing to centralized servers or virtual machines, labs can scale compute resources as needed without overloading acquisition systems, risking&nbsp;data loss or even sample loss from crashes during data collection.<h3>Supporting collaborative review and shared methods</h3>Decoupled environments allow multiple users to access and process the same dataset simultaneously, without file duplication or manual data transfer. Analysts can apply shared processing templates, compare runs side-by-side, and annotate results in a common workspace.This is especially valuable for QA/QC teams performing multi-batch comparisons, method validation, or deviation investigations.Enhancing compliance and traceability.From a compliance standpoint, separating acquisition and processing improves data governance. Centralized environments can enforce version control, restrict unauthorized edits, and capture complete audit trails for every action taken—critical for GxP labs and 21 CFR Part 11 compliance. Review workflows can also be digitally signed and locked, ensuring that once approved, the data and metadata are still immutable<h3>Conclusion</h3>Decoupling acquisition from processing is a foundational change in how modern analytical labs operate. It reduces bottlenecks, increases system uptime, availability and enables advanced processing workflows to run on purpose-built infrastructure—without compromising compliance or productivity.<blockquote>You can see how Thermo Fisher Scientific is solving these challenges in <a target="_blank" rel="noopener noreferrer" href="https://www.thermofisher.com/content/lifetech/global/en/home/digital-solutions/ardia-platform/proteomics.html">Proteomics software solution</a>.</blockquote><blockquote>Visit us on&nbsp;<a target="_blank" rel="noopener noreferrer" href="https://www.linkedin.com/showcase/72003132/admin/dashboard/">LinkedIn:</a>&nbsp;#Ardia #ChromatographyDataSystemSoftware, #DataManagement</blockquote></div></article><section><blockquote><h4>Chris Knowles</h4>Chris Knowles began his career with a PhD in mammalian cell line engineering and over a decade as a Principal Scientist and applications specialist in biopharma and bioprocessing. With deep expertise in software solutions for LC-MS, MS/MS, and advanced mass spectrometry techniques, he focuses on biologics production and analytics, including emerging areas like gene therapy, oligonucleotide therapeutics, and viral vectors. Now, as a Product Marketing Manager for BioPharma Software at Thermo Fisher Scientific, Chris is dedicated to helping customers overcome analytical challenges by delivering powerful, compliant, and user-friendly software solutions.</blockquote></section>

Decoupling Acquisition From Data Review: Unlocking Flexibility and Efficiency in Chromatography and Mass Spectrometry Workflows

