Chemical degradation of Human p38-MAPK reveals it as a potential host target to combat parasitic infections by Leishmania donovani and Plasmodium falciparum

Abstract

The interplay between host and parasite determines parasite burden and disease outcome. Parasite exploits host signaling pathways like p38-MAPK for its survival and pathogenesis. Here we have used NR-7h, a proteolysis-targeting chimera (PROTAC) targeting human p38-MAPK to assess p38-MAPK’s role in Leishmania donovani and Plasmodium falciparum infection in their respective host cells. NR-7h degrades host p38-MAPK in a time- and dose-dependent manner. Degradation of host p38-MAPK by NR-7h reduces parasite load in host cells dose-dependently, implicating the role of p38-MAPK in parasite survival. During Leishmania infection, the modulation of cytokine profiling and oxidative burst upon NR-7h mediated degradation of host p38-MAPK is further correlated with parasite death. The effect of host p38-MAPK degradation by NR-7h with Amphotericin B enhances the efficacy of parasite-directed therapy. For Plasmodium infection, growth inhibition and invasion assays reveal impaired growth and merozoite invasion, suggesting that host p38-MAPK signaling contributes to both parasite invasion and intraerythrocytic development. This study underscores the importance of host p38-MAPK for L. donovani and P. falciparum progression and highlights NR-7h’s potential in antiparasitic therapy by targeting this pathway.

PROTAC-mediated degradation of host p38-MAPK by NR-7h limits Leishmania donovani and Plasmodium falciparum infection by modulating immune responses, highlighting host-directed signaling pathways as promising antiparasitic therapeutic targets.

Introduction

Leishmania donovani (L. donovani), a protozoan, causes Visceral Leishmaniasis (VL) that has been classified as a threat to human beings, causing loss of weight and fever with spleen and liver swelling^1,2. Annually, around 50,000–90,000 new cases of VL are reported worldwide³. The disease outcome always depends upon the early response from the host cells to encounter the pathogen, including the production of pro-inflammatory responses^4,5, generation of oxidative bursts^6,7, and phagocytosis^8,9. However, it is well reported that Leishmania parasites evade host immune responses by modulating host factors/signaling that includes more production of anti-inflammatory cytokines^10,11, induction of autophagy^12–15, and hijacking of post-translational modifications like Sumoylation¹⁶. Plasmodium falciparum (P. falciparum) parasitizes human erythrocytes and causes malaria, which is a global health burden with an estimated 249 million cases and about 6 million deaths in 2023¹⁷. The complex life cycle, high polymorphism, immune evasion, and host modulation are some of the challenges that impede the Malaria elimination program. Many efforts have been made to develop therapeutics targeting parasites, however, due to the development of drug resistance, these therapeutics are not effective^18–20. To overcome this issue, there is an urgent need to investigate host targets to develop effective therapeutics.

The human p38-MAPK pathway plays a critical role in various cellular processes such as inflammation, cell growth, differentiation, and cell death^21,22. Upon activation, p38-MAPK modulates pro-inflammatory cytokines, chemokines, and adhesion molecules that are responsible for recruiting the immune cells at the site of infection²³. Also, p38-MAPK modulates the expression of the antimicrobial peptides to improve the phagocytosis process of immune cells and thus a better presentation of antigens to major histocompatibility complexes²⁴. Moreover, p38-MAPK has served as an adjunctive therapy for the management of inflammatory disorders and immune responses induced to combat infections^25,26. There are reports on the inhibition or modulation of p38-MAPK activity as a potential enhancing agent for an appropriate immune response²⁷. At the cellular level, host p38-MAPK signaling is known to be modulated during parasite infections, which further emphasizes the need to study the disease pathogenesis in the absence of host p38-MAPK. In the context of Leishmaniasis, it is reported that human p38-MAPK also plays a role in inflammation and immunomodulation, thus regulating the progression and severity of infection of the Leishmania parasites^28,29. Studies have shown that p38-MAPK activity modulates the intracellular survival and multiplication of Leishmania parasites in human macrophages^30–33. p38-MAPK is a key stress-activated kinase involved in macrophage immune responses, regulating cytokine production, inflammation, and host defense mechanisms during intracellular parasitic infections. Since Leishmania actively modulates host signaling pathways to ensure its survival, targeting p38-MAPK offers a promising strategy to disrupt parasite persistence.

Plasmodium falciparum infection in human erythrocytes also involves constant interaction between host and parasite. Multiple studies have shown that during the asexual stage, the parasite modulates host signaling pathways for the disease progression^34–36, highlighting the targets for host-directed therapies. The parasite exports its protein into the erythrocytes, resulting in remodeling of the erythrocyte and alteration in the architecture of the erythrocyte membrane³⁷. By doing so, the parasite exploits the host cell for its own growth and survival, including nutrient uptake³⁸, presentation of parasite antigen, for instance, the presentation of PfEMP1 on erythrocyte membrane and its interaction with human endothelial receptor ICAM-1 resulting in increased cytoadherence of parasite-infected erythrocytes³⁹, and interaction with cytoskeletal proteins⁴⁰, affecting the normal erythrocyte physiology. These modulations help the parasite to evade the immune system, increase its virulence, and support its survival within the host cell. Mature human erythrocytes lack nuclei and organelles and thus fail to maintain homeostasis by genetic regulation. In normal erythrocyte physiology, p38-MAPK maintains membrane stability and responds to oxidative or osmotic stress. Under hyperosmotic stress, p38-MAPK activation results in the induction of eryptosis, and its inhibition by p38-MAPK inhibitors results in the rescue of erythrocytes from classical hallmarks of eryptosis, including phosphatidylserine exposure, intracellular calcium levels, and ROS⁴¹. Its activation is tightly regulated and involves phosphorylation by upstream kinases, including MAP kinase kinase(MKK3/6)⁴², in response to stress signals, leading to downstream effects such as phosphorylation of cytoskeletal and membrane proteins. Under pathological conditions such as malaria, erythrocyte p38-MAPK assumes a more dynamic and stage-responsive role, as parasite development induces substantial remodeling of host red-cell signaling networks. Phospho-proteomic studies of Plasmodium falciparum–infected erythrocytes have shown that several host kinases, including stress-activated MAPKs, undergo temporal, stage-specific changes in phosphorylation during the ring, trophozoite, and schizont stages³⁴, indicating an active rewiring of host signaling pathways rather than a static stress response. These findings collectively suggest that p38-MAPK functions beyond its classical physiological role and participates in parasite-modulated host-cell remodeling, further supporting its consideration as a host-directed therapeutic target^43,44. Therefore, targeting human p38-MAPK could be a potential therapeutic strategy to impede the disease progression caused by the sister parasites Leishmania and Plasmodium.

