Pyrimethamine

Detailed Mechanism of Competitive Inhibition of DHFR by this compound

This compound acts as a competitive inhibitor of DHFR, meaning it competes with the enzyme's natural substrate, dihydrofolate (DHF), for binding to the active site. patsnap.comasm.org The high affinity of this compound for the DHFR active site allows it to effectively block the binding of DHF. patsnap.com This inhibitory action is highly selective for the plasmodial and Toxoplasma gondii forms of the enzyme over the human counterpart. drugbank.com The binding of this compound to DHFR is a reversible process. nih.gov

Impact on Tetrahydrofolic Acid Regeneration and Downstream Biosynthetic Pathways

The inhibition of DHFR by this compound directly halts the reduction of dihydrofolate (DHF) to tetrahydrofolic acid (THF). wikipedia.orgpatsnap.com THF and its derivatives are crucial one-carbon donors for the synthesis of essential biomolecules. patsnap.com Specifically, the lack of THF disrupts the de novo synthesis of purines and the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), a necessary step in pyrimidine synthesis. nih.govlibretexts.org The ultimate consequence of this disruption is the impaired synthesis of DNA, RNA, and proteins, which are all vital for cell division and growth. patsnap.comgoogle.com

Structural Biology of this compound-DHFR Interactions

The selective toxicity of this compound against parasites like Plasmodium falciparum compared to humans is rooted in the structural differences between their respective DHFR enzymes. aacrjournals.org

In Plasmodium species, this compound binds effectively to the active site of DHFR (pDHFR). The binding is characterized by the pyrimidine ring of the inhibitor being buried within a deep cleft of the enzyme. nih.gov This interaction is stabilized by a combination of hydrogen bonds and van der Waals forces. nih.gov However, mutations in the dhfr gene, particularly at codons 51, 59, 108, and 164, can lead to resistance by introducing steric hindrance and altering the active site's conformation, thereby reducing the binding affinity of this compound. nih.govmdpi.comcambridge.org For instance, the S108N mutation, where serine is replaced by asparagine at position 108, creates steric clashes with the rigid p-chlorophenyl group of this compound, significantly weakening its binding. cambridge.orgproteopedia.org

The binding of this compound within the DHFR active site is a delicate interplay of molecular interactions. Hydrogen bonds are crucial for the initial anchoring of the inhibitor. In Plasmodium vivax DHFR, the N1 and 2-amino nitrogen atoms of this compound's pyrimidine ring form hydrogen bonds with the carboxylate oxygen atoms of Asp-53. nih.gov The 4-amino nitrogen also forms a hydrogen bond with the main chain carbonyl oxygen of Ile-14. nih.gov

Steric hindrance plays a pivotal role in drug resistance. Mutations that introduce bulkier amino acid side chains into the active site can physically obstruct the binding of this compound. mdpi.com The S108N mutation in P. falciparum DHFR is a classic example, where the larger asparagine residue clashes with the p-chlorophenyl group of the drug. cambridge.org This steric conflict can lead to a dislocation of the nicotinamide ring of the NADPH cofactor, further compromising the inhibitor's binding. cambridge.org The loss of hydrogen bonds and the introduction of steric clashes are the primary mechanisms by which mutations in the DHFR gene confer resistance to this compound. wikipedia.org

Interactive Data Table: Key Amino Acid Interactions in this compound Binding

Interactive Data Table: Impact of DHFR Inhibition on Cellular Processes

Comparative Analysis of this compound Binding to Human DHFR (hDHFR)

Thymidine Phosphorylase (TP) Inhibition and Dual Targeting Mechanisms

Recent studies have identified Thymidine Phosphorylase (TP) as a direct target of this compound, unveiling a dual-targeting mechanism that contributes to its antitumor effects. aacrjournals.org While both this compound and the classic DHFR inhibitor, Methotrexate, can suppress cancer cell proliferation by targeting human dihydrofolate reductase (hDHFR), only this compound demonstrates the ability to inhibit epithelial-mesenchymal transition (EMT), metastasis, and invasion of lung cancer cells. aacrjournals.orgnih.gov This distinction suggested that DHFR is not the sole target of this compound's anticancer activity. aacrjournals.org

Further investigation revealed that this compound also targets TP, an enzyme implicated in tumor migration and invasion. aacrjournals.org The structural similarity between this compound and thymidine supports the hypothesis that TP is another target of this drug. aacrjournals.org Molecular docking simulations have shown favorable binding scores for this compound with TP. aacrjournals.org This dual inhibition of both DHFR and TP by this compound plays a synergistic role in its antitumor proliferation and anti-metastatic activities. aacrjournals.orgnih.gov Notably, the overexpression of TP has been associated with a poor prognosis in lung cancer patients, highlighting the clinical relevance of this target. aacrjournals.orgnih.gov

Table 1: Comparative Effects of this compound and Methotrexate on Cancer Cell Processes

Inhibition of Signal Transducer and Activator of Transcription 3 (STAT3) Pathway

This compound has been identified as a novel and specific inhibitor of the Signal Transducer and Activator of Transcription 3 (STAT3) transcriptional activity. nih.govnih.govresearchgate.net STAT3 is a key transcription factor that, when persistently activated, promotes many tumorigenic processes. nih.govnih.govoup.com Interestingly, this compound's inhibitory effect on the STAT3 pathway is not direct. It does not significantly affect the primary steps of STAT3 activation, such as STAT3 phosphorylation, its translocation to the nucleus, or its binding to DNA, at concentrations sufficient to block its transcriptional activity. nih.govnih.govresearchgate.net

The mechanism underlying STAT3 inhibition by this compound is a consequence of its primary action on DHFR. nih.govnih.gov By inhibiting DHFR, this compound leads to a deficiency in reduced folates, which are crucial for the synthesis of purines and pyrimidines. nih.govnih.gov This disruption of folate metabolism, specifically the reduction in the intracellular pool of 5,10-methylene-THF needed for thymidylate synthesis, ultimately results in decreased STAT3 transcriptional activity. nih.gov This finding reveals an unexpected link between folate metabolism and the regulation of the STAT3 signaling pathway. nih.govnih.gov The combination of this compound with other STAT3 inhibitors or therapies targeting downstream effectors may offer novel strategies for treating STAT3-driven cancers. nih.govoup.com