Multidrug-resistant Acinetobacter baumannii (MDR AB) poses a critical global health challenge due to limited treatment options and robust resistance mechanisms. Targeting bacterial bioenergetics, particularly ATP synthase, represents a promising alternative approach inspired by the success of bedaquiline against Mycobacterium tuberculosis.In this study, a library of trisubstituted pyridines was synthesized and evaluated for ATP synthase inhibition in A. baumannii. Four lead compounds demonstrated potent and selective inhibition, exhibiting strong antibacterial activity against both susceptible and MDR clinical isolates. Furthermore, these compounds acted synergistically with colistin, underscoring their potential as novel antibiotics to combat resistant A. baumannii infections.<blockquote><h4>The original article</h4><h5><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380">Synthesis and Evaluation of Pyridine-Based Antibacterial Agents that Inhibit ATP Synthase in Acinetobacter baumannii</a></h5>Angelina L. Dennison, Armaan Singh, Toni A. Marchlewski, Sierra N. Ghee, Kevin C. Gence, lAva G. Hammock, Sabrina Liu, Shaylla Wilson, Alexander P. L. Williams, Katie T. Ward, Thomas E. Meigs, P. Ryan Steed*, and Amanda L. Wolfe*ACS Omega 2025, 10, 39, 45823–45839<a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.5c06380">https://doi.org/10.1021/acsomega.5c06380</a><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/">CC-BY 4.0</a></h6></blockquote>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.Antimicrobial resistance─a leading cause of death globally─was responsible for &gt;4 million premature deaths in 2019, with projections suggesting this number could rise to over 10 million by 2050. <a href="javascript:void(0);">(1,2)</a> The nosocomial ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) contribute significantly to these deaths. Multidrug resistant A. baumannii (MDR AB), an opportunistic Gram-negative pathogen, poses a serious challenge due to a multitude of resistance pathways, including a robust outer membrane (OM), active efflux pumps, and exiguous aperture porins. Moreover, AB is considered one of the most difficult-to-treat ESKAPE pathogens due to its resistance to safety net antibiotics (including colistin, tigecycline, and carbapenems), which demonstrates the dire need for development of novel antimicrobials. <a href="javascript:void(0);">(3)</a>Recently, efforts in antibiotic discovery and development have shifted toward identifying new bacterial targets to overcome current resistance mechanisms in pathogens. One emerging target is ATP synthase, an essential enzyme in bacterial bioenergetics and all life (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#fig1">Figure 1</a>A). <a href="javascript:void(0);">(4−6)</a> F1Fo ATP synthase is a protein complex comprised of two rotary motors that catalyzes the final step in oxidative phosphorylation. <a href="javascript:void(0);">(7)</a> In bacteria, the membrane-embedded Fo motor is composed of stator ab2 subunits adjacent to a homooligomeric ring of c subunits (c10 in AB). <a href="javascript:void(0);">(8)</a> Rotation of the c-ring, driven by an electrochemical gradient of H+, is coupled to rotation of a central stalk within the cytoplasmic F1 motor (composed of α3β3γδε), which catalyzes phosphorylation of ADP to ATP. ATP synthase has proven to be a druggable antibiotic target through the success of the FDA-approved antitubercular drug bedaquiline (BDQ). <a href="javascript:void(0);">(9)</a> BDQ inhibits ATP production in Mycobacterium tuberculosis (MT) by binding to the H+ binding site at the subunit ac interface in Fo, halting ATP synthesis in the F1 complex and causing cell death. <a href="javascript:void(0);">(10−12)</a> BDQ was the first antibiotic to target bacterial energy metabolism and the first drug approved for MDR MT in over four decades. <a href="javascript:void(0);">(13)</a>Due to the success of BDQ against MT, our group <a href="javascript:void(0);">(14−17)</a> and others <a href="javascript:void(0);">(18−21)</a> have begun to explore whether functionalized quinolines can inhibit ATP synthase in other bacterial pathogens via a similar binding interaction with the Fo c subunit and act as antibiotics against resistant strains. One challenge of targeting ATP synthase in Gram-negative pathogens, like AB specifically, is that the Fo complex of ATP synthase is embedded in the inner membrane. Therefore, the drugs must first penetrate the negatively charged OM and then embed in the hydrophobic inner membrane to inhibit the enzyme. Recently, we have developed and