Due to the selectivity issues and high toxicity, many p38-MAPK inhibitors have been unsuccessful at the clinical trial stage⁴⁵. Recently, proteolysis-targeting chimeras or PROTACs have emerged as a new therapeutic strategy for targeted protein degradation owing to their high specificity. A PROTAC is an innovative therapeutic modality that takes advantage of bifunctional molecules that selectively target proteins for degradation. PROTACs recruit an E3 ubiquitin ligase to bind with the target protein; it ubiquitinates and degrades it through the proteasome^46,47. PROTACs fundamentally differ from traditional inhibitors in that, while a conventional inhibitor can inhibit the protein’s function, PROTACs degrade the protein inside the cell. This can, therefore, allow disease treatment by degrading targeted proteins rather than suppressing their activity. Moreover, PROTACs are beneficial to target proteins conventionally considered “undruggable”⁴⁸. While originally developed for cancer, PROTAC technology has recently been repurposed to develop multiple therapeutic interventions, including cardiovascular and metabolic disorder⁴⁹, neurodegenerative disorders^50,51, and antiviral therapies⁵². For example, a PROTAC based on the CDK inhibitor SNS032 (THAL-SNS032) successfully degraded the host kinase CDK9 and inhibited replication of Human cytomegalovirus (HCMV), as well as other viruses including SARS-CoV-2, highlighting the potential of host-targeted degradation to block viral replication⁵³. In another case, PROTACs engineered from the HCV protease inhibitor VX-950 (targeting the viral NS3 protease) were able to degrade NS3 protease, including drug-resistant NS3 mutants in human cell culture, offering a route to overcome viral resistance⁵⁴.

Using the PROTAC technology, we have explored a one-of-its-kind molecule, NR-7h PROTAC, for targeted degradation of p38-MAPK⁵⁵. NR-7h utilizes pomalidomide for the recruitment of cereblon E3 ubiquitin ligase⁵⁶ and a selective and potent inhibitor of p38-MAPK, PH797804⁵⁷, for targeting and subsequent degradation of p38-MAPK⁵⁵, which plays a vital role in the invasion and survival of the L. donovani and P. falciparum in their respective hosts. This approach might be more effective with specificity compared to present modes of treatment for visceral leishmaniasis and malaria, as it reduces the risk associated with potential drug resistance and toxicity in side effects.

Results

Degradation of human macrophage p38-MAPK via PROTAC molecule, NR-7h

To investigate the activation levels of human macrophage p38-MAPK upon L. donovani infection in macrophages, we infected THP-1 macrophages with 20 MOI of L. donovani promastigotes over varying time intervals. The activation levels of phospho-p38-MAPK (P-p38-MAPK) were monitored by western blotting at different time points. The data show no significant alterations in the activation levels of P-p38-MAPK in the infected macrophages compared to the uninfected macrophages at 2 h and 4 h post-infection. However, a significant upregulation (~1.5-fold) in the P-p38-MAPK activation levels was observed in infected macrophages at 6 h post-infection. Interestingly, after 12 h of infection, the activation levels of P-p38-MAPK were found to be significantly downregulated (~1.5-fold) in the infected macrophages compared to uninfected macrophages (Fig. 1A), suggesting the activation of human macrophage p38-MAPK only during the early stages of L. donovani invasion that was subsequently inactivated at later time points of infection.

Fig. 1. Infection-induced activation and NR-7h-mediated proteasomal degradation of p38-MAPK.

A Activation of p38-MAPK in infected macrophages. PMA-differentiated THP-1 macrophages were infected with 20 MOI of L. donovani for varying durations (2, 4, 6, and 12 h). Cell lysates were prepared from the infected macrophages. Western blotting was employed using a specific antibody to quantify the expression of P-p38-MAPK and p38-MAPK. Data presented is the mean from n = 2 biologically independent experiments. B–D Degradation of p38-MAPK by NR-7h. PMA differentiated THP-1 macrophages were treated with different concentrations of NR-7h concurrently with the stimulation of 100 ng/mL of LPS for 24 h (B) and 1, 3, and 6 h (C). Western blotting was used to evaluate the levels of p38-MAPK expression. Untreated cells were used as a control. Data presented is the mean from n = 3 biologically independent experiments. D Imaging flow cytometry was used to evaluate the fluorescence levels of (i) p38-MAPK, (ii) P-p38-MAPK (iii) an image panel of macrophages. Side Scatter channel (SSC) showing light scattered at 90° from the laser beam, representing the internal complexity and granularity of the cell. The SSC signal is pseudo-colored pink in the Amnis IDEAS software and is used for morphological visualization, not as a fluorescence control. Data presented is the mean from n = 2 biologically independent experiments. B, C GAPDH served as the loading control. Data from one of three experiments were presented. Band intensities were analyzed using ImageJ software and plotted using GraphPad Prism 8. Statistical analysis utilized the unpaired t-test with Welch’s correction, with significance levels denoted as *, **, and *** for p-values less than 0.05, 0.01, and 0.001, respectively. E The half-maximal degradation concentration (DC₅₀) of NR-7h was calculated and plotted in GraphPad Prism 8. F Macrophages were treated with NR-7h (1 µM) in the presence and absence of 20 µM MG132 proteasome inhibitor. Western blotting was used to evaluate p38-MAPK expression with GAPDH as the loading control. Data presented is the mean from n = 2 biologically independent experiments.

To study the functional role of human macrophage p38-MAPK during L. donovani infection, we applied an advanced approach of utilizing the PROTAC molecule, NR-7h. THP-1 macrophages were treated with a range of concentrations of NR-7h for 24 h with the stimulation of 100 ng/mL of LPS. Since p38-MAPK is a stress-responsive kinase, LPS stimulation provides a physiological inflammatory trigger that increases its expression/activation, enabling the measurement and interpretation of the pathway’s functional role.

To check the degradation ability, p38-MAPK protein levels were monitored by western blotting. Significant degradation of p38-MAPK (~2-fold) was observed upon the treatment of NR-7h across concentrations ranging from 0.25 μM to 10 μM at 24 h post-treatment (Fig. 1B) while no effect was observed in the level of a closely-related kinase, Extracellular signal-Regulated Kinase 1/2 (ERK1/2) in 0.01 μM to 10 μM NR-7h treated macrophages (Supplementary Fig. 1), thus validating the specificity of NR-7h. Furthermore, we evaluated the degradation of human macrophage p38-MAPK upon the treatment of NR-7h at shorter time points (Fig. 1C). We found that upon treatment of NR-7h at the concentrations of 0.25 μM and 1 μM for 1 h, 3 h, and 6 h in the macrophages, the maximal degradation of p38-MAPK was observed upon treatment of 1 μM of NR-7h for 6 h in a concentration- and time-dependent manner. We also observed a lower level of p38-MAPK and P-p38-MAPK in 1 μM NR-7h-treated macrophages compared to untreated macrophages using imaging flow cytometry (Fig. 1D). The half-maximal degradation concentration (DC50) that reduce the target protein level by 50% relative to untreated control was noted to be 162.9 nM in NR-7h treated macrophages (Fig. 1E). Further, to confirm the ubiquitin-proteasome-dependent mechanism of NR-7h, THP-1 macrophages were treated with 1 µM NR-7h in the absence and presence of 20 µM MG132⁵⁵, a proteasomal inhibitor. Immunoblotting revealed that the p38-MAPK degradation was significant (~2-fold) in NR-7h-treated macrophages in the absence of MG132 (Fig. 1F).

