Identification of PKIS compounds inhibiting intracellular growth of Stm and Mtb. (a) Gating strategy for DsRed-bright Stm-infected HeLa cells to determine the percentage of infected cells. The negative control treated with DMSO and positive control treated with 97i are depicted in grey. (b) Rescreen of 201 PKIS compounds, 30 of which appear twice, to assess their impact on Stm bacterial burden, expressed as average z-scores of the DsRed-bright population. (c) Hit compounds with z-scores < − 2. PKIS compounds that reduced bacterial burden in both the primary screen and rescreen are depicted in green, while black dots represent compounds that were only effective in one of the two screens. Controls are depicted in grey. (d) Gating strategy for DsRed+ Mtb-infected MelJuSo cells. (e) Re-screen on Mtb-infected MelJuSo cells. (f) Hit compounds with z-scores < − 2 for Mtb. The screens were performed with three technical replicates, and error bars show standard deviations.

Identification of HDT targets against intracellular Stm through combined chemical and genetic inhibition of host kinases. (a) Each circle in the phylogenetic tree depicts host kinases that are relevant for HDT against intracellular Stm by combining data from chemical19,20 and genetic6 kinase inhibition screens. The effect of chemical inhibition of kinase targets is represented by circle size, which corresponds to the number of Stm hit compounds that inhibit a kinase by > 50%51. The effect of genetic inhibition of kinase targets, using a siRNA knockdown screen of host kinases in Stm-infected HeLa cells, is shown by color. The most relevant targets for Stm survival are shown in green, while likely off-targets are shown in black. White circles represent targets for which genetic inhibition data were not available. (b) All 11 Stm hit compounds were clustered according to chemotype and chemical similarity, using the Tanimoto coefficient as a similarity measure with cut-off value of 0.551. (c) Chemical structures of the 2-anilino-4-pyrrolidinopyrimidine Stm hit compounds GSK1379738A and GSK1379760A. (d) The level of inhibition of kinase targets shared by GSK1379760A and GSK1379738A at 1 μM. (e) Effect of knockdown of JAK2 and AAK1 on the bacterial burden of Stm-infected HeLa cells. (f) Chemical structures of the 4-anilinoquinoline Stm hit compounds GW557777X and GW560116X. (g) The level of inhibition of kinase targets shared by GW557777X and GW560116X at 1 μM. (h) Effect of knockdown of the same kinase targets on the bacterial burden of Stm-infected HeLa cells.

Identification of HDT targets against intracellular Mtb through combined chemical and genetic inhibition of host kinases. (a) Each circle in the phylogenetic tree depicts host kinases that are relevant for HDT against intracellular Mtb by combining data from chemical19,20 and genetic6 kinase inhibition screens. The effect of chemical inhibition of kinase targets is represented by circle size, which corresponds to the number of Mtb hit compounds that inhibit a kinase by > 50%51. The effect of genetic inhibition of kinase targets, using a siRNA knockdown screen of host kinases in Mtb-infected MelJuSo cells, is shown by color with the most relevant targets in Mtb survival shown in green in green for relevant targets involved in Mtb survival, while likely off-targets are shown in black. White circles represent targets for which genetic inhibition was not available. (b) All 17 Mtb hit compounds were clustered according to chemotype and chemical similarity, using the Tanimoto coefficient as a similarity measure with cut-off value of 0.551. (c) Chemical structures of Mtb hit compounds belonging to the chemotype of morpholino-imidazo/triazolo-pyrimidinones. (d) Inhibition of phosphatidyl inositol 3-kinases by morpholino-imidazo/triazolo-pyrimidinone hit compounds. (e) Effect of genetic inhibition of phosphatidyl inositol 3-kinases on the bacterial burden of Mtb-infected MelJuSo cells. (f) Chemical structures of Mtb hit compounds belonging to the chemotype of 2-aminobenzimidazoles. (g) Inhibition of selected kinases by 2-aminobenzimidazole Mtb hit compounds. Only the five shared kinases that gave the strongest effect on intracellular Mtb upon knockdown are shown (see Supplementary Figure 4 for the whole list of 51 shared targets). (h) Effect of knockdown of the same kinase targets on the bacterial burden of Mtb-infected MelJuSo cells.