interrogated a series of functionalized quinolines that are able to inhibit ATP synthase in AB and penetrate the OM to act as antibiotics against MDR AB strains. <a href="javascript:void(0);">(17)</a> Specifically, the quinoline core was substituted with a benzyl sulfide at the C1 position and a flexible and basic nitrogen-containing side chain at C2. Functionalization on the western portion of the quinoline increased enzyme inhibition and overall antibacterial activity against both susceptible and MDR AB clinical isolates. However, increasing steric bulk beyond a certain threshold decreased both enzymatic and bacterial activity significantly, which was attributed to overall larger steric bulk limiting the interactions with the c subunit and reducing membrane permeability. Quinoline WSA 261 (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#fig1">Figure 1</a>B) demonstrated the most potent antibacterial activity against both susceptible and MDR AB of the series. <a href="javascript:void(0);">(17)</a> To explore the necessity of the quinoline core and to interrogate the limitations on molecular flexibility and size on AB ATP synthase inhibition more broadly, we have synthesized a library of 34 novel trisubstituted pyridine analogs (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#fig1">Figure 1</a>C) and evaluated this library in enzymatic, antibacterial, and cytotoxicity assays to assess the viability of this series as potential antibacterial agents for treating AB infections.<h4>Methods</h4>Reagents and solvents were purchased reagent-grade and used without further purification. All reactions were performed in flame-dried glassware under an Ar or N2 atmosphere. Evaporation and concentration in vacuo was performed at 40–45 °C. TLC was conducted using precoated SiO2 60 F254 glass plates from EMD with visualization by UV light (254 or 366 nm). NMR (1H or 13C) were recorded on an Varian INOVA-400 MHz spectrometer or a Bruker AVANCE-400 MHz spectrometer at 298 K. Residual solvent peaks were used as an internal reference (CDCl3 with 0.1% TMS). Coupling constants (J) (H,H) are given in Hz. Coupling patterns are designated as singlet (s), doublet (d), triplet (t), multiplet (m) or quintet (qu). IR spectra were recorded on a <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library?mainauthorItems=1&amp;search=IRSpirit">Shimadzu IRSpirit FT-IR spectrophotometer</a> and measured neat. Low-resolution mass spectral data were acquired on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-2020">Shimadzu single quadrupole LCMS-2020.</a> High-resolution mass spectral Samples were analyzed with a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=3&amp;search=Q+Exactive+HF-X">Q Exactive HF-X</a> (ThermoFisher, Bremen, Germany) mass spectrometer. Samples were introduced via a heated electrospray source (HESI) at a flow rate of 10 μL/min. HESI source conditions were set as: nebulizer temperature 400 °C, sheath gas (nitrogen) 20 arb, auxiliary gas (nitrogen) 0 arb, sweep gas (nitrogen) 0 arb, capillary temperature 320 °C, RF voltage 45 V. The mass range was set to 100–1000 m/z. All measurements were recorded at a resolution setting of 120,000. Solutions were analyzed at 0.1 mg/mL or less based on responsiveness to the ESI mechanism.<a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1758"> Xcalibur</a> (ThermoFisher, Breman, Germany) was used to analyze the data. Molecular formula assignments were determined with Molecular Formula Calculator (v 1.3.0). All observed species were singly charged, as verified by unit m/z separation between mass spectral peaks corresponding to the 12C and 13C12Cc-1 isotope for each elemental composition.<h4>Results and Discussion</h4><h5>ATP Synthase Inhibition</h5>Once synthesized, the pyridine compounds were tested for their ability to inhibit ATP synthase activity using our previously reported luciferin/luciferase assay. <a href="javascript:void(0);">(17)</a> Briefly, endogenous electron transport chains of inverted inner membrane vesicles were energized with NADH to generate a proton gradient and to drive ATP synthesis. ATP was measured via luminescence from the luciferase-catalyzed oxidation of d-luciferin. Luminescence activities across increasing inhibitor concentrations were corrected for gradient independent background sources of ATP by comparison with a control containing the protonophore CCCP (carbonyl cyanide 3-chlorophenylhydrazone). The data were then fit to a four-parameter logistic dose–response curve, from which IC50 values and Hill coefficients were determined using a nonlinear least-squares