NR-7h-mediated degradation of human macrophage p38-MAPK reduces the parasitic burden of L. donovani

To check the cytotoxic effect of NR-7h on macrophage viability, we performed an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and found no significant alterations in the viability of macrophages upon treatment with various concentrations of NR-7h (Fig. 2A). As depicted in Fig. 1A, the expression level of P-p38-MAPK was upregulated at 6 h post-L. donovani infection, therefore, we further investigated the role of human macrophage p38-MAPK in the invasion of L. donovani. Different concentrations of NR-7h were employed to degrade human macrophage p38-MAPK for 6 hr followed by the infection of L. donovani parasites. The intracellular parasite load was calculated by quantifying the relative expression level of the kinetoplastid minicircle housekeeping gene (JW gene) of L. donovani using quantitative real-time PCR (qRT-PCR). The maximum clearance of the intracellular parasites was observed at the concentration of 0.75 μM (~16-fold), and 1 μM (~8-fold) of NR-7h in infected macrophages. (Fig. 2B). Confocal microscopy was also used to count the number of intracellular amastigotes and to calculate the infectivity rate in NR-7h-treated-infected macrophages as compared to the control group (untreated-infected macrophages) (Fig. 2C(i)). A significant reduction of L. donovani infected cells was observed upon the treatment of NR-7h on the concentrations of 0.5 μM and 1 μM (Fig. 2C(ii)). Similarly, a significant reduction in intracellular amastigotes was observed in a dose-dependent treatment of NR-7h (Fig. 2C(iii)). These results underscore the pivotal role of human macrophage p38-MAPK in favoring L. donovani invasion dynamics.

Fig. 2. Effect of NR-7h on the parasite burden in infected macrophages.

A PMA-differentiated THP-1 macrophages were treated with varying concentrations of NR-7h for 72 h. The viability of cells was assessed using the MTT assay. B, C PMA-differentiated THP-1 macrophages were treated with different concentrations of NR-7h in conjunction with the stimulation of 100 ng/ml LPS for 6 h, followed by infection with 20 MOI of L. donovani for 24 h. Untreated infected macrophages served as the control group. B RNA was isolated to quantify the expression of the JW gene using qRT-PCR. C (i) Parasite load was measured by using Propidium iodide (PI) staining via confocal microscopy. Scale bar, 20 µm. (ii) Percentage of infected macrophages and (iii) Percent parasitemia were plotted using GraphPad Prism 8. Data presented is the mean from n = 2 biologically independent experiments.

Further to confirm the role of human p38-MAPK in L. donovani pathogenesis, a specific siRNA was used to knock down p38-MAPK. Transfection efficiency was checked on both transcript and protein levels. The maximum knockdown of p38-MAPK levels was observed with 50 nM of sip38-MAPK, with >3-fold at transcript (Fig. 3A(i)) and >2-fold at protein level (Fig. 3A(ii)) compared to control (Mock siRNA). The effect of sip38-MAPK (50 nM)-mediated knockdown, NR-7h (1 μM) mediated degradation, and the chemical inhibition by a p38-MAPK specific inhibitor PD 169316⁵⁸ (1 μM) of p38-MAPK on the parasite load was also observed in L. donovani infected macrophages using confocal microscopy (Fig. 3B) and at transcript level (Fig. 3C). A significant reduction in parasite load was observed in both sip38-MAPK-mediated knockdown and NR-7h mediated degradation of p38-MAPK while no changes were observed with the inhibitor PD 169316 compared to control (Untreated-infected macrophages). The data suggest that the depletion of p38-MAPK, either by siRNA or degradation by PROTAC, significantly impairs parasite survival.

Fig. 3. Role of human p38-MAPK on the parasite burden in infected macrophages.

A PMA-differentiated THP-1 macrophages were transfected with two concentrations of siP38-MAPK for 36 h. The transcript levels (i) and the protein levels (ii) of p38-MAPK were assessed. B (i) Parasite load was measured by using Propidium iodide (PI) staining via confocal microscopy. Scale bar, 20 µm. (ii) Percentage of infected macrophages and (iii) percent parasitemia were plotted using GraphPad Prism 8. Data presented is the mean from n = 2 biologically independent experiments. C Parasite load was measured at the transcript level. Data represent the mean ± standard deviation (SD) from n = 3 biologically independent experiments. Statistical analysis utilized the unpaired t-test with Welch’s correction, with significance levels denoted as *, **, and *** for p-values less than 0.05, 0.01, and 0.001, respectively.

Next, we investigated the effect of NR-7h treatment on the ability of human macrophage p38-MAPK degradation and subsequently on the parasite load till 60 h of L. donovani infection. We observed the significant degradation of human macrophage p38-MAPK in terms of less fluorescence intensity at 12, 36, and 60 h post-infection. Also, the reduction in the intracellular parasite load was observed upon NR-7h treatment till 60 h of infection. The data shows the efficacy of NR-7h treatment on p38-MAPK degradation and subsequent reduction of the parasite load in a time-dependent manner (Fig. 4A). Further, to be sure that the effect of NR-7h is host-mediated, we performed promastigote proliferation assay and observed no significant changes in the survival or proliferation of L. donovani promastigotes upon the treatment of various concentrations of NR-7h (Fig. 4B).

Fig. 4. NR-7h drives sustained degradation of p38-MAPK, reduces intracellular parasite burden without affecting promastigote proliferation.

A Long-term effect of NR-7h treatment on p38-MAPK degradation as well as on parasite burden. PMA-differentiated THP-1 macrophages were treated with 1 μM of NR-7h in conjunction with the stimulation of 100 ng/mL LPS for 6 h, followed by infection with 20 MOI of L. donovani for 12, 36, and 60 h. Untreated infected macrophages served as the control group. (i) The green fluorescence intensity of p38-MAPK was monitored by staining with Alexa Fluor 488, while parasite load by staining with propidium iodide (PI) was monitored by confocal microscopy. Scale bar, 20 µm. Data from one of three experiments were presented. (ii) The mean fluorescence intensity of p38-MAPK and (iii) parasite burden were plotted using GraphPad Prism 8. Data represent the mean ± standard deviation (SD) from n = 3 biologically independent experiments. B Effect of NR-7h treatment on parasite proliferation. L. donovani promastigotes were treated with various concentrations of NR-7h. After 72 h of treatment, the survival of parasites was recorded using the MTT assay, and the parasite viability percentage was calculated. Data presented is the mean from n = 2 biologically independent experiments. Statistical significance was determined using Student’s unpaired 2-tailed t-test, with significance levels denoted as *, **, and *** for p-values less than 0.05, 0.01, and 0.001, respectively.

Degradation of human macrophage p38-MAPK by NR-7h modulates host immune mechanisms during L. donovani infection

The p38-MAPK pathway is an essential modulator of numerous cellular signaling processes, including inflammation and cytokine production^59,60. Here, we explored how the degradation of human p38-MAPK alters cytokine secretion and presents complex dynamics of regulation. Treatment of human macrophages with different doses of NR-7h drastically suppressed the production of TNFα, IL-12, IL-32γ, the proinflammatory cytokine, as well as the anti-inflammatory cytokine, IL-10, compared to the untreated control at the transcript level. Interestingly, when L. donovani was introduced to NR-7h-pretreated macrophages, a higher upregulation in the production of TNFα (~5-fold), IL-12 (~3-fold), and IL-32γ (~3-fold) was observed compared to the untreated infected macrophages. However, a significant decrease in the production of IL-10 (~4-fold) was also observed compared to the untreated infected macrophages (Fig. 5A). These findings reveal the interplay between human p38-MAPK activity and cytokine profile that regulates the infection of L. donovani, thereby suggesting the pathogen’s adaptive mechanisms for developing susceptibility and pathogenicity in macrophages.

