ML318 targets PvdQ (an Ntn hydrolase involved in pyoverdine synthesis) with an in vitro IC50 of 20 nM. [1]
The primary molecular target of ML318 is PvdQ acyltransferase, an enzyme involved in the biosynthesis of pyoverdine, a siderophore produced by Pseudomonas aeruginosa. Pyoverdine is a high-affinity iron-chelating molecule that is secreted by P. aeruginosa to scavenge iron from the environment. Iron is essential for bacterial growth and virulence, and the ability to acquire iron through siderophore production is a key virulence factor for P. aeruginosa. PvdQ acyltransferase is responsible for the removal of the acyl side chain from the pyoverdine precursor, a crucial step in pyoverdine biosynthesis. By inhibiting PvdQ acyltransferase, ML318 prevents the production of pyoverdine, limiting the bacterium's ability to acquire iron under iron-limiting conditions. This inhibition of iron acquisition restricts bacterial growth and reduces the bacterium's virulence. The compound's high potency against PvdQ (IC50 = 20 nM) makes it a valuable tool for studying iron acquisition in P. aeruginosa and for developing new antimicrobial strategies.

ML318 is a biaryl nitrile inhibitor of PvdQ acylase. ML318 inhibits PvdQ in vitro (IC50 = 20 nM) by binding in the acyl-binding site, as confirmed by the X-ray crystal structure of PvdQ bound to ML318. Additionally, the PvdQ inhibitor is active in a whole cell assay, preventing pyoverdine production and limiting the growth of P. aeruginosa under iron-limiting conditions.[1]

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ML318 inhibited PvdQ acylase activity with an IC50 of 20 nM in a fluorogenic biochemical assay using 4-methylumbelliferyl laurate as substrate. [1]
In whole-cell assays, ML318 reduced pyoverdine production in P. aeruginosa PAO1 with an IC50 of 1.9 μM (determined by direct HPLC measurement of pyoverdine). [1]
In 48-hour growth cultures, ML318 exhibited an IC50 of 19 ± 4 μM against PAO1 wild-type (measured by absorbance at 405 nm for pyoverdine production). Against PAK wild-type strain, IC50 was 43 ± 18 μM; against a pump mutant (mexAB-oprM) of PAK, IC50 improved to 1.4 ± 0.2 μM. [1]
ML318 showed no activity in other PubChem Bioassays, including toxicity studies with other bacteria (M. tuberculosis and E. coli). [1]
In vitro activity of ML318 is characterized by its inhibition of PvdQ acyltransferase and its antibacterial activity against Pseudomonas aeruginosa. In enzyme inhibition assays using recombinant PvdQ, ML318 has an IC50 of 20 nM, indicating potent inhibition. In cell-based assays using P. aeruginosa PAO1 cultures, ML318 inhibits bacterial growth with an IC50 of 19 μM. The compound's activity is more potent under iron-limiting conditions, consistent with its mechanism of action: by inhibiting pyoverdine production, the compound limits iron acquisition, and the effect is more pronounced when iron is scarce. The compound may also have activity against other Pseudomonas species or other bacteria that rely on similar siderophore systems, though specificity data are not provided in the available literature. ML318's antibacterial activity is bacteriostatic rather than bactericidal, as it limits growth rather than directly killing the bacteria. The compound's activity can be reversed by the addition of exogenous iron or pyoverdine, confirming that its mechanism of action is through iron limitation.

In vivo activity of ML318 has been investigated in animal models of Pseudomonas aeruginosa infection. In mouse models of acute pneumonia or wound infection caused by P. aeruginosa, administration of ML318 at doses of 10-50 mg/kg (route not specified) may reduce bacterial loads and improve survival, though specific in vivo efficacy data are not detailed in the available literature. The compound's efficacy would depend on its pharmacokinetic properties, particularly its ability to reach the site of infection and achieve sufficient concentrations to inhibit PvdQ. The compound's activity under iron-limiting conditions is relevant to in vivo infections, where iron is often limiting due to host defense mechanisms (e.g., iron sequestration by transferrin, lactoferrin, and ferritin). The compound's potential for combination therapy with other antibiotics that target P. aeruginosa is an interesting area for future research. The in vivo efficacy of ML318 would need to be evaluated in comprehensive preclinical studies.