regression.Assessing the SAR of ATP synthase inhibition, pyridines with H or Br at C4 exhibited the lowest inhibitory activity across all amine series (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsomega.5c06380/suppl_file/ao5c06380_si_001.pdf">Table S1</a>, <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#fig2">Figure 2</a> and <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsomega.5c06380/suppl_file/ao5c06380_si_001.pdf">S1</a>). Among compounds having C4 phenyl groups, the 4-methoxyphenyl, 3-fluoro-4-methoxyphenyl, and 4-methylphenyl analogs showed potent IC50 values across all amine types, while the 2,4-difluorophenyl, phenyl, and 4-fluorophenyl substituents generally demonstrated weaker inhibitory activity. Compounds WSA 290, 289, 275, 288, and 305 showed the strongest inhibitory activity among the pyridines, with IC50 values between 190 and 270 ng/mL. These in vitro IC50 values are significantly lower than that of WSA 261 (IC50 = 770 ng/mL), suggesting that the pyridine scaffold improves AB ATP synthase inhibitory activity. WSA 290, 289, and 288 all belonged to the C2 cyclopentyl piperidine substituted series, while WSA 275 belonged to the methyl piperazine series and WSA 305 belonged to the ethyl piperidine series. WSA 290 and WSA 275, the strongest performing compounds in their amine series, contained 3-fluoro-4-methoxyphenyl at C4; similarly, WSA 297 (IC50 = 270 ng/mL) with the same 3-fluoro-methoxyphenyl C4 substituent was the strongest performing compound within the benzyl pyrrolidine series. WSA 288 and WSA 305 had methoxyphenyl C4 substituents, and WSA 289 contained a methylphenyl group off C4. The presence of a methoxy group, in both ortho disubstituted and monosubstituted forms, on the C4 phenyl seems to confer stronger inhibition.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2025_10_39_45823_45839_Figure_2_Potent_inhibition_of_ATP_synthesis_activity_86ae703368.jpg" alt="ACS Omega 2025, 10, 39, 45823–45839: Figure 2. Potent inhibition of ATP synthesis activity. ATP synthesis activities of inverted membrane vesicles in the presence of 0–16 μg/mL inhibitor (log2 scale) are plotted relative to the DMSO control containing no inhibitor. Replicate measurements are shown as dots. The fitted dose response curves are shown as solid lines, and the dashed lines indicate the 95% confidence bounds of the fit." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2025_10_39_45823_45839_Figure_2_Potent_inhibition_of_ATP_synthesis_activity_86ae703368.jpg 245w," sizes="100vw" width="4151" height="2101"><h5>Computational Docking</h5>Computational docking was performed using GNINA and PLIP (see <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#sec4">Methods</a>). The protein (PDB ID: <a target="_blank" rel="noopener noreferrer" href="http://doi.org/10.2210/pdb7P2Y/pdb">7P2Y</a>) <a href="javascript:void(0);">(22)</a> was assigned protonation states using the H++ server, <a href="javascript:void(0);">(23)</a> compounds were protonated at pH 7 using Open Babel, <a href="javascript:void(0);">(24)</a> and both were prepared for docking using Autodock Tools. Docking was performed using GNINA with a fixed grid box centered around cAsp60. Poses from GNINA were then input into PLIP 2.3.0 to extract interaction level data.The strong inhibitors WSA 290, 289, 275, 288, and 305 all formed salt-bridges between their secondary or tertiary amines and cAsp60 (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/suppl/10.1021/acsomega.5c06380/suppl_file/ao5c06380_si_001.pdf">Table S2</a>). The conformations adopted also allowed for hydrogen bonding with the backbones of cPhe53, cGly57 (WSA 290), cMet64 (WSA 275), and aLeu257 (WSA 305). Additionally, cIle65, cLeu71, aIle242, aLeu245, aIle246, and aLeu257 formed a hydrophobic binding pocket that consistently interacts with these compounds. The enriched binding pocket for high affinity compounds is shown in <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#fig3">Figure 3</a>A, where residue-ligand contacts were computed using PLIP on the chosen GNINA poses for each compound. This binding pocket overlaps substantially with that predicted for our previous quinoline scaffold (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c06380#fig1">Figure 1</a>A), supporting a conserved binding mode centered around the cAsp60-mediated salt-bridge and a hydrophobic cavity across both chemotypes.