Fig. 5. Effect of NR-7h treatment on the immune mechanisms in infected macrophages.

A PMA-differentiated THP-1 macrophages were treated with 1 μM of NR-7h, alongside stimulation with 100 ng/mL LPS for 6 h followed by infection with 20 MOI of L. donovani for 24 h. RNA was isolated to quantify the expression level of inflammatory markers using qRT-PCR. Untreated macrophages served as the control group. Data presented is the mean from n = 2 biologically independent experiments. B PMA-differentiated THP-1 macrophages were treated with 1 μM of NR-7h, along with stimulation with 100 ng/mL LPS for 6 h, followed by infection with L. donovani for different time intervals from 10 min to 120 min, with the addition of 10 μM DCFDA. Samples were analyzed using fluorimetry. Data represent the mean ± standard deviation (SD) from n = 3 biologically independent experiments. C PMA-differentiated THP-1 macrophages were treated with 1 μM of NR-7h, in combination with 100 ng/mL LPS and 20 ng/mL of hIFNγ for 6 h, followed by infection with L. donovani for 24 h. (i) Nitrite levels were measured using the supernatant with Griess reagent. (ii) Cell lysates were prepared to assess the expression level of NOS2, with GAPDH utilized as the loading control. Data represents one of the n = 2 biologically independent experiments. Band intensities were quantified using ImageJ software and plotted using GraphPad Prism 8. Statistical significance was assessed using the unpaired t-test with Welch’s correction, with significance levels denoted as *, **, and *** for p-values less than 0.05, 0.01, and 0.001, respectively.

Oxidative burst-mediated responses of human macrophages are critical regulatory events determining L. donovani survival and infectivity, and the generation level of reactive oxygen species (ROS) is closely linked with p38-MAPK activation. We performed a kinetic analysis of ROS generation from 10 min to 120 min post-L. donovani infection. Using fluorimetry, we observed an upregulation of ROS generation in human macrophages when infected with L. donovani, however a spectacular upregulation of ROS generation amounts when the macrophages were pretreated with NR-7h for 6 h, followed by the infection of Leishmania parasites for 30 min and 90 min (Fig. 5B). Consequently, nitric oxide (NO) production was observed wherein a significant reduction (~1.5-fold) in nitrite levels was noticed in human macrophages post 24 h of L. donovani infection, however, NR-7h-treated-L. donovani infected macrophages showed a significant increase (~2-fold) in nitrite levels (Fig. 5C(i)). Since Nitric Oxide Synthase 2 (NOS2) catalyzes the production of NO, its expression levels were also monitored using immunoblotting and observed to be upregulated (~1.5-fold) in NR-7 h-treated-infected macrophages (Fig. 5C(ii)), compared to untreated-infected macrophages.

Altogether, the data obtained provide new insights into complex interactions between L. donovani and the human macrophage p38-MAPK-mediated immune response during invasion, thus opening new perspectives in the parasite–human macrophage interaction and therapeutic strategies.

Combinatorial efficacy of NR-7h and Amphotericin B against intra-macrophagic amastigotes in L. donovani-infected macrophages

Our study demonstrates the anti-leishmanial effect of p38-MAPK degradation by NR-7h in L. donovani-infected macrophages. Next, we investigated the anti-leishmanial ability of NR-7h in combination with Amphotericin B. Amphotericin B, though largely known as an antifungal drug, has well-documented anti-leishmanial activity and is commonly used as a first-line therapy against visceral leishmaniasis⁶¹. Amphotericin B works by binding to ergosterol-like lipids in the membrane of Leishmania, leading to changes in the membrane integrity. The change lets ions leak out, ultimately causing cell death. Macrophages were pre-conditioned with diverse concentrations of NR-7h for 6 h before infection with L. donovani and concurrently treated with Amphotericin B at a concentration of 10 nM for 24 h. Intracellular parasite burden in form of measuring the relative expression levels of the kinetoplastid minicircle housekeeping gene (JW gene) of Leishmania was quantified at transcript level (Fig. 6A) and also with staining of PI using confocal microscopy (Fig. 6C). The parasite load was reduced by approximately 70%, in a dose-dependent manner, when infected cells were treated with either 10 nM Amphotericin B alone or the different doses (0.5 μM/1 μM) of NR-7h alone. Treatment of L. donovani-infected macrophages with NR-7h alone resulted in a dosage-dependent reduction of parasite burden. We intended to test a conceptual framework in combining Amphotericin B with the NR-7h PROTAC molecule to observe whether simultaneous targeting of the parasite (via Amphotericin B) and the host-cell pathway manipulated by the parasite (via NR-7h PROTAC-mediated degradation) produces an enhanced reduction in parasite burden compared to single agents. Since the PROTAC used in this study targets a host protein (p38-MAPK), it does not kill the parasite directly, but rather alters the parasite’s intracellular environment. Therefore, testing it in combination with a known anti-parasitic agent provides a biologically relevant context to understand whether host-targeted modulation can potentiate conventional therapy. We observed that the combination of Amphotericin B (10 nM) and NR-7h (0.5 μM/1 μM) significantly enhanced the clearance of intracellular parasites. These combined effects translated to a more significant decrease in parasite load than either modality alone and made the possibility of integrating human macrophage-directed strategies, such as p38-MAPK degradation via NR-7h, with conventional anti-leishmanial therapeutics. No cytotoxicity effect was observed upon the treatment of both compounds in the macrophages (Fig. 6B).

Fig. 6. Effect of the combination of NR-7h and Amphotericin B treatment in L. donovani-infected macrophages.

A PMA-differentiated THP-1 macrophages were treated with various concentrations of NR-7h along with the stimulation with 100 ng/mL LPS for 6 h, while another group received only 10 nM Amphotericin B (Amp B) treatment for 24 h with the infection of L. donovani. For the combination treatment group, macrophages pre-treated with NR-7h were infected with L. donovani at 20 MOI and simultaneously treated with 10 nM Amphotericin B. RNA was isolated to assess the expression level of the JW gene using qRT-PCR. C Parasite load was measured with PI staining. Scale bar, 20 µm. B The viability of uninfected THP-1 macrophages treated by the combination of NR-7h and Amphotericin B was assessed using the MTT assay. The data presented are the mean from n = 2 biologically independent experiments.

NR-7h mediated degradation of p38-MAPK in human erythrocytes and its antimalarial potential

To investigate the effect of erythrocyte p38-MAPK degradation on Plasmodium infection, NR-7h PROTAC was deployed. The erythrocytes were treated with different concentrations of NR-7h (1, 2.5, and 5 μM), and the level of p38-MAPK was assessed by immunoblotting. The results suggested that NR-7h significantly degraded erythrocyte p38- MAPK (~2-fold) compared to untreated control at all the concentrations (Fig. 7A). Further, to confirm the ubiquitin-proteasome-dependent mechanism of NR-7h, erythrocytes were treated with NR-7h (5 µM) in the absence and presence of MG132, a proteasomal inhibitor. Immunoblotting revealed that the p38-MAPK degradation was significant (>~3-fold) in NR-7h-treated erythrocytes in the absence of MG132 (Fig. 7B). The degradation of p38-MAPK under the treatment of NR-7h was also assessed by imaging flow cytometry. Reduced level of p38-MAPK in NR-7h(1 μM) erythrocytes was observed (Fig. 7C.(i)) while no effect was observed in the level of a closely-related kinase, ERK1/2 MAPK in NR-7h(1 μM) erythrocytes (Fig. 7C.(ii)), thus validating the specificity of NR-7h. Additionally, the ability of NR-7h to degrade host erythrocyte p38-MAPK in P. falciparum-infected erythrocytes was also assessed by immunoblotting (Supplementary Fig. 2A).

