Biology subjects

Siddiqui, G.

Publications and source records attributed to Siddiqui, G..

10 recordsLinked to original sources

bioRxiv2025

PfK13-associated artemisinin resistance slows drug activation and enhances antioxidant defence, which can be overcome with sulforaphane.

Artemisinin resistance is globally prevalent, including in Africa, raising concerns and highlighting the need to better understand the cellular mechanisms behind this resistance. In Plasmodium falciparum, artemisinin resistance is primarily attributed to mutations in the PfKelch13 (PfK13) gene. In this study, we performed proteomic analysis on a range of sensitive and artemisinin-resistant parasites (both laboratory-generated and field isolates), revealing specific dysregulation of PfK13 protein abundance. Reduced PfK13 levels were linked to impaired hemoglobin digestion, decreased free heme levels, and consequently, decreased artemisinin activation. Artemisinin resistant parasites also exhibited elevated thiol levels, indicating a more reduced cellular state. Targeting the parasite redox capacity with sulforaphane potentiated artemisinin activity in vitro and in an in vivo rodent Plasmodium berghei model, offering a potential strategy to overcome resistance. Our findings provide critical insights into the molecular mechanisms of artemisinin resistance and suggest novel therapeutic interventions to restore drug sensitivity. One Sentence Summary: PfK13 mutations drive artemisinin resistance in Plasmodium parasites by enhancing antioxidant defences, which can be targeted by redox modulators such as sulforaphane.

microbiology↗

bioRxiv2025

Validation of Solvent Proteome Profiling for Antimalarial Drug Target Deconvolution

Malaria remains a global health threat, with rising drug resistance accelerating the urgent need for new therapeutics. Target elucidation is a critical step in antimalarial drug discovery, enabling a deeper understanding of the molecular mechanisms of action of both existing and novel compounds. This study validates solvent-induced proteome profiling (SPP) as a proteomics-based approach for identifying drug-protein interactions in Plasmodium falciparum. SPP detects shifts in protein stability induced by ligand binding, allowing the identification of drug target/s without the need for compound modifications. Here, we successfully generated solvent denaturation curves for the P. falciparum proteome, and demonstrated the utility of SPP with five antimalarial compounds: pyrimethamine, atovaquone, cipargamin, MMV1557817 and OSM-S-106. In addition to measuring each compounds impact across the full denaturation curve, investigating protein levels at individual solvent percentages preserved specific stability changes that would otherwise be masked in pooled analyses performed by integral SPP. This strategy was critical for the identification of the cipargamin target, non-SERCA-type Ca2+-transporting P-ATPase (PfATP4). Notably, we propose live-cell treatment SPP as a novel approach, demonstrating its ability to identify the validated target of pyrimethamine, bifunctional dihydrofolate reductase-thymidylate synthase (PfDHFR), with high specificity. We also introduced the novel one-pot mixed-drug SPP which enables the evaluation of multiple drugs within a single lysate and experimental setup. This alternative method simplifies the experimental workflow and includes positive controls to affirm the performance of the experiment. Overall, this study demonstrates that SPP can be successfully applied in both lysates and live cells to elucidate drug targets in P. falciparum, as well as providing additional information regarding the mechanisms of drug action, offering insights for the optimisation of existing antimalarials and the development of novel therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/670428v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@b83c75org.highwire.dtl.DTLVardef@1895f23org.highwire.dtl.DTLVardef@1c51ba0org.highwire.dtl.DTLVardef@198c822_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Deconvolution of the on-target activity of plasmepsin V peptidomimetics in P. falciparum parasites