Induction of Cathepsin B-Dependent and Caspase-Dependent Apoptotic Pathways

This compound has been shown to induce apoptosis in various cancer cells, including melanoma and pituitary adenoma cells, through a multifaceted mechanism that involves both cathepsin and caspase cascades. aacrjournals.orgmedicinacomplementar.com.brnih.govresearchgate.netaacrjournals.org This dual-edged mechanism allows this compound to bypass or overcome the resistance to apoptosis often observed in cancer cells. nih.gov

In melanoma cells, this compound treatment leads to the activation of both initiator caspases (caspase-8 and caspase-9) and the executioner caspase-3. medicinacomplementar.com.brnih.govnih.gov The activation of caspase-9 suggests the involvement of the intrinsic, mitochondria-mediated apoptotic pathway. medicinacomplementar.com.br Concurrently, this compound induces an early and significant increase in the expression and activity of cathepsin B. medicinacomplementar.com.br The use of specific inhibitors has confirmed the functional roles of both pathways. A pan-caspase inhibitor (Z-VAD-FMK) and a cathepsin B inhibitor (CA-074-Me) can both individually reduce this compound-induced apoptosis, with a more pronounced protective effect observed when both inhibitors are used simultaneously. medicinacomplementar.com.brnih.gov This indicates that both caspase-dependent and cathepsin B-dependent pathways are actively involved in the execution of apoptosis following this compound treatment. medicinacomplementar.com.brnih.gov

Table 2: Key Mediators in this compound-Induced Apoptosis

Modulation of p38-NF-κB Axis

In the context of castration-resistant prostate cancer (CRPC), this compound has been shown to exert its antitumor effects by inhibiting the p38-NF-κB signaling axis. researchgate.netnih.gov The p38 mitogen-activated protein kinase (MAPK) and the nuclear factor-κB (NF-κB) pathways are crucial in cancer development and progression, regulating processes like proliferation, inflammation, and apoptosis. nih.gov

Research has demonstrated that this compound significantly decreases the phosphorylation of both p38 and the p65 subunit of NF-κB in a dose-dependent manner in CRPC cells, without altering the total protein levels of p38. researchgate.net This suggests that this compound acts as an inhibitor of p38 MAPK activity. nih.gov The inhibition of p38 by this compound subsequently impairs the activity of p65/NF-κB. This was further supported by experiments where both this compound and a specific p38 MAPK inhibitor (SB202190) could prevent both basal and TNF-α-induced NF-κB activity. nih.gov Therefore, this compound negatively regulates p65/NF-κB activity in CRPC cells through a mechanism dependent on the inhibition of p38 MAPK. nih.gov

Regulation of Cell Cycle Progression (e.g., G0/G1 and S-phase arrest)

This compound has been demonstrated to inhibit cancer cell proliferation by inducing cell cycle arrest at different phases, depending on the cancer type. nih.govnih.goviiarjournals.orgnih.gov In human metastatic melanoma cells and castration-resistant prostate cancer cells, this compound treatment leads to a significant accumulation of cells in the S-phase of the cell cycle. nih.govnih.govnih.gov This S-phase arrest is a notable mechanism contributing to its antiproliferative effects in these cancers. nih.govnih.gov

Conversely, in non-small cell lung cancer (NSCLC) A549 cells, this compound induces cell cycle arrest at the G0/G1 phase. iiarjournals.org This G0/G1 arrest is associated with the downregulation of key cell cycle regulatory proteins, including cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), and CDK4, along with the upregulation of the CDK inhibitor p21. iiarjournals.org In JAR trophoblastic cells, treatment with this compound also resulted in G0/G1 and S phase arrest. nih.gov The ability of this compound to halt cell cycle progression at these critical checkpoints is a key component of its anticancer activity. iiarjournals.orgnih.gov

Table 3: Cell Cycle Effects of this compound in Different Cancer Cell Lines

Induction of Ubiquitination-Mediated Degradation of AIMP2-DX2

A novel anticancer mechanism of this compound involves the targeted degradation of AIMP2-DX2, a splice variant of the aminoacyl-tRNA synthetase-interacting multifunctional protein 2 (AIMP2). frontiersin.orgmdpi.comnih.gov While AIMP2 acts as a tumor suppressor, AIMP2-DX2 is a tumorigenic factor that is frequently overexpressed in lung cancer, and its ratio to AIMP2 correlates with cancer progression. frontiersin.orgmdpi.comnih.gov

This compound has been shown to dose-dependently decrease the protein level of AIMP2-DX2 without affecting the level of the tumor-suppressing AIMP2. mdpi.com Further investigation revealed that this compound promotes the ubiquitination of AIMP2-DX2, leading to its subsequent degradation by the proteasome. mdpi.comnih.govbiorxiv.org In lung cancer cells with high expression of AIMP2-DX2, this compound potently suppresses cell growth. mdpi.com This targeted degradation of a pro-tumorigenic protein represents a specific and potent mechanism of this compound's antitumor activity, suggesting its potential as a therapeutic agent for lung cancers characterized by high levels of AIMP2-DX2. mdpi.comnih.gov

Suppression of NRF2 through DHFR Inhibition and One-Carbon Metabolism

This compound has been identified as an indirect inhibitor of the Nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway. nih.gov Its mechanism of action is not through direct binding to NRF2 or its negative regulator, Kelch-like ECH-associated protein 1 (KEAP1), but rather through the inhibition of dihydrofolate reductase (DHFR). nih.govmdpi.com DHFR is a crucial enzyme in one-carbon metabolism, responsible for regenerating tetrahydrofolate from dihydrofolate. cellsignal.com Tetrahydrofolate is essential for the synthesis of purines and pyrimidines, which are the building blocks of DNA and RNA. mdpi.comcellsignal.com