Fig. 7. Degradation of erythrocyte p38-MAPK by NR-7h and its antimalarial potency.

A Uninfected erythrocytes were treated with different concentrations of NR-7h. Western blotting was used to evaluate the levels of p38-MAPK expression, with untreated erythrocytes serving as the control and GAPDH as the loading control. B Erythrocytes were treated with NR-7h (2.5 µM) in the presence and absence of MG132 proteasome inhibitor. Western blotting was used to evaluate p38-MAPK expression with GAPDH as the loading control. Data represent the mean ± standard deviation (SD) from n = 3 biologically independent experiments. C Imaging flow cytometry was used to evaluate the fluorescence levels of (i) p38-MAPK and (ii) ERK 1/2 in the NR-7h (1 µM) treated erythrocytes. Untreated erythrocytes were used as the control. D Pf3D7 infected erythrocytes (at ring stage) were treated with different concentrations of NR-7h and then for 72 h. (i) Growth inhibition curve—%inhibition against log concentration of NR-7h. (ii) Giemsa image panel of P. falciparum-infected erythrocytes (Scale bar: 10 µm). E Growth inhibition curve of PD 169,316 representing %inhibition against log concentration of PD 169,316. F Erythrocytes were pre-treated with 1 µM and 5 µM NR-7h and then infected with purified Pf3D7 schizonts. (i) Parasitemia plotted as relative invasion plotted against each NR-7h concentration, (ii) confocal microscopy image panel (Scale bar: 10 µm), and (iii) Giemsa staining image (Scale bar: 5 µm) of P. falciparum-infected erythrocytes post 4 h of invasion. Untreated infected erythrocytes served as the control group. Band intensities were analyzed using ImageJ software and plotted using GraphPad Prism 8. Statistical analysis utilized the unpaired t-test with Welch’s correction, with significance levels denoted as *, **, and *** for p-values less than 0.05, 0.01, and 0.001, respectively.

To determine the in vitro antimalarial potency of NR-7h, Pf3D7 culture at the ring stage was treated with a range of NR-7h concentrations for 72 h. Post-treatment, parasitemia was calculated for each group by Giemsa staining. NR-7h showed a concentration-dependent anti-malarial effect on Pf3D7 culture and the half-maximal inhibitory concentration (IC₅₀) of NR-7h was determined to be 1.047 µM by the growth inhibition curve (Fig. 7D). Additionally, in-vitro antimalarial potency of a known p38-MAPK inhibitor, PD 169316, was also assessed to validate the role of p38-MAPK during Plasmodium infection and a concentration-dependent anti-malarial effect of PD 169316 was observed by Giemsa staining (Supplementary Fig. 2B) with an IC₅₀ of 3.67 µM (Fig. 7E). Further, to determine the effect of NR-7h mediated p38-MAPK degradation on parasite invasion the NR-7h pre-treated erythrocytes were infected with purified mature Pf3D7 schizonts and the formation of rings was observed after 4 h by confocal microscopy and Giemsa staining. A significant reduction was observed in the Plasmodium parasite’s ability to invade the NR-7h-treated erythrocytes at both 1 µM (~1.5-fold) and 5 µM (~3-fold) as compared to the untreated erythrocytes (Fig. 7F).

Discussion

p38-MAPK is one of the critical members of the family of MAPKs and comprises four isoforms: p38α, p38β, p38γ, and p38δ, which differ in tissue expression and functional roles⁶². It is regulated by dual phosphorylation on its Thr-Gly-Tyr motif by upstream kinases that include MKK3 and MKK6 in response to stressors such as UV light, cytokines (eg, TNF-α, IL-1β), and PAMPs⁶³. It influences many signaling pathways, including inflammatory response, stress, apoptosis, differentiation, and transcription factor-mediated via ATF2, NF-κB, and proteins with the help of post-translational modification^64–67. p38-MAPK has a dual role in cancer as a tumor suppressor and promoter⁶⁸. It has been linked to chronic inflammatory diseases⁶⁹, including rheumatoid arthritis and psoriasis⁷⁰, and neurodegenerative disorders⁷¹, including Alzheimer’s⁷². Despite its value as a therapeutic target, clinical development of p38-MAPK inhibitors has been slow, limited by such factors as off-target effects and toxicity⁶⁹.

Several previous studies have reported a host-protective role for p38-MAPK activation during L. donovani infection. Notably, Junghae and Raynes³⁰ demonstrated that activation of p38-MAPK attenuates intracellular parasite survival in macrophages, while Kar et al.⁷³ and Gupta et al.⁷⁴ showed that p38-MAPK signaling promotes pro-inflammatory cytokine production and enhances parasite clearance. These studies primarily focused on early stages of infection and relied on pharmacological modulation or signaling readouts of p38-MAPK activity, leading to the conclusion that p38 activation supports host defense mechanisms.

While transient or early activation of p38-MAPK may facilitate immediate inflammatory responses required for parasite control, our data indicate that the continued presence of p38-MAPK during established infection is exploited by L. donovani to support intracellular survival and suppress effective immune clearance.

We observed the higher activation of human p38-MAPK at 6h of L. donovani infection in human macrophages, which further downregulates at later time points of infection, suggesting the involvement of human p38-MAPK activation at the early time of parasite infection. Importantly, prior studies largely employed kinase inhibitors, which block catalytic activity but leave the p38-MAPK protein intact. To investigate the role of p38-MAPK beyond transient activation, we employed NR-7h, a PROTAC molecule that induces sustained degradation of the p38-MAPK protein itself, thereby eliminating both its kinase activity and its non-catalytic scaffold functions. This distinction is critical, as removal of the protein can yield biological outcomes that are not captured by catalytic inhibition alone. Using this approach, we degraded human p38-MAPK by NR-7h followed by L. donovani infection. We observed a reduction of infected macrophages as well as the reduced number of parasites in those infected macrophages, which indicates the requirement of human p38-MAPK by L. donovani not only for its invasion but also for its disease progression. These findings were further validated using complementary strategies: siRNA-mediated knockdown of p38-MAPK also led to a reduction in parasite load, whereas treatment with the p38 kinase inhibitor PD169316 did not significantly alter parasite load. These results suggest that the presence of the p38-MAPK protein itself, rather than its catalytic activity alone, is important for supporting parasite survival.