<img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2025_10_39_45823_45839_Figure_3_A_The_putative_binding_site_in_the_ac_interface_of_A_baumannii_ATP_synthase_25731daecd.jpg" alt="ACS Omega 2025, 10, 39, 45823–45839: Figure 3. (A) The putative binding site in the ac interface of A. baumannii ATP synthase (PDB ID: 7P2Y) is shown at the interface of subunit a (left, pale cyan) and the c-ring (right, light blue). Residues in the binding pocket are colored by enrichment score, defined here as frequency of binding to strong inhibitors divided by frequency of binding to weak inhibitors, where strong inhibitors are those with IC50 &lt; 500 ng/mL and weak inhibitors are those with IC50 &gt; 5000 ng/mL. The residues shown as sticks are those with enrichment &gt;1 (i.e., frequent interaction with strong inhibitors) and cAsp60 (a key salt-bridge forming residue). (B) Computed binding poses of compounds WSA 276 (cyan), WSA 278 (yellow), and WSA 280 (magenta) in the methyl piperazine series with 4-dimethylamino, 4-methoxyphenyl, and 4-methylphenyl western fronts. (C) Computed binding poses of compounds WSA 288 (cyan), WSA 289 (magenta), and WSA 302 (yellow) in the cyclopentyl piperidine series with 4-methoxyphenyl, 4-methylphenyl, and phenyl western fronts, respectively. (D) Computed binding poses of compounds WSA 297 (yellow), WSA 298 (cyan), WSA294 (orange), and WSA296 (purple) of the benzyl pyrrolidine series, with 2,4-fluoromethoxyphenyl, 4-fluorphenyl, 4-phenyl, and 4-methoxyphenyl C4 substituents respectively." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2025_10_39_45823_45839_Figure_3_A_The_putative_binding_site_in_the_ac_interface_of_A_baumannii_ATP_synthase_25731daecd.jpg 159w," sizes="100vw" width="1868" height="1838"><h4>Conclusions</h4>As discussed, development of new antibiotics that overcome bacterial resistance in MDR AB is a critical need globally due to the rising rate of bacterial infections caused by this pathogen. Bacterial bioenergetics are underexplored drug targets that if properly and selectively inhibited could provide a robust area for antibiotic development, helping to slow the rate of resistance emergence. While the quinoline scaffolds derived from the antitubercular ATP synthase inhibitor BDQ have been the standard for inhibitor development of this type to date, limitations in molecular design and synthesis require new scaffolds to be explored.Through biochemical and antibacterial evaluation of our trisubstituted pyridine library we have demonstrated that the traditional quinoline core is not required for ATP synthase inhibition in AB, opening a new area of drug development from a more readily available starting material. Specifically, we explored the SAR of AB ATP synthase inhibition and found that enzyme inhibition is driven by both molecular size and interaction with key residues in the proposed binding site of the ac interface of Fo. However, despite the increase in enzyme inhibition compared to the traditional quinoline series, antibacterial activity was limited, and the most potent compounds were cytotoxic against mammalian cells indicating that there is room for improvement using strategic molecular design. Four pyridines, WSA 276, WSA 288, WSA 289, WSA 298, with variations at C2 and C4 but similar overall molecular size and flexibility demonstrated potent antibacterial activity against both susceptible and MDR AB clinical isolates due to selective inhibition of AB ATP synthase in ng/mL concentrations. Additionally, this set had the most favorable preliminary toxicity ratio when dosed in combination with colistin, making them the basis for future studies to improve activity and selectivity of the class.

Synthesis and Evaluation of Pyridine-Based Antibacterial Agents that Inhibit ATP Synthase in Acinetobacter baumannii

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Synthesis and Evaluation of Pyridine-Based Antibacterial Agents that Inhibit ATP Synthase in Acinetobacter baumannii

Decoupling Acquisition From Data Review: Unlocking Flexibility and Efficiency in Chromatography and Mass Spectrometry Workflows

5 Things to Consider When Selecting Software for Targeted MS Quantitation

Folin-Ciocâlteu, RP-HPLC (reverse phase-high performance liquid chromatography), and LC-MS (liquid chromatography-mass spectrometry) provide complementary information for describing cider (Malus spp.) apple juice

LC-MS-based metabolomics for detecting adulteration in Tribulus terrestris-derived dietary supplements

Upcoming events

Understanding quantification algorithms

36th MassSpec Forum Vienna 2026

Analytical Strategies for Peptide & Protein Therapeutics

Webinars

High-throughput stability screening strategies for antibody formulation

Big Scale, Big Wins: Prep Chromatography Explained

PFAS in Food & Food Contact Material