By inhibiting human DHFR (hDHFR), this compound disrupts the one-carbon transfer reactions necessary for nucleotide synthesis. nih.govmdpi.com This disruption of one-carbon metabolism is the key to its NRF2-suppressive activity. nih.gov Studies have shown that the inhibition of DHFR is a prerequisite for the suppression of NRF2 by this compound. nih.gov Metabolomic analyses have revealed strong similarities between the effects of this compound, its potent analogue WCDD115, and the classical DHFR inhibitor methotrexate. nih.gov Mechanistically, this compound has been shown to reduce the half-life of the NRF2 protein by promoting its ubiquitination and subsequent degradation, a process that occurs independently of KEAP1. researchgate.net This highlights the critical role of one-carbon metabolism in maintaining the stability and function of the NRF2 signaling pathway. nih.gov The suppression of NRF2 and its antioxidant functions represents a therapeutic strategy for cancers where NRF2 is overactive. nih.govmdpi.com

Table 1: Comparative Inhibitory Activity of this compound and Analogue WCDD115

Data sourced from Chembo et al., 2025. nih.gov

Mitochondrial Dysfunction and Cytochrome c Release

This compound can induce apoptosis in certain cells through a mechanism involving direct action on mitochondria. researchgate.netmedicinacomplementar.com.br A key event in this process is the disruption of the mitochondrial membrane potential (MMP). researchgate.netmedicinacomplementar.com.br Studies using the A549 non-small cell lung cancer cell line demonstrated that this compound treatment leads to a reduction in MMP. researchgate.net This depolarization of the mitochondrial membrane is a critical step that precedes the release of pro-apoptotic factors from the mitochondrial intermembrane space into the cytosol. researchgate.netembopress.org

One of the most significant of these factors is cytochrome c. embopress.org Following the loss of MMP, this compound enhances the release of cytochrome c into the cytosol. researchgate.netresearchgate.net Once in the cytosol, cytochrome c participates in the formation of the apoptosome, a protein complex that activates downstream caspases and executes the apoptotic program. embopress.org The release of cytochrome c is therefore a hallmark of mitochondrially-driven apoptosis and is a direct consequence of this compound-induced mitochondrial dysfunction. researchgate.netresearchgate.net This pathway highlights a mechanism of action for this compound that is central to programmed cell death. researchgate.net

Table 2: Effects of this compound on Mitochondrial Apoptotic Events in A549 Cells

Data sourced from Lin et al., 2018. researchgate.net

Immunomodulatory Activities, including Apoptosis in Peripheral Blood Lymphocytes

Beyond its antiparasitic functions, this compound possesses immunomodulatory properties, notably the ability to induce apoptosis in peripheral blood lymphocytes (PBLs). nih.govresearchgate.net This effect has been observed in both resting and activated T lymphocytes. researchgate.netresearchgate.net The induction of apoptosis in these immune cells is a key aspect of its immunomodulating activity. medicinacomplementar.com.br

The mechanism underlying this apoptosis involves the intrinsic pathway, characterized by direct effects on mitochondria, rather than relying on death receptors like CD95/Fas. medicinacomplementar.com.brresearchgate.net this compound treatment leads to the activation of upstream caspases, specifically caspase-8 and caspase-10, which subsequently activate effector caspases like caspase-3. researchgate.net Furthermore, this compound has been shown to down-regulate the expression of Bcl-2, an anti-apoptotic protein that normally functions to protect mitochondrial integrity. medicinacomplementar.com.brresearchgate.netresearchgate.net The reduction in Bcl-2 levels facilitates mitochondrial membrane depolarization and the release of cytochrome c, thereby promoting the apoptotic cascade. researchgate.net Studies have shown that in activated PBLs, the apoptotic effect of this compound is dose-dependent. researchgate.net In the context of malarial infections, this compound treatment has been found to increase apoptosis in B lymphocytes and modulate the expression of cytokines, such as upregulating interferon-gamma (IFN-γ) and transforming growth factor-beta (TGF-β). nih.gov

Table 3: Summary of this compound's Apoptotic Effects on Peripheral Blood Lymphocytes

Molecular Basis of this compound Resistance in Plasmodium falciparum

Point Mutations in P. falciparum Dihydrofolate Reductase (Pfdhfr) Gene

Specific Amino Acid Substitutions and Their Impact on Binding Affinity

Several key amino acid substitutions in the Pfdhfr gene have been identified as direct contributors to this compound resistance. The foundational mutation is typically a change from serine to asparagine at codon 108 (S108N). asm.orgird.frird.frtandfonline.com This single mutation is strongly associated with in vitro resistance to this compound. ird.frnih.gov The S108N mutation is thought to cause steric hindrance with the chlorine atom of this compound's aryl group, thereby reducing the drug's binding affinity. researchgate.net

Additional mutations can build upon the S108N background to confer higher levels of resistance. These include:

N51I (Asparagine to Isoleucine at codon 51) : This mutation, in conjunction with S108N, further increases resistance. asm.orgasm.org

C59R (Cysteine to Arginine at codon 59) : Similar to N51I, the C59R mutation adds to the level of resistance when present with other mutations. asm.orgasm.org

I164L (Isoleucine to Leucine at codon 164) : The I164L mutation, when combined with others, leads to a very high level of this compound resistance and can significantly compromise the clinical efficacy of drugs containing it. ird.frasm.org

The binding affinity of this compound is progressively reduced as these mutations accumulate. pnas.org For instance, parasites with a single S108N mutation show a significant increase in resistance, and this is further amplified by the addition of N51I or C59R mutations. asm.org

Table 1: Key Amino Acid Substitutions in Pfdhfr and their Impact on this compound Resistance

Accumulation of Multiple Mutations and Exacerbated Resistance

The development of high-level this compound resistance is a stepwise process involving the accumulation of multiple mutations in the Pfdhfr gene. springermedizin.de While the S108N mutation alone confers a moderate level of resistance, the presence of double, triple, or quadruple mutations leads to a significant increase in the parasite's ability to withstand the drug. asm.orgird.fr

The "triple mutant" (N51I, C59R, and S108N) is strongly associated with a high degree of this compound resistance and is a reliable predictor of treatment failure in vivo. asm.orgmdpi.com The addition of the I164L mutation to this combination, creating a "quadruple mutant," results in even higher levels of resistance, often rendering this compound-based therapies ineffective. ird.frasm.org Studies have shown a direct correlation between the number of Pfdhfr point mutations and the level of both in vitro and in vivo resistance to this compound. asm.org

This compound is frequently used in combination with sulfadoxine, a drug that inhibits a different enzyme in the same folate pathway, dihydropteroate synthase (DHPS). fda.govnih.gov This combination is designed to be synergistic, meaning its combined effect is greater than the sum of its individual effects. oup.comsci-hub.se However, resistance can also develop to this combination therapy.