Together, these findings suggest that p38-MAPK plays distinct, time-dependent roles during Leishmania infection. While early activation of p38-MAPK appears to support host defense, the continued presence of p38-MAPK at later stages may be exploited by the parasite to modulate host immunity and promote intracellular survival. Thus, our results do not contradict previous studies but rather extend them, highlighting the importance of timing and the mode of p38-MAPK regulation when interpreting host signaling responses during infection.

p38-MAPK is a classical regulator of various cellular and immune mechanisms. For a better understanding of human p38-MAPK-mediated host responses during L. donovani infection, we further investigated the effect of p38-MAPK degradation on host immune mechanisms in L. donovani-infected macrophages. Human p38-MAPK regulates cytokine production as its downstream process⁶⁰, we observed that degradation of human p38-MAPK led to a significant reduction in the levels of TNFα and IL-10, a pro-inflammatory and an anti-inflammatory cytokine, respectively. In the context of the Leishmania parasite infection, it is well-documented that parasites suppress host pro-inflammatory cytokine production^75,76 and induce host anti-inflammatory cytokine production^77,78 to facilitate their survival. Interestingly, when Leishmania parasites infected NR-7h-pre-treated macrophages, a significant upregulation of TNFα and a concomitant downregulation of IL-10 were observed. We also evaluated other pro-inflammatory markers, IL-12 and IL-32γ, to obtain a broader understanding of macrophage immune polarization following p38-MAPK degradation during Leishmania infection. IL-12 is a key Th1-promoting cytokine that drives IFN-γ-mediated macrophage activation⁷⁹, while IL-32γ acts as an upstream amplifier of TNFα and IL-12 production, enhancing pro-inflammatory signaling⁸⁰. Although IL-12 expression is generally regulated by p38-MAPK activation and TNFα signaling³¹, in our study, p38-MAPK degradation led to elevated TNFα levels, which can induce IL-12 through TNFα–NF-κB–dependent activation, thereby reinforcing the pro-inflammatory cascade. Moreover, increased TNFα may also stimulate IL-32γ expression, which further potentiates TNFα and IL-12 production. The coordinated upregulation of TNFα, IL-12, and IL-32γ, along with reduced IL-10⁸¹, indicates that p38-MAPK degradation reprograms macrophages toward a robust pro-inflammatory phenotype, disrupting the parasite’s immune evasion strategy, and suggests that Leishmania parasites modulate the p38-MAPK pathway for their own survival advantage.

Additionally, p38-MAPK also regulates oxidative stress⁸², therefore, we next observed ROS and NO levels, the two key processes to control the parasite. Degradation of human p38-MAPK induced generation levels of ROS and NO during L. donovani infection that might contribute to the reduction of parasite load. Since targeting human p38-MAPK is a host-directed therapeutic approach, we further evaluated the synergistic effect of NR-7h with the known antileishmanial drug Amphotericin B that acts directly on Leishmania parasites⁶¹. The combination of both compounds led to the near-complete clearance of L. donovani parasites.

In context to Plasmodium falciparum infection in human erythrocytes, a substantial reduction in parasitic load was observed in NR-7h treated erythrocytes with degraded p38-MAPK. During its intraerythrocytic stages of development, the Plasmodium parasite introduces osmotic changes by altering the permeability of the erythrocyte membrane⁸³ and oxidative stress⁸⁴ in the host erythrocytes, which serve as triggers for activation and downstream signaling of p38-MAPK. Activation of p38-MAPK under oxidative stress results in downstream phosphorylation of Src tyrosine kinase and Band 3, ultimately leading to lysis of erythrocytes⁸⁵. Since the p38-MAPK activation is linked to erythrocyte lysis, its inhibition is correlated with reduced lysis and parasite egress. Additionally, human erythrocytes with mutant Band 3, an invasion ligand of Plasmodium parasite⁸⁶, lacking the N-terminal 11 amino acids which otherwise carry the phosphorylation site show inhibited parasite invasion and maturation⁸⁷. This further establishes the importance of the activation and signaling of p38-MAPK in infected erythrocytes. Previous studies have also shown that human p38-MAPK inhibitors, including RWJ67657 and RWJ68198, affect the parasite development within the human erythrocytes⁴³. Altogether, p38-MAPK degradation-mediated inhibition in parasite egress, invasion, and survival contributes to the overall reduction in parasitic load. Further investigation of the host p38-MAPK’s role in parasite infection can be validated by developing genetically modified systems. Similar strategies have been applied in the malaria field using genetically tractable erythroid systems to test host determinants of parasite invasion and growth. More recently, Kell-null erythrocytes generated via CRISPR/Cas9 in the BEL-A erythroid progenitor cell line showed that P. falciparum invasion is substantially reduced in these genetically modified cells⁸⁸. Beyond erythrocytes, macrophage studies in Leishmania infection provide additional precedents for the utility of host genetic modification. Conditional deletion of TAK1 in macrophages impaired parasite control by skewing effector responses⁸⁹ while inducible knockdown of HO-1⁹⁰ or NRF2⁹¹ reduced parasite survival by enhancing oxidative stress. Taken together, these examples establish that targeted genetic perturbation of host cells can yield direct and mechanistic insights into host–pathogen interactions.

To conclude, this study establishes that NR-7 h-mediated degradation of host p38-MAPK significantly reduces Leishmania donovani and Plasmodium falciparum infections. p38-MAPK degradation modulates immune responses and stress response pathways that effectively promote parasite clearance (Fig. 8). Furthermore, host p38-MAPK degradation synergizes with Amphotericin B to effectively improve the clearance of L. donovani parasites. These findings highlight host p38-MAPK as a promising therapeutic target and underscore the potential of targeted protein degradation strategies as a new avenue for host-directed antiparasitic therapy.

Fig. 8. A schematic representation of the activation and role of p38-MAPK in macrophages infected with Leishmania donovani and Plasmodium falciparum-infected erythrocytes.

Under normal conditions (left panels), infection triggers MAPKKK/MAPKK-dependent phosphorylation of p38-MAPK. In macrophages, p38-MAPK activation modulates inflammatory cytokine production and oxidative stress responses that collectively support intracellular parasite survival. In infected erythrocytes, hypo-osmotic shock induces p38-MAPK and Src activation, leading to Band 3 phosphorylation and membrane destabilization to facilitate parasite exit. Upon treatment with NR-7h, a p38-MAPK targeting PROTAC molecule (right panels), p38-MAPK is ubiquitinated and degraded via recruitment of E3 ligase, suppressing its phosphorylation. Depletion of p38-MAPK suppresses downstream signaling rather than merely inhibiting its catalytic activity. In macrophages, this leads to reduced parasite survival, while in erythrocytes it blocks Band 3 modification and hemolysis, thereby inhibiting Plasmodium entry and egress.

Materials and methods

Cell and Leishmania parasite culture

THP-1 cells, obtained from the National Center for Cell Science (NCCS, Pune, India), were cultured in RPMI-1640 media (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine, 10 mM HEPES buffer, 20 mM sodium bicarbonate, 1 mM sodium pyruvate, and penicillin/streptomycin (10,000 units/ml) at 37 °C in a humidified incubator with 5% CO₂. To induce differentiation into macrophages, Phorbol 12-myristate 13-acetate (PMA) (Sigma) was added at a concentration of 50 ng/mL for 24 h. L. donovani Bob promastigotes were cultured in M199 medium (Gibco) supplemented with 10% FBS (Gibco) and 10 µg/mL gentamycin at 26 °C. Infections were performed using the metacyclic stage of L. donovani promastigotes at a multiplicity of infection (MOI) of 20.

