Rational design of biodegradable sulphonamide candidates treating septicaemia by synergistic dual inhibition of COX-2/PGE2 axis and DHPS enzyme

Abstract A new series of co-drugs was designed based on hybridising the dihydropteroate synthase (DHPS) inhibitor sulphonamide scaffold with the COX-2 inhibitor salicylamide pharmacophore through biodegradable linkage to achieve compounds with synergistic dual inhibition of COX-2/PGE2 axis and DHPS enzyme to enhance antibacterial activity for treatment of septicaemia. Compounds 5 b, 5j, 5n and 5o demonstrated potent in vitro COX-2 inhibitory activity comparable to celecoxib. 5j and 5o exhibited ED50 lower than celecoxib in carrageenan-induced paw edoema test with % PGE2 inhibition higher than celecoxib. Furthermore, 5 b, 5j and 5n showed gastric safety profile like celecoxib. Moreover, in vivo antibacterial screening revealed that, 5j showed activity against S.aureus and E.coli higher than sulfasalazine. While, 5o revealed activity against E.coli higher than sulfasalazine and against S.aureus comparable to sulfasalazine. Compound 5j achieved the target goal as potent inhibitor of COX-2/PGE2 axis and in vivo broad-spectrum antibacterial activity against induced septicaemia in mice.


2.4.
In silico prediction of the physicochemical properties, drug likeness score, pharmacokinetics, toxicity profile and ligand efficiency metrics.
Early prediction of the physicochemical and pharmacokinetic properties of new drug candidates is an infrastructure for lead optimization as well as drug development process [1].
The values presented in the topographical polar surface area (TPSA; a sum of polar atoms' surfaces: a descriptor for drug absorption, penetrability and bioavailability) showing a value of146.45 Å 2 for each compound (≤140 Å 2 ). These results indicate good oral bioavailability and an acceptable molecular flexibility.
Additionally, the percentage of absorption (%ABS; calculated as %ABS = 109-0.345 × TPSA) [5], as well as the solubility of the compounds were determined. all the tested compounds showed % ABS range 58.47-63.07%, which indicated a considerable bioavailability upon oral administration and their solubility values ranging from 0.16-11.51mg/L, satisfying the solubility requirement (>0.0001 mg/L) [6,7]. The drug-likeness score values showed that all the evaluated compounds gave positive values ranging from 0.05 to 0.07 which were comparable with sulfasalazine (0.07).
Furthermore, ADME properties were also predicted using Pre-ADMET software and the results revealed that all the compounds showed low cell permeability in Caco-2 cell model (

In vitro COX-1 and COX-2 enzymatic assay
All the synthesized target compounds were screened for their COX-1 and COX-2 inhibitory activities using Cayman colorimetric COX (ovine) inhibitor screening assay kit (Catalog No. 560131) supplied by Cayman chemicals, Ann Arbor, MI, USA. According to the manufacturer instructions and a previously reported method[55,56].The testing procedures for determining IC50 values of the tested compounds and reagents were prepared.

carrageenan-induced paw edema in mice
Swiss mice of both sexes; males and females excluding pregnant females weighing about (20-25 g) were purchased from EgyVac (Giza, Egypt) and kept in a 12 h light/dark cycle under standard conditions of temperature (24 -26 ˚C) and humidity (50 ± 10%) with free access to food and water for 1 week before starting the experiments for adaptation. All the experimental procedures were carried out regarding to the ethical principles of the Institutional Animal Care and Use Committee (IACUC) of faculty of science, Helwan University, Egypt (approval no. hU2021/Z/AEA121-02).
Mice were divided into seven groups of five mice per each; the first group acted as a negative control whose mice were administered 0.5% sodium carboxy methyl cellulose solution while the second group was given celecoxib (10 µmol/kg) as a positive control in addition to the third group

Determination of ED50
Compounds 5b, 5j, 5n and 5o were further screened at doses of 5, 10, 20, 30 and 40µmol/kg body weight and the dose-response curves was used to determine the ED50 (median effective dose) values by measuring the inhibition of the edema volume after 8 hours of carrageenan injection[58].