The synergistic action of the two drugs means that resistance often requires mutations in both target genes: Pfdhfr for this compound and Pfdhps (dihydropteroate synthase gene) for sulfadoxine. oup.com Point mutations in the Pfdhps gene, such as A437G and K540E, are associated with sulfadoxine resistance. asm.orgspringermedizin.de

The presence of mutations in both genes leads to synergistic resistance. For example, a parasite with triple mutations in Pfdhfr (conferring high-level this compound resistance) combined with double mutations in Pfdhps (the "quintuple mutant") is strongly associated with treatment failure of the sulfadoxine-pyrimethamine combination. plos.orgspringermedizin.de The accumulation of mutations appears to follow an ordered pathway, often with initial mutations occurring in Pfdhfr before accumulating in Pfdhps. oup.com This combined resistance undermines the efficacy of the synergistic drug combination. asm.orgasm.org

Molecular Epidemiology of this compound Resistance

The emergence and spread of this compound resistance in Plasmodium falciparum, the parasite responsible for the most severe form of malaria, is a significant public health concern. This resistance is primarily associated with specific point mutations in the parasite's dihydrofolate reductase (dhfr) gene, the target of this compound.

This compound resistance has been observed to originate in specific focal points and subsequently spread globally. oup.com Highly resistant parasite strains, characterized by three or more mutations in the dhfr gene, are thought to have emerged from a limited number of geographical locations, possibly one in Asia and two in South America, before disseminating worldwide. oup.com The Thailand-Cambodia border is considered a primary epicenter for the origin of this compound resistance. nih.gov From there, resistant alleles have migrated to other parts of Southeast Asia and Africa, following a pattern similar to that of chloroquine resistance. oup.comnih.gov

The prevalence of specific this compound resistance-conferring mutations varies geographically. In Central Africa, a high prevalence of Pfdhfr single-nucleotide polymorphisms (SNPs) has been observed, with the triple mutation haplotype (I51R59N108) being particularly frequent in countries like Cameroon, Equatorial Guinea, and Gabon. malariaworld.org Studies in southern China have revealed distinct distribution patterns of pfdhfr alleles between the Yunnan-Burma border and Hainan Island, reflecting their different histories of drug administration. nih.gov

In eastern Africa, there has been a significant increase in mid-level resistance to sulfadoxine-pyrimethamine (SP), a combination therapy, in several countries, including Tanzania, Sudan, Mozambique, and Kenya. bmj.com The prevalence of the pfdhps 540E mutation, a marker for SP resistance, has expanded from limited foci in East Africa to exceed 50% in most of East and South East Africa. plos.org While western Africa has generally maintained low levels of resistance, some countries like Mali have seen an increase. bmj.com

The global migration of these resistant parasites has been a decisive factor in the establishment of drug-resistant P. falciparum populations. oup.com

Longitudinal studies are crucial for monitoring the evolution of this compound resistance over time. In Yaoundé, Cameroon, surveillance from 1994 to 2001 showed a significant and progressive increase in the proportion of this compound-resistant P. falciparum isolates. ird.fr The prevalence of isolates with mutant dhfr alleles rose from 42–45% in 1994–1995 to 88–92% in 2000–2001, with a predominance of triple dhfr mutations in the later years. ird.fr

In Maputo Province, Mozambique, annual surveillance between 2004 and 2008, following the implementation of artesunate plus SP, revealed that the dhfr triple mutation remained close to fixation. lshtm.ac.uk Furthermore, the "quintuple" mutation (dhfr triple mutant combined with dhps double mutant) increased dramatically from 11.0% in 2004 to 75.0% by 2008. lshtm.ac.uk

Conversely, in regions where SP drug pressure has been removed, a decline in resistance markers has been observed. In northwestern Ethiopia, after the withdrawal of SP, there was a significant decrease in the frequency of the triple Pfdhfr mutation from 50.8% to 15.9%. plos.org This suggests that in the absence of drug pressure, resistant strains may be less fit and are gradually replaced by sensitive ones. plos.org However, other long-term studies, such as one in Tanganyika (now part of Tanzania), showed that this compound-resistant parasites could still be detected 20 years after the initial observations of their decline in the absence of drug pressure. who.int

A study in Kilifi, Kenya, revealed that mutations in dhfr associated with this compound resistance were present at high frequencies even before the widespread use of the drug and remained high after its replacement with another first-line treatment. wellcomeopenresearch.org This highlights the complexity of resistance dynamics and the persistence of resistance mutations in a population.

A strong correlation exists between the presence of specific mutations in the dhfr gene and the in vitro sensitivity of P. falciparum to this compound. The 50% inhibitory concentration (IC50), a measure of drug effectiveness in vitro, increases with the number of dhfr mutations.

Studies have consistently shown that isolates with wild-type dhfr alleles are sensitive to this compound, exhibiting low IC50 values. ird.frasm.org In contrast, isolates with single, double, or triple mutations in the dhfr gene show progressively higher IC50 values, indicating increased resistance. ird.frasm.org

For instance, Kenyan field isolates could be categorized into three distinct groups based on their this compound IC50s and dhfr genotypes. asm.org Wild-type isolates had the lowest IC50s, while those with mutations at codon 108 (with or without mutations at codons 51 or 59) showed a significant increase in resistance. asm.org The highest level of resistance was observed in parasites with the triple mutation (Ile-51, Arg-59, and Asn-108). asm.org

Similarly, in Cameroon, a clear distinction was observed between isolates with pure wild-type dhfr alleles (IC50 < 100 nM) and those with pure mutant alleles (IC50 ≥ 100 nM). ird.fr This high correlation between in vitro results and molecular markers validates the use of molecular surveillance to predict this compound resistance levels in a population. ird.fr

The presence of the dhfr triple mutant has been suggested as a useful predictor of treatment failure in some studies. ajtmh.org However, the predictive value can vary regionally. researchgate.net In some regions, the combination of the dhfr triple mutant and specific dhps mutations (such as the Gly-437 + Glu-540 double mutant) is a better predictor of SP treatment failure. ajtmh.org