Validation of PKIS hit compounds in cell lines and primary human macrophages. (a, b) The efficacy of the hit compounds was validated in CFU assays using lysates from Stm-infected HeLa cells (a) and Mtb-infected MelJuSo cells (b). The bacterial burden is expressed as a percentage of CFUs compared to the DMSO control. (c, d) Compound safety was assessed using an LDH-release assay with supernatant from Stm-infected HeLa cells (c) and Mtb-infected MelJuSo cells (d), with cell viability expressed as a percentage of the DMSO control and with 1% Triton X-100-treated cells corresponding to 0%. (e, f) To assess whether hit compounds act as antibiotics or host-directed therapeutics, direct antimicrobial effects were evaluated in cell-free cultures of Stm (e) and Mtb (f). The turbidity of the bacterial suspensions, as measured by absorbance at OD600, is given as a percentage of the DMSO control. (g) The efficacy of the hit compounds was validated in CFU assays using lysates from Stm-infected M1 (black circles, grey bars) and M2 (white circles, open bars) primary human macrophages. (h) An LDH-release assay was performed using supernatant from Stm-infected macrophages. (i) The efficacy of the hit compounds was validated in CFU assays using lysates from Mtb-infected M1 (black circles) and M2 (white circles) primary human macrophages. (j) An LDH-release assay was performed using supernatant from Mtb-infected macrophages.

Time kinetics and dose–response relationship of Stm hit compounds in Stm-infected HeLa cells. (a) HeLa cells were infected with bioluminescent Stm-lux, treated with Stm hit compounds or DMSO at different concentrations and followed over time by measuring emitted light, expressed as relative light units (RLU). DMSO was used at equal % (v/v). Gompertz growth curves were fitted using non-linear regression. (b) Bioluminescence, shown in blue, was measured for HeLa cells infected with Stm-lux after 18 h of treatment with Stm hit compounds at indicated concentrations. RLUs were normalized to the untreated control. Four-parameter logistic regression was used to determine the dose–response relationship between compounds and inhibition of Stm growth, and to determine the IC50 values of compounds. In addition, the cell supernatant was used to determine host cell viability, shown in grey, using LDH-release assays. Again, four-parameter logistic regression was performed to determine the LD50 values of compounds. The data comprises four independent technical replicates.

Testing of Stm and Mtb hit compounds in vivo in zebrafish embryo models. (a) Schematic representation of the zebrafish embryo toxicity model. Embryos were visibly inspected and scored for health with a score of 5 representing healthy embryos, 0 representing dead embryos, and scores in between representing embryos with one to four of the following conditions: malformed tail curvature, oedema, cranial malformations, no response to physical stimulation. (b) Representative images of embryos with malformations. (c-d) Zebrafish embryos were treated with Stm (c) and Mtb (d) hit compounds at different concentrations. Each square of the heat map represents the average health score of 20 embryos. (e) Schematic representation of the zebrafish embryo Stm-infection model. Embryos were infected with ~ 200 CFUs at 52 h post fertilization (hpf). (f) Representative image of Stm-infected zebrafish embryos treated with DMSO at 72 hpf. (g) Stm-infected zebrafish were treated with 0.1% (v/v) DMSO, 1 µM moxifloxacin, 3 µM GSK1379738A and other compounds at 10 µM. Each group comprises 38–42 embryos. (h) Schematic representation of the zebrafish embryo Mmar-infection model. Embryos were infected with ~ 200 CFUs at 28 hpf. (I) Representative image of Mmar-infected zebrafish embryos treated with DMSO at 120 hpf. (J) Mmar-infected embryos were treated with 0.1% DMSO, 200 µM rifampicin and other compounds at 10 µM. Each group comprises 21–32 embryos. Statistically significant differences are indicated by *p < 0.05 and ***p < 0.001.