Estimation of rat plasma prostaglandin E2 (PGE2)
Five blood samples were collected from rats 8hours after carrageenan injection and centrifuged to separate plasma, immediately frozen and stored until use. A competitive immunoassay procedure was applied for quantitative determination of PGE2 in biological fluids with EIA PGE2 kit (Aldrich, Steinheim, Germany). After incubation at room temperature, a monoclonal antibody to PGE2 in kit was used to bind to PGE2 in the sample competitively. Excess reagents were washed away, and then the substrate was added and incubated for short time until generation of yellow color. The optical density was recorded using a microplate reader DYNATech, MR 5000 (Dynatech Industries Inc., McLean, VA) at 450nm, and expressed in pg/ml[59].

S36
The acute gastric ulcerogenic effect of compounds 5b, 5j, 5n and 5o in adult male Wistar rats was evaluated [58]. Rats weighing from 200 to 250 g were divided into seven groups of five rats each and fasted for 12 h before carrying out the experiment. Control group received only the vehicle saline, while other groups received celecoxib and Diclofenac sodium as reference drugs as well as the test compounds orally at a dose of 30µmol/kg body weight, dosed twice at 4h interval. After Six hours of the treatment of the last dose they were killed by ether inhalation, their stomachs were removed, opened along the greater curvature, washed under running water, fixed in saline solution and examined for the presence of hyperemia, hemorrhage or gastric ulcers. In addition, histopathological examination was also carried out to confirm the inflammatory reaction degree in the gastric layers of the treated rats' stomachs.  (Table 4, page S5 supplementary file).

Minimal inhibitory concentration (MIC) measurement
Two fold serial broth dilution method[60] was used to measure the minimal inhibitory concentrations (MIC) of the test compounds. A suitable broth: 24 h at 37˚C was used to grow the test organisms. Two fold serial dilutions of solutions of the test compounds were prepared using 200, 100, 50, 25, 12.5, 6.25 and 3.125 µg/mL. The tubes were then inoculated with the test S37 organisms; each 5 mL received 0.1 mL of the above inoculum and were incubated at 37˚C for 48 h.
Then, the tubes were observed for the presence or absence of microbial growth. The MIC values of the prepared compounds are listed in (Table 5, page S5 supplementary file).

Minimal bactericidal concentration (MBC) measurement
MBC tests were always measured following to the MIC as follows[63]: A loop-full from the tube that did not show visible growth (MIC) was spread over a quarter of Müller-Hinton agar plate.
After incubation for 18 h, the plates were examined for growth. Again, the tube containing the lowest concentration of the test compound that failed to yield growth on subculture plates was judged to contain the MBC of that compound for the respective test organism ( Table 5, page S5 supplementary file).

In vivo antibacterial screening in mice (Bacteremic infection)
To investigate the in vivo antibacterial activity of compounds 5b, 5j, 5n and 5o against E.coli and S. aureus using sulfasalazine as a reference drug, 352 mice of both sexes (except pregnant females) with weights ranging from 20 to 25gm were divided into 14 groups; 7 groups for E.coli (5b group, 5j group, 5n group, 5o group, sulfasalazine group, positive control and negative control) and the other 7 groups for S. aureus. The 32 mice in 5b group were divided into 4-dose group each group containing 8 mice of equal numbers of males and females. Likewise for 5j group, 5n group, 5o group and sulfasalazine, for positive and negative control groups, each contains 8 mice; half males and half females. The mice in each dose group were subjected to intragastric administration of different dosage (100, 300, 500, 600 µmol/kg) of compound 5b and so on for 5j, 5n, 5o and Results were displayed as ED50 and 95% confidence limit calculated for each group using doseresponse curves.

Molecular
Operating  RMSD values; conformity of the binding forces with the co-crystalized ligand, celecoxib. Finally, the best pose was isolated, placed in the active site, and saved as a picture to get it exported as JPEG file.

4.4.
In silico prediction of physicochemical properties, drug likeness score, pharmacokinetics, toxicity profile and ligand efficiency metrics S39 In the present study, prediction of the physicochemical properties was performed using Molinspiration chemoinformatic server, pharmacokinetics by Pre-ADMET calculator, drug likeness score and toxicological effects by Osiris property explorer.