Table 1: Correlation of P. falciparum dhfr Genotype with In Vitro this compound Resistance

Longitudinal Surveillance of Resistance Trends

Strategies to Circumvent this compound Resistance

The widespread resistance to this compound has necessitated the development of strategies to overcome this challenge. A primary approach is the use of combination therapies. patsnap.comtandfonline.com Combining this compound with other antimalarial drugs that have different mechanisms of action can enhance efficacy and delay the development of resistance. patsnap.com The most well-known combination is sulfadoxine-pyrimethamine (SP), where sulfadoxine inhibits another enzyme in the folate pathway, dihydropteroate synthase (DHPS). patsnap.com

Another strategy is the development of novel antifolates that are effective against this compound-resistant strains. mdpi.comoup.com P218 is a new dihydrofolate reductase (DHFR) inhibitor that has shown high selectivity and activity against this compound-resistant P. falciparum. mdpi.combiorxiv.org It has demonstrated potent inhibition of both the asexual and sexual stages of this compound-resistant parasites. biorxiv.orgresearchgate.net

The combination of a short-acting antifolate, such as chlorproguanil, with dapsone has also been explored as an alternative to SP. oup.com This combination is thought to select less efficiently for resistance due to the shorter half-life of the drugs. oup.com

Furthermore, the use of triple combination therapies is being investigated. tandfonline.com However, the effectiveness of this approach may be limited if significant resistance to the component drugs already exists. tandfonline.com

In some regions where this compound resistance is high, the drug has been replaced by artemisinin-based combination therapies (ACTs) as the first-line treatment for uncomplicated malaria.

This compound's Role in Inhibition of T. gondii Growth

This compound effectively inhibits the growth of Toxoplasma gondii by targeting a crucial metabolic pathway. patsnap.comnih.gov The parasite, like Plasmodium, relies on the synthesis of folic acid for its survival and replication. patsnap.comnih.gov

The mechanism of action of this compound against T. gondii is the inhibition of the enzyme dihydrofolate reductase (DHFR). patsnap.comnih.govoup.com DHFR is essential for the conversion of dihydrofolate to tetrahydrofolate, a vital cofactor in the synthesis of nucleic acids (DNA and RNA) and certain amino acids. patsnap.com By blocking DHFR, this compound disrupts these synthetic processes, leading to an inhibition of parasite growth and ultimately cell death. patsnap.comdrugbank.com

This compound exhibits a higher affinity for the Toxoplasma DHFR enzyme compared to the mammalian host's enzyme, which contributes to its therapeutic effect. oup.com However, this selectivity is not absolute. patsnap.com

In clinical practice, this compound is often used in combination with a sulfonamide, such as sulfadiazine. patsnap.com This combination provides a synergistic effect because sulfadiazine inhibits dihydropteroate synthase (DHPS), another enzyme in the folate biosynthesis pathway. nih.govasm.org The simultaneous blockage of two steps in this pathway is highly effective at inhibiting the proliferation of T. gondii. nih.gov Studies have shown that combining this compound with other drugs can significantly reduce the parasite load in experimental models. brieflands.com In vitro studies have demonstrated that the addition of even low concentrations of this compound can dramatically increase the inhibitory effect of sulfamethoxazole. oup.com

Mechanisms of Resistance in T. gondii to this compound

Resistance to this compound in the protozoan parasite Toxoplasma gondii is a significant concern in the management of toxoplasmosis. The primary mechanism of this resistance involves specific genetic mutations in the parasite's dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. nih.govfrontiersin.org this compound functions by inhibiting the DHFR enzyme, which is crucial for the synthesis of nucleic acids and some amino acids, thereby halting parasite replication. frontiersin.orgpatsnap.com

Research has identified that point mutations within the DHFR coding region can significantly reduce the binding affinity of this compound to the enzyme, allowing the parasite to continue its metabolic functions even in the presence of the drug. patsnap.com These mutations are analogous to those observed in the malaria parasite, Plasmodium falciparum, which also exhibits resistance to this compound. nih.govpnas.org

Several key amino acid substitutions in the T. gondii DHFR enzyme have been identified as conferring resistance. In vitro studies involving random mutagenesis have pinpointed mutations at various residues. For instance, substitutions such as Tryptophan to Arginine at position 25 (W25R), Leucine to Serine at position 98 (L98S), and Leucine to Hisitidine at position 134 (L134H) have been shown to produce drug resistance. nih.govasm.orgnih.govfrontiersin.org

Furthermore, site-directed mutagenesis experiments have revealed other critical mutations. A T83N mutation (Threonine to Asparagine at codon 83) has been found to likely confer resistance to this compound. frontiersin.org The level of resistance can be further amplified when multiple mutations are present. For example, the T83N mutation, when combined with S36R (Serine to Arginine at codon 36) and F245S (Phenylalanine to Serine at codon 245), results in increased resistance. frontiersin.org The accumulation of multiple mutations in the DHFR-TS gene is associated with high-level resistance. nih.gov

It is important to note that different strains of T. gondii may exhibit varying intrinsic sensitivities to this compound, even without mutations in the predicted DHFR-TS protein sequences. nih.govasm.org While the primary focus of resistance research has been on target site modifications, the adaptive potential of the T. gondii parasite suggests that other mechanisms may also contribute to drug resistance, although these are not as well understood. nih.gov

Effects on Cell Proliferation and Viability in Various Cancer Cell Lines

This compound has been shown to effectively reduce the viability of a wide range of cancer cell lines in a dose-dependent manner. nih.govaacrjournals.org For instance, in non-small cell lung cancer (NSCLC) A549 cells, this compound treatment led to a gradual decrease in cell viability with increasing concentrations and longer exposure times. iiarjournals.org This was accompanied by morphological changes indicative of cell distress, such as shrinkage and rounding. iiarjournals.org

Similarly, studies on colorectal cancer (CRC) cell lines, including HCT116, DLD1, RKO, SW480, and HT29, demonstrated that this compound significantly inhibited their growth. nih.gov The inhibitory effect of this compound has also been observed in ovarian cancer cell lines (SKOV3, A2780, OVCAR8, and ES2), where it suppressed cell viability with IC50 values ranging from 20 to 60 µM. techscience.com In prostate cancer, this compound has been found to inhibit the proliferation of DU145 and PC3 cells by inducing S-phase cell cycle arrest. nih.gov