Protein immunoblotting

To prepare cell lysates, RIPA lysis buffer (GBiosciences) supplemented with a protease inhibitor cocktail (Roche) was used. Equal amounts of cell lysates (25 µg for THP-1 macrophages and 100 µg for erythrocytes) were loaded and separated using SDS-PAGE. The proteins were then transferred onto a nitrocellulose immunoblot membrane (Bio-Rad). The membrane was blocked overnight at 4 °C in 5% BSA (SRL) in 1× PBS containing 0.05% Tween20 (Sigma) (wash buffer), followed by three washes with the wash buffer. Subsequently, the membrane was incubated with primary antibodies for 2 h at room temperature, followed by three additional washes and incubation with HRP-conjugated secondary antibodies for another 1 h at room temperature. After three washes, the blots were visualized using a densitometric ECL kit (Bio-Rad) on the ChemiDoc Imaging System (Bio-Rad). The data were analyzed using ImageJ software.

Imaging flow cytometry

THP-1 macrophages were treated with 1 μM NR-7h (kindly provided by Prof. Angel R. Nebreda and Prof. Antoni Riera, Institute for Research in Biomedicine, Barcelona) along the co-stimulation of LPS (100 ng/mL) for 6 h. Cells were fixed, permeabilized, and then stained with 1:200 of each human p38-MAPK (Biolegend #622403), human ERK1/2 (Affinity Biosciences # AF6687), and human Phospho-p38-MAPK (Biolegend #690201) antibody, for an hour followed by the secondary antibodies, anti-rabbit Alexa fluor 488 (Invitrogen #A11008) and anti-mouse Alexa fluor 546 (Invitrogen #A11030), respectively for half an hour. Similarly, erythrocytes were treated with 2.5 µM NR-7h to analyze the p38-MAPK degradation by imaging flow cytometry. The cells were fixed in cold 2% paraformaldehyde and permeabilized in 0.001% Triton X-100. Subsequently, the cells were stained with human p38-MAPK antibody (1:200) and human ERK1/2 antibody (1:200) for 1.5 h, followed by staining with anti-rabbit Alexa 488 antibody (1:200) for 45 minutes. After washing, the cells were processed in a Cytek Amnis FlowSight imaging flow cytometer. The events were first visualized on a brightfield Area versus Aspect Ratio plot, and the population of interest corresponding to single cells was gated, thereby excluding debris and cell aggregates. The gated population was subsequently used for downstream analysis using IDEAS software. Fluorescence intensity distributions were generated by plotting histograms of cell count (denoted as normalized frequency) versus fluorescence intensity of the relevant fluorescence channel, and fluorescence profiles were compared across experimental groups.

Cell viability assessment

MTT assay was conducted to evaluate the impact of NR-7h on the viability of THP-1 cells. A solution of MTT dye (Sigma-Aldrich) was prepared by diluting 5 mg of MTT in 1 mL of PBS, which was further diluted to 1:10 in RPMI medium. For the assay, 10,000 THP-1 cells in 100 µL of complete media were seeded in each well of 96-well flat-bottom plates. Following the differentiation of monocytes into macrophages, THP-1 macrophages were treated with various concentrations of NR-7h with the co-stimulation of LPS (100 ng/mL). After 72 h, the MTT assay was carried out according to the manufacturer’s instructions. Specifically, treated or untreated THP-1 macrophages were incubated with MTT dye solution for 2 h at 37 °C, followed by adding a stopping buffer (5% formic acid in isopropanol) to halt the reaction for 20 min at 37 °C. Absorbance was then measured at 570 nm, and the cell viability percentage was calculated. Each experiment was performed in triplicate and repeated three times.

RNA extraction and gene expression profiling

Isolation of total RNA from treated/untreated/uninfected and L. donovani macrophages was performed at an appropriate time point using the TRIZOL reagent (Invitrogen). Subsequently, the quantification of the isolated RNA was conducted using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). For cDNA synthesis, one microgram of total RNA was utilized, and the process was carried out using the First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions, employing random hexamer primers. The levels of p38α-MAPK, JW, TNFα, IL-12, IL-32γ, and IL-10 were quantified using gene-specific primers. The PCR reactions were performed on an Applied Biosystems Real-Time PCR System (ABI) using the PowerUp SYBR Green PCR Master Mix (Thermo Fisher Scientific). Primer sequences for IL-10-Forward 5′-TTAAGGGTTACCTGGGTTGC-3′, Reverse 5′-TGAGGGTCTTCAGGTTCTCC-3′; IL-12-Forward 5′-ATGCCCCTGGAGAAATGGTG-3′, Reverse 5′-GGCCAGCATCTCCAAA CTCT-3′; IL-32γ-Forward 5′-AGGCCCGAATGGTAATGCT-3′, Reverse 5′-CCACAGTG TCCTCAGTGTCACA-3′; TNF-α-Forward 5′-CCTCTCTCTAATCAGCCCTCTG-3′, Reverse 5′-GAGGACCTGGGAGTAGATGAG-3′; JW-Forward 5′-CCTATTTTACACCAACCCCCAGT-3′, Reverse 5′-GGGTAGGGGCGTTCTGCGAAA-3′; p38α-MAPK-Forward 5′-ACTCAGATGCCGAAGATGAAC-3’, Reverse 5′-GTGCTCAGGACTCCATCTCT-3′. The results were expressed as fold change of control (Uninfected samples) using the 2^−ΔΔCTmethod.

Parasite quantification by Propidium Iodide Staining

0.5 × 10⁶ THP-1 cells were cultured on 18 mm diameter round coverslips coated with poly-L-lysine (Sigma) and treated with 50 ng/mL PMA to induce macrophage differentiation for 24 h. Following the incubation, the coverslips were rinsed with RPMI-1640 medium to eliminate non-adherent cells. Subsequently, the adherent THP-1 macrophages were treated with different concentrations of NR-7h, with the co-stimulation of LPS (100 ng/mL) for 6 h, followed by infection with metacyclic L. donovani promastigotes at a multiplicity of infection (MOI) of 20. After 6 h of infection, the cells were washed three times with RPMI medium to remove non-phagocytosed promastigotes. The infected macrophages were then further incubated for 24 h. Following the incubation, the coverslips were washed with PBS and fixed with chilled methanol. Subsequently, the cells were stained with propidium iodide (500 nM in 2× SSC buffer)¹⁶. A minimum of 100 macrophages per coverslip was counted using a confocal microscope to determine the number of resident amastigotes. Confocal imaging was performed using a high-speed laser scanning confocal microscope with a 60× objective magnification, utilizing an excitation/emission wavelength of 535/617 nm.

Parasite quantification by measuring JW gene levels

After the preparation of cDNA, the relative expression level of the kinetoplastid minicircle housekeeping gene (JW gene) of L. donovani was quantified using quantitative Real-Time PCR (qRT-PCR).

siRNA transfection

For siRNA transfections, siRNA specific to human p38 alpha MAPK (sc-29433) and control siRNA (sc-37007) were procured from Santacruz. Briefly, THP-1 macrophages were transfected with 50 nM of siRNA in Opti-MEM medium (GIBCO) using the Hiperfect transfection reagent (Qiagen) as per the manufacturer’s protocol. After 5 h of transfection, cells were supplemented with the complete media containing 20% FBS and further incubated for 36 h. Knockdown efficiency was measured by qRT-PCR and western blotting. For infection studies, these transfected THP-1 macrophages were infected with purified metacyclic L. donovani promastigotes at 20 MOI.

