Changes in the transcriptomics and proteomics landscape, along with the associated gene ontology.

(A-F) These panels highlight the common genes and proteins associated with individual compounds, with different colors indicating the distribution of these genes and proteins across various gene ontology terms.

Proteome Integral Solubility Alteration (PISA) analysis where significant intracellular target alterations are depicted in teal.

For compounds IV, IVa, and IVb, significant solubility changes indicate protein stabilization, whereas for IVj, the significant solubility changes indicate destabilization. (A)Volcano plot of protein solubility alterations for Compound IV at MIC. (B) Scatterplot showing protein solubility changes for Compound IV compared to baseline. (C) Volcano plot of protein solubility alterations for Compound IVa at MIC. (D) Scatterplot displaying protein solubility changes for Compound IVa compared to baseline. (E) Volcano plot of protein solubility alterations for Compound IVb at MIC. (F) Scatterplot indicating protein solubility changes for Compound IVb compared to baseline. (G) Volcano plot of protein solubility alterations for Compound IVj at MIC. (H) Scatterplot highlighting protein solubility changes for Compound IVj compared to baseline. (I) Volcano plot showing no significant alterations in protein solubility for Compound IVk at MIC. (J) Scatterplot displaying no significant alterations in protein solubility for Compound IVk compared to baseline.

Proteins with highest solubility alteration from the PISA assay

Interactions of compounds with essential proteins.

Significant alterations in protein solubility are highlighted in black, compared to baseline protein expression changes. The panel does not include compound IVK as no significant alterations in the solubility of essential proteins were detected for this compound. (A) Intracellular essential protein targets detected for compound IV. (B) Protein co-expression network among the essential proteins with significant alterations induced by compound IV. Highlighted nodes represent the distribution of the primary Gene Ontology (GO) term associated with these proteins. (C) Intracellular essential protein targets detected for compound IVa. (D) Protein co-expression network among the essential proteins with significant alterations induced by compound IVa. Highlighted nodes represent the distribution of the primary Gene Ontology (GO) term associated with these proteins. (E) Intracellular essential protein targets detected for compound IVb. (F) Protein co-expression network among the essential proteins with significant alterations induced by compound IVb. Highlighted nodes represent the distribution of the primary Gene Ontology (GO) term associated with these proteins. (G) Intracellular essential protein targets detected for compound IVj. (H) Protein co-expression network among the essential proteins with significant alterations induced by compound IVj. Highlighted nodes represent the distribution of the primary Gene Ontology (GO) term associated with these proteins.

Weighted Correlation Network Analysis (WGCNA) and the selection of drug-associated pathways.

(A) The clustering dendrogram and expression heatmap of genes are presented, identifying the WGCNA modules. (B) The correlation between the identified modules and different treatments is displayed. Modules that exhibit a significant association with traits, indicated by a correlation greater than 0.5 and a p-value less than 0.05. Red and green colors represent positive and negative correlations with gene expression, respectively. (C) The distribution of different WGCNA modules within the protein co-expression network of H. pylori is depicted.

Identification of target-associated pathways.

(A) Differential expression of proteins influenced by Compound IV. Upregulated proteins are represented in red, while downregulated proteins are represented in blue. The targets associated with Compound IV are highlighted within the brown module(Triangle). (B) Differential expression of proteins influenced by Compound IVa. Upregulated proteins are represented in red, while downregulated proteins are represented in blue. The targets associated with Compound IVa are highlighted within the brown module (Triangle). (C) Differential expression of proteins influenced by Compound IVb. Upregulated proteins are represented in red, while downregulated proteins are represented in blue. The targets associated with Compound IVb are highlighted within the pink module (Triangle). (D) Differential expression of proteins influenced by Compound IVb. Upregulated proteins are represented in red, while downregulated proteins are represented in blue. The targets associated with Compound IVb are highlighted within the megenta module (Triangle). (E) Differential expression of proteins influenced by Compound IVj. Upregulated proteins are represented in red, while downregulated proteins are represented in blue. The targets associated with Compound IVj are highlighted within the brown module (Triangle arrow symbol). (F) Differential expression of proteins influenced by Compound IVk. Upregulated proteins are represented in red, while downregulated proteins are represented in blue. The targets associated with Compound IVk are highlighted within the turquoise module (Triangle arrow symbol).

Biophysical assays validating changes in the targeted pathway.

(A) The time-kill curves were obtained by measuring the bacterial growth inhibition using an initial inoculum of 10^5 CFU/ml at various time points up to 24 hours. We observed bactericidal activity with all of our compounds. (B) Flow cytometry analysis was utilized to quantify the generation of reactive oxygen species (ROS), with fluorescence intensity plotted against counts. The presented data represents the compilation of three independent experiments. (C) Flow cytometry analysis was performed on both control and drug-treated H. pylori cells following the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labelling) assay protocol. The data is presented as a density plot of fluorescence intensity versus the cell counts. The presented data represents the compilation of three independent experiments. (D) Changes in the ATP production rate and The presented data represent eight replicates of a single experiment. (E) OCR of each sample (with 6 replicates) at 60 minutes was related to the basal OCR and compared with the same ratio obtained with the sole DMSO (as the untreated reference sample). Values are the means of the replicates ±SD.