The mechanism behind this anti-proliferative effect is often linked to the inhibition of dihydrofolate reductase (DHFR), an enzyme crucial for DNA synthesis. nih.govaacrjournals.org By blocking this enzyme, this compound disrupts the production of nucleic acids, thereby halting the proliferation of rapidly dividing cancer cells. iiarjournals.orgtechscience.com Some research suggests that the sensitivity of cancer cells to this compound may be correlated with the expression levels of DHFR. aacrjournals.org

Induction of Apoptosis in Cancer Cells

A significant aspect of this compound's anticancer activity is its ability to induce apoptosis, or programmed cell death, in various cancer cell types. researchgate.netiiarjournals.org This process is a key mechanism for eliminating malignant cells.

In non-small cell lung cancer (NSCLC) A549 cells, this compound treatment led to an increase in sub-G1 phase accumulation, a hallmark of apoptosis. iiarjournals.org This was accompanied by the activation of key apoptotic proteins, caspase-9 and caspase-3, and the cleavage of poly (ADP-ribose) polymerase (PARP). iiarjournals.org Furthermore, this compound induced mitochondrial dysfunction, as evidenced by a reduction in mitochondrial membrane potential and the release of cytochrome c. iiarjournals.org The study also noted a decrease in the anti-apoptotic proteins Bcl-2 and Bcl-xL, while the pro-apoptotic protein Bak was significantly increased. iiarjournals.org These findings suggest that this compound triggers the intrinsic, mitochondria-mediated apoptotic pathway in NSCLC cells. iiarjournals.org

Similarly, in castration-resistant prostate cancer (CRPC) cell lines, DU145 and PC3, this compound was shown to promote apoptosis. researchgate.net Flow cytometry analysis revealed a significant increase in apoptotic cells following treatment. researchgate.net This was further supported by a decrease in the expression of the anti-apoptotic protein Bcl-2 and an increase in the cleavage of caspase-3. researchgate.net The pro-apoptotic effects in prostate cancer cells are thought to be mediated through the inhibition of the p38/NF-κB signaling pathway. researchgate.net

Research on melanoma cells has also demonstrated that this compound can induce apoptosis through a mechanism involving both caspases and cathepsins. iiarjournals.orgoup.com In some cancer types, such as ovarian cancer, apoptosis was observed at later time points or higher concentrations, suggesting it may not be the primary mode of cell death in all cases. techscience.com

Suppression of Tumor Growth in Xenograft Models

The antitumor effects of this compound observed in cell cultures have been successfully translated to in vivo animal models. Xenograft studies, where human cancer cells are implanted into immunodeficient mice, have demonstrated this compound's ability to significantly suppress tumor growth.

In a xenograft model using non-small cell lung cancer A549 cells, mice treated with this compound showed a notable reduction in tumor growth compared to the control group. iiarjournals.org After 35 days, the tumors in the this compound-treated mice were significantly smaller and weighed less. iiarjournals.org Similarly, in a Lewis lung cancer (LLC) xenograft model, this compound treatment suppressed tumor growth. aacrjournals.org

Research on prostate cancer has also yielded positive results. In a xenograft model using PC3 prostate cancer cells, oral administration of this compound led to a substantial reduction in tumorigenesis. researchgate.net This was accompanied by a decrease in the expression of Ki-67, a marker of cell proliferation, and phosphorylated p38 in the tumor tissues. researchgate.net Another study using H460 lung cancer cells in a mouse xenograft model showed that this compound significantly reduced both the size and weight of tumors, with an efficacy comparable to the chemotherapy drug taxol. mdpi.com

Furthermore, in a melanoma xenograft model, this compound treatment initiated after the onset of metastatic tumors resulted in a significant reduction in tumor weight over time. researchgate.net These findings from various xenograft models provide strong evidence for the in vivo antitumor efficacy of this compound. researchgate.netiiarjournals.orgnih.gov

Inhibition of Metastasis and Invasion (e.g., Epithelial-Mesenchymal Transition)

Beyond its effects on cell growth and survival, this compound has been shown to inhibit the processes of metastasis and invasion, which are critical for cancer progression and spread. A key mechanism involved in this is the inhibition of the epithelial-mesenchymal transition (EMT). aacrjournals.orgnih.gov EMT is a process where epithelial cells lose their characteristics and gain mesenchymal features, enabling them to become more motile and invasive. mdpi.com

In lung cancer cells, while both this compound and the standard chemotherapy drug methotrexate can inhibit cell proliferation, only this compound was found to inhibit EMT, migration, and invasion. aacrjournals.orgnih.gov This suggests that this compound has an additional target beyond DHFR that is involved in these processes. aacrjournals.orgnih.gov Research has identified thymidine phosphorylase (TP) as another target of this compound. aacrjournals.orgnih.gov TP is an enzyme associated with the EMT of cancer cells. aacrjournals.org By targeting both DHFR and TP, this compound plays a dual role in inhibiting both tumor proliferation and metastasis. aacrjournals.orgnih.gov

Studies on lung cancer cell lines NCI-H460 and A549 demonstrated that this compound inhibited cell migration and invasion. aacrjournals.org Western blot analysis revealed that this compound treatment led to changes in the expression of EMT markers, such as an increase in E-cadherin (an epithelial marker) and a decrease in vimentin (a mesenchymal marker). aacrjournals.org In vivo studies using a Lewis lung cancer xenograft model further confirmed that this compound treatment drastically decreased the number of metastatic tumor nodes on the lung surfaces. aacrjournals.org

Prostate Cancer

This compound has demonstrated significant antitumor effects in preclinical models of prostate cancer, particularly in castration-resistant prostate cancer (CRPC), a more advanced and difficult-to-treat form of the disease. researchgate.netnih.gov

Studies on the metastatic prostate cancer cell lines DU145 and PC3 have shown that this compound inhibits cell proliferation, induces cell cycle arrest in the S phase, and promotes apoptosis. nih.gov The inhibition of cell proliferation is linked to the suppression of DNA synthesis. The pro-apoptotic effect is evidenced by a decrease in the anti-apoptotic protein Bcl-2 and an increase in cleaved caspase-3. researchgate.net