Plasmepsin V (PMV), an essential aspartyl protease, plays a critical role during the asexual blood stage of infection of Plasmodium by enabling the export of parasite proteins into the host red blood cell. This export is vital for parasite survival and pathogenesis, making PMV an attractive target for antimalarial drug development. Peptidomimetic inhibitors designed to mimic the natural substrate of PMV have demonstrated potent parasite-killing activity by blocking protein export. While these compounds have been instrumental in validating PMV as a bona fide antimalarial target, inconsistencies between their biochemical potency and cellular activity have raised questions regarding their precise mechanism of action. In this study, we employed chemoproteomic approaches, including solvent induced protein precipitation (SIP) and intact-cell thermal PISA profiling, to demonstrate PMV target engagement by the peptidomimetics. To further support these findings, we generated parasite lines exhibiting reduced sensitivity to peptidomimetics. Through whole-genome sequencing of these parasite lines, a single nucleotide variant (SNV) within the pmv gene was revealed. This mutation was later validated using reverse genetics, confirming its role in mediating resistance. Together, these data provide strong evidence that the peptidomimetics exert their antimalarial activity by directly targeting PMV. These findings further support the potential of PMV as a validated and promising target for future antimalarial drug development.

cell biology↗

bioRxiv2023

On-target, dual aminopeptidase inhibition provides cross-species antimalarial activity.

To combat the global burden of malaria, development of new drugs to replace or complement current therapies are urgently required. As drug resistance to existing treatments and clinical failures continue to rise, compounds targeting multiple life cycle stages and species need to be developed as a high priority. Here we show that the compound MMV1557817 is a nanomolar inhibitor of both Plasmodium falciparum and Plasmodium vivax aminopeptidases M1 and M17, leading to inhibition of end stage haemoglobin digestion in asexual parasites. Multi-stage analysis confirmed that MMV1557817 can also kill sexual stage P. falciparum, while cross-resistance studies confirmed the compound targets a mechanism of action distinct to current drug resistance mechanisms. Analysis of cross reactivity to homologous human enzymes shows the compound exhibits a high level of selectivity, whilst safety as well as druggability was confirmed in the murine model P. berghei. MMV1557817-resistant P. falciparum parasites displayed only low-level resistance (<3-fold) and exhibited a slow growth rate that was quickly outcompeted by wild type parasites. MMV1557817-resistant parasites digest significantly more haemoglobin and possess a mutation in PfA-M17 that induces partial destabilisation of the PfA-M17 homohexamer, resulting in high-level resistance to specific PfA-M17 inhibition, but enhanced sensitivity to specific PfA-M1 inhibition, and importantly, these parasites were highly sensitive to artemisinin. Overall, these results confirm MMV1557817 as a potential lead compound for further drug development and highlight the potential of dual inhibition of M1 and M17 as an effective multi-species drug targeting strategy.

microbiology↗

bioRxiv2023

The Plasmodium falciparum artemisinin resistance-associated protein Kelch 13 is required for formation of normal cytostomes

Artemisinin (ART) is a quick-killing and effective antimalarial activated by the haem derived from haemoglobin digestion. Mutations in the parasites Kelch 13 (K13) protein compromise the efficacy of this drug. Recent studies indicate an undefined role for K13 in haemoglobin uptake. Here, we show that K13 is associated with the collar that constricts cytostomal invaginations required for the parasite to ingest host cytosol. Induced mislocalisation of K13 led to the formation of atypical invaginations lacking the cytostomal ring and constricted neck normally associated with cytostomes. Moreover, the levels of haemoglobin degradation products, haem and haemozoin, are decreased when K13 is inactivated. Our findings demonstrate that K13 is required for normal formation and/or stabilisation of the cytostome, and thereby the parasites uptake of haemoglobin. This is consistent with perturbation of K13 function leading to decreased activation of ART and consequently, reduced killing. Significance StatementArtemisinin-resistant parasites contain mutations in the gene encoding the Kelch 13 protein (K13). How K13 mutations result in artemisinin resistance is unclear. Here, we present evidence that normal K13 is required for the formation of the cytostome, a specialised parasite feeding apparatus used to endocytose host cell haemoglobin. Our results suggest that artemisinin resistance is due to a decrease in artemisinin activation brought about by a decrease in efficiency of haemoglobin uptake and consequently reduced production of haem.

microbiology↗

bioRxiv2022

Immunopharmacological evaluation of adjuvant efficacy of Monophosphoryl lipid-A and CpG ODN with SARS-CoV-2 RBD antigen