L. donovani promastigotes proliferation assay

L. donovani promastigotes were treated with various concentrations of NR-7h. After 72 h of treatment, the survival of parasites was recorded using the MTT assay. Treated or untreated parasites were incubated with MTT dye solution for 2 h at 37 °C, followed by adding a stopping buffer (5% formic acid in isopropanol) to halt the reaction for 20 min at 37 °C. Absorbance was then measured at 570 nm, and the parasite viability percentage was calculated.

Immunofluorescence assay

THP-1 cells were seeded on 18-mm-diameter round coverslips coated with poly-L-lysine (Sigma) and were stimulated with 50 ng/mL of PMA to differentiate into macrophages for 24 h. THP-1 macrophages were treated with 1 μM NR-7h with the co-stimulation of LPS (100 ng/mL) for 6 h, followed by infection with metacyclic L. donovani promastigotes at an MOI of 20 for 12, 36, and 60 h. Cells were washed with RPMI followed by fixation with 2% paraformaldehyde (PFA) for 15 min. Cells were washed with 1× PBS and were permeabilized with a permeabilization buffer (0.1% BSA and 0.2% saponin in 1× PBS). Then, cells were washed with washing buffer (0.1% BSA and 0.1% saponin in 1× PBS) followed by the incubation of 1:200 dilution of primary antibody for p38-MAPK at room temperature for 1 h. Following washes, a secondary antibody, anti-rabbit Alexa Fluor 488 at a dilution of 1:250 was introduced to the cells for 30 min at room temperature. Cells were washed and stained with propidium iodide (500 nM in 2× SSC buffer). The coverslips were mounted on the slides with a mounting medium, Fluoroshield with DAPI (Sigma). The fluorescence intensity of p38-MAPK and the number of resident amastigotes were determined using confocal microscopy.

Quantification of reactive oxygen species (ROS)

THP-1 differentiated human macrophages were treated with 1 μM NR-7h along with the co-stimulation of LPS (100 ng/mL) for various time intervals ranging from 10 min to 120 min in 96-well plates (Corning #CLS3603). Following the treatment, macrophages were infected with L. donovani promastigotes at a multiplicity of infection (MOI) of 20 for 30 min at 37 °C. Simultaneously, cells were loaded with 10 µM DCFH-DA for 30 min, following the guidelines provided by the manufacturer (Abcam #ab113851). Once the incubation period concluded, the cells were promptly analyzed for levels of ROS generation using fluorimetry with an excitation/emission of 485 nm/535 nm.

Nitric oxide (NO) quantification

PMA differentiated THP-1 macrophages were treated with 1 μM NR-7h along with the co-stimulation of LPS (100 ng/mL) for a duration of 6 h, followed by infection with L. donovani promastigotes at an MOI of 20 along with the co-stimulation with LPS (100 ng/mL) and hIFNγ (20 ng/mL). After 24 h post-infection, the supernatant was collected to determine the level of NO (in the form of nitrite) using the Griess reagent kit (Invitrogen #G-7921), following the instructions provided by the manufacturer.

Plasmodium parasite culture and in-vitro assays

P. falciparum 3D7 (Pf3D7) was cultured in O+ human erythrocytes (obtained from Blood Bank Organization, Pusa Road, New Delhi, India with prior declaration of their intended use for research purposes. Blood units categorized as “quantity not sufficient” (QNS) were used for in vitro Plasmodium culture), and RPMI 1640 (Gibco) supplemented with 0.5% albumax (Gibco), 2 g/L sodium bicarbonate (Biobasic), 30 mg/L hypoxanthine (Sigma), and 10 mg/L gentamycin (SRL) under mixed gas conditions. An in vitro growth inhibition assay was carried out in synchronized Pf3D7 culture at the ring stage at 0.8% parasitemia and 2% hematocrit. Pf-infected erythrocytes were treated with different concentrations of NR-7h (20 µM, 10 µM, 5 µM, 2.5 µM, 1.25 µM, 625 nM, 312 nM, and 156 nM) and PD 169316 (40 µM, 20 µM, 10 µM, 5 µM, 2.5 µM, 1.25 µM and 625 nM) for 72 hr in a total volume of 100 µL in each well of 96-well microtiter plate. Post 72 h, Giemsa smears were prepared, and parasitemia for each group was calculated by counting at least 2000 cells. Growth inhibition ability of NR-7h and PD 169316 was calculated as % inhibition, and it was plotted against logarithmic NR-7h concentration and PD 169316, respectively. For the in vitro Pf3D7 invasion assay, erythrocytes were pre-treated with 1 µM and 5 µM. Post-incubation with NR-7h, the erythrocytes were washed with 1× iRPMI to remove the residual drug. Mature and rupturing Pf3D7 schizonts were purified and subsequently incubated with pre-treated erythrocytes at 0.8% parasitemia and 1% hematocrit in a total volume of 100 µL in each well of a 96-well microtiter plate. After 2 h, the erythrocytes were washed with 1× iRPMI and further incubated for 2 h in a total volume of 100 µL in each well of a 96-well microtiter plate. Post incubation of a total of 4 h, thin smears were prepared for each group and fixed in 100% methanol. Slides were subsequently processed either for Giemsa staining or mounted with an anti-fade solution containing DAPI (Sigma) for confocal microscopy. Formation of ring in the infected erythrocyte was observed, and parasitemia was plotted as % rings against each NR-7h concentration. Untreated infected erythrocytes served as the control group in the in vitro assays. For the in-vitro assays, parasitemia was calculated by the formula: % Parasitemia = (Infected erythrocytes/total number of erythrocytes) × 100, while the % inhibition was calculated as % inhibition = [(Parasitemia_control − Parasitemia_Treated)]/Parasitemia_control × 100.

Statistics and reproducibility

The data were displayed as the mean (n = 2) and mean ± SD (standard deviation) for n ≥ 3. Every experiment was replicated twice/thrice in distinct batches. All charts were produced, and the corresponding statistical evaluations were carried out utilizing GraphPad Prism (GraphPad Software, USA). The statistical significance was determined using the unpaired t-test with Welch’s correction. The significance level was achieved with p-values < 0.05. The p-values were denoted as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

A.R. and S.S. conceptualized the idea, designed the experiments, executed the data interpretation, and analyzed the experimental data. A.R. and S.S. reviewed the work and corrected the paper. J.S., N.R. and S.S. wrote the paper. J.S. and N.R. designed the study, conducted the investigation, curated, analyzed, and interpreted the data. J.S. performed degradation and immune modulation studies in macrophages. N.R. performed degradation and anti-malarial studies in RBC. R.B. and J.S. performed invasion studies. A.G., P.S., E.M., N.J. and G.D.G.P. helped in the Leishmania-related experimental methodology. R.P.P. assisted in the interpretation of Leishmania-related experimental data. J.S., N.R., A.R. and S.S. critically analyzed the data. All authors contributed to the article and approved the submitted version.

Peer review

Peer review information

Communications Biology thanks Christian Doerig, Rui Zhou, and Anindita Ukil for their contribution to the peer review of this work. Primary Handling Editors: Bang Shen and David Favero. A peer review file is available.

Data availability

The original contributions presented in the study are included in the article. The source data for the figures are provided in the supplementary data file. Uncropped and unedited blot/gel images corresponding to the figures in the paper are provided as Supplementary Figs. 3–8 in the Supplementary Information. Further inquiries can be directed to the corresponding authors.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s42003-026-10096-0.