The mechanism of action in prostate cancer has been linked to the inhibition of the p38-NF-κB signaling pathway. researchgate.netnih.gov In vivo studies using a xenograft model with PC3 cells showed that oral administration of this compound suppressed tumor growth. nih.gov This was associated with reduced expression of the proliferation marker Ki-67 and phosphorylated p38 in the tumors. researchgate.net

Furthermore, this compound has been shown to reduce the activity of telomerase, an enzyme important for cancer cell immortality, in PC3 cells. researchgate.net These findings highlight the multifaceted ways in which this compound exerts its antitumor effects on prostate cancer, suggesting its potential as a novel therapeutic strategy for CRPC. nih.gov

Lung Cancer (e.g., Non-Small Cell Lung Carcinoma)

This compound has demonstrated notable antitumor effects in non-small cell lung cancer (NSCLC). iiarjournals.orgiiarjournals.org Its primary mechanism involves the inhibition of dihydrofolate reductase (DHFR), an enzyme essential for DNA synthesis. aacrjournals.orgdrugbank.comwikipedia.org By blocking DHFR, this compound disrupts the folate metabolism, leading to cell cycle arrest and apoptosis (programmed cell death). wikipedia.orgnih.govnih.gov

In vitro studies on the A549 human NSCLC cell line have shown that this compound reduces cell viability and induces G0/G1 phase arrest in the cell cycle. iiarjournals.orgnih.gov This is associated with the downregulation of key cell cycle proteins such as cyclins D1 and E, as well as cyclin-dependent kinases (CDK) 4 and 2, and the upregulation of the cell cycle inhibitor p21. iiarjournals.orgnih.gov Furthermore, this compound treatment leads to an accumulation of cells in the sub-G1 phase, a hallmark of apoptosis, and triggers mitochondrial dysfunction. iiarjournals.orgiiarjournals.org This is evidenced by the activation of caspases-9 and -3 and the cleavage of poly (ADP-ribose) polymerase (PARP). iiarjournals.orgnih.gov

Another significant finding is this compound's ability to inhibit the AIMP2-DX2 protein, a splice variant of AIMP2 that is highly expressed in lung cancer and its levels increase with tumor progression. mdpi.com this compound induces the ubiquitin-mediated degradation of AIMP2-DX2, thereby suppressing tumor growth. mdpi.com In a mouse xenograft model using H460 lung cancer cells, which have high levels of AIMP2-DX2, this compound administration significantly reduced tumor size and weight, comparable to the effects of the chemotherapy drug taxol. mdpi.com

Moreover, research has identified thymidine phosphorylase (TP) as another target of this compound in lung cancer cells. aacrjournals.orgnih.gov While both this compound and the standard chemotherapy drug methotrexate inhibit DHFR, only this compound was found to also inhibit the epithelial-mesenchymal transition (EMT), metastasis, and invasion of lung cancer cells, suggesting a dual targeting mechanism involving both DHFR and TP. aacrjournals.orgnih.gov In vivo studies using a Lewis lung cancer xenograft model in mice confirmed that this compound suppressed tumor growth and drastically decreased the number of metastatic tumor nodes on the lung surfaces. aacrjournals.org

Table 1: Research Findings of this compound in Lung Cancer

Melanoma

In the context of melanoma, this compound has been shown to induce apoptosis in cancer cells through a dual mechanism involving both caspases and cathepsins. researchgate.net Studies have demonstrated that this compound treatment leads to a significant increase in the activities of caspases-3/7, -8, and -9 in melanoma cell lines. researchgate.net

Furthermore, this compound has been observed to work synergistically with the chemotherapy drug temozolomide, enhancing its cytotoxic effects on melanoma cells. nih.govnih.gov This suggests a potential for combination therapies to improve treatment outcomes for melanoma patients. Research on derivatives of this compound, such as methylbenzoprim, has also shown marked tumor regression in mouse models of melanoma. researchgate.net

Table 2: Research Findings of this compound in Melanoma

Breast Cancer

This compound has shown promise in preclinical models of breast cancer by inhibiting the STAT3 signaling pathway. nih.govoup.com STAT3 is a transcription factor that, when activated, promotes tumor cell proliferation, invasion, and migration. nih.govoup.com

In vitro studies using metastatic breast cancer cell lines demonstrated that this compound inhibited STAT3 activity, leading to reduced tumor cell proliferation and invasion. nih.gov In tumor-bearing mice, treatment with this compound resulted in reduced STAT3 activation within the tumor cells, attenuated tumor growth, and decreased tumor-associated inflammation. nih.gov An interesting finding was the enhanced cytotoxic activity of tumor-infiltrating CD8+ T cells, suggesting that this compound may also have an immune-stimulatory effect. nih.gov The potential therapeutic relevance of this compound may extend to triple-negative breast cancer, a subtype where STAT3 activation is known to support cancer stem cells. nih.gov

Table 3: Research Findings of this compound in Breast Cancer

Acute Myeloid Leukemia

In the context of acute myeloid leukemia (AML), this compound has been identified as a potent inducer of apoptosis and differentiation in leukemia cells. nih.gov High-throughput drug screening identified this compound as an effective agent against AML cell lines, with its antileukemic effects partially attributed to its function as a dihydrofolate reductase (DHFR) antagonist. nih.gov

Oral administration of this compound was effective in two different xenograft mouse models of AML, where it specifically targeted leukemic cells while leaving healthy CD34+ cells unaffected. nih.govresearchgate.net The cytotoxic effects of this compound on AML cells could be partially reversed by the addition of excess folic acid, highlighting the importance of folate metabolism as a therapeutic target in this disease. nih.gov Further studies have shown that STAT3 is upregulated in the hematopoietic stem cells of MDS and AML patients, and its increased expression is associated with a poorer prognosis. ashpublications.org this compound, acting as a STAT3 inhibitor, has been shown to inhibit the proliferation of leukemic cell lines by inducing apoptosis. ashpublications.org