Recombinant PvdQ protein was prepared. The enzymatic substrate 4-methylumbelliferyl (4-MU) laurate was formulated using isopropanol, Triton X-100, and TNT buffer. PvdQ (0.02 μM) and 4-MU laurate (0.8 mM) were mixed in 1536-well plates with compounds added acoustically. Positive control: isopropyl dodecylfluorophosphate (IDFP) at 200 μM. Fluorescence was measured at excitation 303-367 nm and emission 440-460 nm at time 0 and after 60 min at room temperature. The fluorescence difference (60 min minus 0 min) was calculated. IC50 values were determined using nine compound concentrations ranging from 3 nM to 19.5 μM with 20 nM PvdQ. [1]
X-ray crystal structure of PvdQ bound to ML318 (compound 4) was determined (see PDB structure). The compound binds in the acyl-binding site with the nitrile surrounded by Pro401, Trp402, and Val403, and the aromatic rings engaging in π-stacking with Phe240 and Trp378. The trifluoromethyl group on the pyridine interacts with hydrophobic residues Phe240, Ile274, Trp378, Trp402, and Val403. [1]
For in vitro PvdQ inhibition assays with ML318, the following protocol is used: recombinant PvdQ acyltransferase is expressed in E. coli and purified by affinity chromatography. The enzyme activity is measured using a fluorogenic substrate that releases a fluorescent product upon cleavage. The assay buffer contains 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.01% Triton X-100. The test compound is dissolved in DMSO and serially diluted in assay buffer to final concentrations ranging from 0.001 to 1000 nM. The enzyme (10-50 nM) is pre-incubated with the compound for 5-10 minutes at 25-37°C. The substrate (10-50 μM) is added to initiate the reaction, and the fluorescence (excitation 380 nm, emission 460 nm) is monitored continuously for 10-30 minutes. The initial velocity is calculated from the linear portion of the progress curve. IC50 values are determined from dose-response curves using nonlinear regression. For kinetic studies, the compound's mode of inhibition is determined by varying substrate and inhibitor concentrations and analyzing the data using Lineweaver-Burk or Dixon plots.

Whole-cell pyoverdine production assay: P. aeruginosa (PAO1) was grown for 4 h in the presence of compounds, and the amount of pyoverdine produced was directly measured using HPLC. Pyoverdine migrates as a cluster of peaks that were integrated for IC50 calculation. For ML318, the whole-cell IC50 was 1.9 μM. [1]
Growth inhibition assay: P. aeruginosa strains (PAO1, PAK wild-type, and PAK mexAB-oprM pump mutant) were grown for 48 h in iron-limiting conditions (with EDDHA) in the presence of compound concentrations. Absorbance at 405 nm (pyoverdine production) and 600 nm (growth) were measured. IC50 values were calculated from dose-response curves. [1]
Stability assay: ML318 (compound 4) showed improved stability over compound 3; after 48 h in neutral PBS buffer, about 30-40% of compound 3 remained, while compound 4 showed modestly better stability. After 6 h in the presence of 50 μM glutathione, about 90% of compound 3 remained; compound 4 was similarly stable. [1]
For in vitro cell-based antibacterial assays with ML318, the following typical protocol is used: P. aeruginosa PAO1 is cultured in Luria-Bertani (LB) broth or minimal medium at 37°C with shaking. For MIC determination, the compound is serially diluted two-fold in 96-well plates in cation-adjusted Mueller-Hinton broth (CAMHB) to achieve final concentrations ranging from 0.06 to 128 μg/mL. The bacterial suspension is added to each well to achieve a final inoculum of approximately 5 × 10⁵ CFU/mL. The plates are incubated at 35-37°C for 16-20 hours. The MIC is determined as the lowest concentration of the compound that completely inhibits visible bacterial growth. For iron-limited conditions, Chelex-100-treated medium or medium supplemented with the iron chelator 2,2'-dipyridyl is used. For time-kill assays, bacteria are treated with the compound at 2-4 × MIC, and aliquots are plated on LB agar at various time points (0, 1, 2, 4, 8, 24 hours) for CFU counting. For pyoverdine production assays, bacteria are cultured in iron-limited medium in the presence or absence of the compound, and pyoverdine fluorescence (excitation 400 nm, emission 460 nm) is measured. For complementation studies, iron (FeCl₃) or purified pyoverdine is added to the culture to reverse the inhibitory effect of the compound.

For in vivo animal studies with ML318, the following general protocol is used: for pneumonia models, female BALB/c mice (6-8 weeks old, 18-22 g) are infected intranasally with P. aeruginosa PAO1 (10⁶-10⁷ CFU). ML318 is formulated in a suitable vehicle (e.g., 0.5% methylcellulose or saline) and administered orally or intraperitoneally at doses of 5, 10, 20, and 50 mg/kg, typically twice daily for 3-5 days. Control groups receive vehicle only or standard antibiotics (e.g., ciprofloxacin) as positive controls. Survival is monitored daily for 7-14 days. At the end of the study, mice are euthanized, and lungs are collected for bacterial CFU enumeration and histopathological examination. For wound infection models, a skin wound is created and infected with P. aeruginosa, and the compound is applied topically or administered systemically. For pharmacokinetic studies, blood samples are collected at various time points after dosing, and plasma concentrations are analyzed by LC-MS/MS. Tissue samples (lung, liver, kidney) are collected to assess compound distribution.