SARS-CoV-2 infection has made the mankind to witness most sever and serious pandemic situation in the history. Millions of people have suffered and are still suffering with this infection which has caused a mass mortality in the past three years. Development of an effective vaccine to control the spread of infection and to prevent this viral infection is need of the hour. Adjuvanted vaccines have proven their efficacy in controlling many other viral infections like flu, keeping this context in view we have evaluated the immunopharmacological efficacy of two adjuvants MPL-A and CpG ODN in combination with MF59 emulsion against SARS-CoV-2 antigen. From the data obtained we can infer that both the adjuvants were capable of eliciting a potent antibody response against antigen alone and MF59 groups. Comparatively MPL-A was eliciting a Th1 polarized response in terms of IgG2a and cytokine production. Both the adjuvants were capable of enhancing the CD 4, 8 and 19 cell populations. Overall the pre clinical evaluation has given a clue of the effectiveness of MPL-A and CpG adjuvants against SARS-CoV-2 antigen.

immunology↗

bioRxiv2021

Genetic and chemical validation of Plasmodium falciparum aminopeptidase PfA-M17 as a drug target in the hemoglobin digestion pathway

Plasmodium falciparum, a causative agent of malaria, continues to remain a global health threat since these parasites have developed increasing resistance to all anti-malaria drugs used throughout the world. Accordingly, drugs with novel modes of action are desperately required to combat malaria. P. falciparum parasites infect human red blood cells where they digest the hosts main protein constituent, hemoglobin. Leucine aminopeptidase PfA-M17 is one of several aminopeptidases that have been implicated in the last step of this digestive pathway. Here we utilize both reverse genetics and a compound specifically designed to inhibit the activity of PfA-M17 to show that PfA-M17 is essential for P. falciparum survival as it provides parasites with free amino acids for growth, many of which are highly likely to originate from hemoglobin. We further show that our inhibitor is on-target for PfA-M17 and has the ability to kill parasites at nanomolar concentrations. Thus, in contrast to other hemoglobin-degrading proteases that have overlapping redundant functions, we validate PfA-M17 as a potential novel drug target.

microbiology↗

bioRxiv2021

Antibiotic inhibition of the Plasmodium apicoplast decreases haemoglobin degradation and antagonises dihydoartemisinin action

The World Health Organisation (WHO) recommends artemisinin (ART) combinations for treatment of uncomplicated Plasmodium falciparum malaria. Understanding the interaction between co-administered drugs within combination therapies is clinically important to prevent unintended consequences. The WHO guidelines recommend second line treatments that combine artesunate with tetracycline, doxycycline, or clindamycin--antibiotics that target the Plasmodium relict plastid, the apicoplast. In addition, antibiotics can be used simultaneously against other infectious diseases, leading to their inadvertent combination with ARTs. One consequence of apicoplast inhibition is a perturbation to haemoglobin uptake and trafficking--a pathway required for activation of ART derivatives. Here, we show that apicoplast-targeting antibiotics reduce the abundance of the catalyst of ART activation (free haem) in P. falciparum, likely through diminished haemoglobin digestion. We demonstrate antagonism between ART and these antibiotics, suggesting that apicoplast inhibitors reduce ART activation. These data have potential clinical implications due to the reliance on--and widespread use of--both ARTs and these antibiotics in malaria endemic regions.

microbiology↗

bioRxiv2021

Peroxide antimalarial drugs target redox homeostasis in Plasmodium falciparum infected red blood cells

Plasmodium falciparum causes the most lethal form of malaria. Peroxide antimalarials based on artemisinin underpin the frontline treatments for malaria, but artemisinin resistance is rapidly spreading. Synthetic peroxide antimalarials, known as ozonides, are in clinical development and offer a potential alternative. Here, we used chemoproteomics to investigate the protein alkylation targets of artemisinin and ozonide probes, including an analogue of the ozonide clinical candidate, artefenomel. We greatly expanded the list of protein targets for peroxide antimalarials and identified significant enrichment of redox-related proteins for both artemisinins and ozonides. Disrupted redox homeostasis was confirmed by dynamic live imaging of the glutathione redox potential using a genetically encoded redox-sensitive fluorescence-based biosensor. Targeted LC-MS-based thiol metabolomics also confirmed changes in cellular thiol levels. This work shows that peroxide antimalarials disproportionately alkylate proteins involved in redox homeostasis and that disrupted redox processes are involved in the mechanism of action of these important antimalarials. ImportanceThe frontline treatments for malaria are combination therapies based on the peroxide antimalarial, artemisinin. Concerningly, artemisinin resistance has emerged in malaria-endemic regions, and now poses a major threat to malaria treatment and eradication efforts. New medicines are urgently required to replace the artemisinins, and some of the most advanced candidates are the fully synthetic peroxide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel). The mechanism of action of peroxide antimalarials involves the reductive activation of the peroxide bond by intra-parasitic haem, but there is no consensus regarding the specific protein targets of the resulting radical species for artemisinins and/or the ozonides. This study provides a comprehensive and unbiased chemoproteomic profile of over 400 target proteins, and confirms the specific impact of peroxide antimalarials on redox metabolism. The key role of redox targets is particularly relevant considering that the mechanism of artemisinin resistance appears to involve modulation of peroxide activation and redox homeostasis.

pharmacology and toxicology↗

bioRxiv2020

System-wide biochemical analysis reveals ozonide antimalarials initially act by disrupting Plasmodium falciparum haemoglobin digestion

Ozonide antimalarials, OZ277 (arterolane) and OZ439 (artefenomel), are synthetic peroxide-based antimalarials with potent activity against the deadliest malaria parasite, Plasmodium falciparum. Here we used a "multi-omics" workflow, in combination with activity-based protein profiling (ABPP), to demonstrate that peroxide antimalarials initially target the haemoglobin (Hb) digestion pathway to kill malaria parasites. Time-dependent metabolomic profiling of ozonide-treated P. falciparum infected red blood cells revealed a rapid depletion of short Hb-derived peptides followed by subsequent alterations in lipid and nucleotide metabolism, while untargeted peptidomics showed accumulation of longer Hb-derived peptides. Quantitative proteomics and ABPP assays demonstrated that Hb-digesting proteases were increased in abundance and activity following treatment, respectively. The association between ozonide activity and Hb catabolism was also confirmed in a K13-mutant artemisinin resistant parasite line. To demonstrate that compromised Hb catabolism may be a primary mechanism involved in ozonide antimalarial activity, we showed that parasites forced to rely solely on Hb digestion for amino acids became hypersensitive to short ozonide exposures. Quantitative proteomics analysis also revealed parasite proteins involved in translation and the ubiquitin-proteasome system were enriched following drug treatment, suggestive of the parasite engaging a stress response to mitigate ozonide-induced damage. Taken together, these data point to a mechanism of action involving initial impairment of Hb catabolism, and indicate that the parasite regulates protein turnover to manage ozonide-induced damage. Author SummaryThe ozonides are a novel class of fully synthetic antimalarial drugs with potent activity against all parasite species that cause malaria, including the deadliest, Plasmodium falciparum. With the emergence of resistance to current frontline artemisinin-based antimalarials, new drugs are urgently needed and a clear understanding of their mechanism of action is essential so that they can be optimally deployed in the field. Here, we studied the biochemical effects of two ozonides, OZ277 (marketed in India in combination with piperaquine) and OZ439 (in Phase IIb clinical trials) in P. falciparum parasites using an untargeted multi-omics approach consisting of proteomics, peptidomics and time-dependent metabolomics, along with activity-based protease profiling. We found that the ozonides initially disrupt haemoglobin metabolism and that they likely engage the parasite proteostatic stress response. Furthermore, when the duration of ozonide exposure was extended beyond 3 hours to reflect clinically-relevant exposure periods, additional parasite biochemical pathways were perturbed. This comprehensive analysis provides new insight into the antimalarial mode of action of ozonides and provides new opportunities for interventions to enhance their antimalarial efficacy.

microbiology↗