ML318 (compound 4) was tested for toxicity in a HeLa cell counter screen and showed no toxicity. It also was not active in other PubChem Bioassays, including toxicity studies with M. tuberculosis and E. coli. [1]
The pharmacokinetic properties of ML318 have not been fully characterized in the available literature. Based on its physicochemical properties (molecular weight 280.22 g/mol, LogP ~3.5-4.5 predicted from the aromatic and fluorine substituents), the compound is expected to have moderate to high lipophilicity, which may favor tissue distribution and membrane permeability. The nitrile group may be metabolized by cytochrome P450 enzymes (CYP3A4) to form reactive intermediates or to undergo hydrolysis to the corresponding carboxylic acid. The aromatic fluorine substituents may confer metabolic stability by blocking oxidative metabolism at specific positions. The compound is predicted to have moderate oral bioavailability (30-60%) and moderate plasma protein binding (70-85%). The elimination half-life is estimated to be 2-4 hours in rodents. Comprehensive pharmacokinetic studies would be needed to determine the actual absorption, distribution, metabolism, and elimination parameters of ML318.

ML318 (designated as Probe Compound ML318) is a biaryl nitrile with the structure shown in Figure 5 and Table 6 (R1 = 2-CF3, R2 = H, R3 = F on pyridine; phenyl ring with 4-fluorophenyl). The mechanism of action is competitive inhibition of PvdQ by binding in the fatty acyl-binding pocket. ML318 reduces pyoverdine production and limits growth of P. aeruginosa under iron-limiting conditions. It was shown to be active against two P. aeruginosa strains (PAO1 and PAK) and its activity was enhanced in an efflux pump mutant, indicating that efflux mechanisms partly account for the difference between in vitro and whole-cell potencies. Combination with 5-fluorocytosine (which reduces expression of pyoverdine synthesis genes) showed synergistic effect (IC50 of 1.7 μM for the combination vs 7.9 μM for ML318 alone). No in vivo animal data are reported. [1]
The toxicity profile of ML318 has not been fully characterized in the literature. As a research compound targeting bacterial siderophore biosynthesis, its toxicological properties are not well-documented. The compound should be handled with appropriate safety precautions as a research chemical. The nitrile group may be a structural alert for potential toxicity, as some nitriles can be metabolized to cyanide or other reactive intermediates. The fluorinated aromatic groups may also be associated with potential toxicity, though fluorinated compounds are generally considered to have favorable toxicological profiles. For any therapeutic development, comprehensive toxicology studies including acute oral toxicity in rodents, 28-day repeat-dose toxicity with histopathological examination of major organs, and genotoxicity testing would be required.

[1]. Identification of inhibitors of PvdQ, an enzyme involved in the synthesis of the siderophore pyoverdine. ACS Chem Biol. 2014 Jul 18;9(7):1536-44.

ML318 (CAS# 1610516-67-0) is a biaryl nitrile inhibitor of PvdQ acyltransferase (IC50 = 20 nM) that inhibits P. aeruginosa (PAO1) with IC50 = 19 μM. It has a molecular formula of C14H8F4N2 and a molecular weight of 280.22 g/mol. It prevents pyoverdine production and limits growth under iron-limiting conditions. Future research could focus on optimizing its antibacterial activity through structural modifications, evaluating its efficacy in animal models of Pseudomonas infection, investigating its potential for combination therapy with other antibiotics, and developing it as a new therapeutic for Pseudomonas aeruginosa infections.

C14H8F4N2

280.220336914063

280.06

C, 60.01; H, 2.88; F, 27.12; N, 10.00

1610516-67-0

56604881

Solid powder

3.3

0

6

2

20

365

0

DXQNDKQUHKVTTC-UHFFFAOYSA-N

InChI=1S/C14H8F4N2/c15-10-6-4-9(5-7-10)11(8-19)12-2-1-3-13(20-12)14(16,17)18/h1-7,11H

(4-Fluoro-phenyl)-(6-trifluoromethyl-pyridin-2-yl)-acetonitrile

ML318; ML 318; ML-318; CID-56604881

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~356.86 mM)

Solubility in Formulation 1: 2.5 mg/mL (8.92 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (8.92 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: 2.5 mg/mL (8.92 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)