Table 4: Research Findings of this compound in Acute Myeloid Leukemia

Pituitary Adenomas

Research has indicated that this compound can sensitize pituitary adenoma cells to the chemotherapeutic agent temozolomide (TMZ). nih.gov Invasive pituitary adenomas are often resistant to conventional therapies, making the synergistic effect of this compound and TMZ a promising therapeutic strategy. nih.gov

In vitro studies on various rat and mouse pituitary adenoma cell lines showed that the combination of this compound and TMZ synergistically inhibited cell proliferation and invasion, and induced apoptosis. nih.gov This combination treatment also led to cell cycle arrest and increased DNA damage. nih.gov The mechanism appears to involve the upregulation of cathepsin B and BAX, an increase in the activity of caspases-3/7, -8, and -9, and the release of cytochrome c from mitochondria. nih.gov In a xenograft tumor model using GH3 pituitary adenoma cells, the combination therapy produced synergistic antitumor activity and improved the survival rate of the animals without increasing systemic side effects. nih.gov

Table 5: Research Findings of this compound in Pituitary Adenomas

Ovarian Cancer

This compound has been investigated for its potential therapeutic effects in ovarian cancer. mdpi.com It has been shown to inhibit the proliferation of ovarian cancer cells and reduce angiogenesis in mouse models. techscience.com The primary mechanism of action in ovarian cancer appears to be the induction of lethal mitophagy, a process of selective degradation of mitochondria by autophagy. techscience.com

In vitro studies have shown that this compound suppresses the proliferation of ovarian cancer cells. techscience.com While it does induce apoptosis, as indicated by the cleavage of caspase-3, this appears to be a secondary effect that occurs after prolonged exposure. techscience.com The more immediate and primary mechanism of cell death is lethal mitophagy, which is triggered by the activation of the p38/JNK/ERK signaling pathway. techscience.com These findings suggest that inducing mitochondrial autophagy could be an effective strategy for treating ovarian cancer. techscience.com

Table 6: Research Findings of this compound in Ovarian Cancer

Triple-Negative Breast Cancer

The anticancer potential of this compound extends to triple-negative breast cancer (TNBC), a particularly aggressive subtype with limited treatment options. mdpi.com The inhibition of STAT3, a key mechanism of this compound, is highly relevant for TNBC as STAT3 activation is known to support the survival and proliferation of cancer stem cells in this subtype. nih.gov While direct studies on this compound in TNBC models are emerging, the established role of STAT3 in TNBC pathogenesis suggests that this compound could be a valuable therapeutic agent. mdpi.comnih.gov

Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia (CLL) is a type of cancer characterized by the excessive production of lymphocytes in the bone marrow. A key molecular feature of CLL is the constitutive activation of the STAT3 (Signal Transducer and Activator of Transcription 3) protein, which plays a crucial role in tumor cell growth. clinicaltrials.govashpublications.org Laboratory studies have demonstrated that this compound can induce the death of CLL cells. clinicaltrials.gov This has led to clinical trials aimed at evaluating its therapeutic potential in patients with this disease. clinicaltrials.govdana-farber.orgcancer.govclinicaltrials.gov

A phase I clinical trial was conducted to determine the safety and optimal dosage of this compound in patients with relapsed CLL. ashpublications.org The study enrolled sixteen patients who had undergone multiple prior therapies. ashpublications.org While no objective responses were observed according to the 2008 International Workshop on Chronic Lymphocytic Leukemia (IW-CLL) criteria, half of the patients achieved stable disease. ashpublications.org One patient, receiving a 50 mg daily dose, remained on therapy for 12 months. ashpublications.org The trial concluded that this compound is safe for CLL patients and that higher doses may be necessary to achieve a therapeutic effect. ashpublications.org Further research in a Phase II study is planned to assess the efficacy of this compound in treating CLL/SLL (Small Lymphocytic Lymphoma). clinicaltrials.govclinicaltrials.gov

Table 1: Clinical Trial of this compound in Chronic Lymphocytic Leukemia

Liver Cancer

Preclinical studies have suggested that this compound may have a therapeutic role in liver cancer. nih.govnih.gov The rationale for its use in this context is linked to the observation that high levels of activated STAT3 are associated with more aggressive liver tumors. researchgate.net A systematic review of preclinical cancer models identified one study on liver cancer as being well-designed enough to potentially be translated into a clinical trial. nih.govnih.gov This was based on the inclusion of appropriate control and treatment groups, as well as comparison with established chemotherapy drugs. nih.govnih.gov

Esophageal Cancer

Research has also explored the potential of this compound in treating esophageal squamous cell carcinoma (ESCC). aacrjournals.orgresearchgate.net In ESCC, the transcription factor NRF2 is often mutated and overactive, leading to resistance to chemotherapy and radiation. aacrjournals.orgresearchgate.netnih.govaacrjournals.org A small molecule screen identified this compound as an inhibitor of NRF2. aacrjournals.orgresearchgate.netnih.govaacrjournals.org

In preclinical studies using mouse models with a specific NRF2 mutation, oral administration of this compound was shown to suppress the development of esophageal abnormalities associated with high NRF2 activity, without causing any observable toxicity. nih.govaacrjournals.org Further investigation has suggested that this compound acts as a "molecular glue," promoting the binding of NRF2 to its suppressor protein KEAP1, specifically in NRF2-mutated ESCC cells. aacrjournals.orgresearchgate.net This action enhances the sensitivity of cancer cells to chemotherapy and radiation. aacrjournals.orgresearchgate.net These findings have provided the basis for a Phase I clinical trial to investigate this compound in NRF2-mutant ESCC. aacrjournals.orgresearchgate.net A systematic review also concluded that there is potential for this compound to be used in clinical trials for esophageal cancer, particularly in combination with existing chemotherapy drugs. nih.gov

DHFR Inhibition in Human Cancer Cells

This compound is a known inhibitor of dihydrofolate reductase (DHFR), an enzyme critical for the synthesis of DNA precursors. nih.govnih.govaacrjournals.orgpatsnap.com By blocking DHFR, this compound disrupts the folate metabolic pathway, leading to a deficiency in reduced folate. nih.govnih.gov This, in turn, inhibits the synthesis of purines and pyrimidines, which are essential building blocks for DNA. nih.gov The effect is particularly pronounced in rapidly dividing cells like cancer cells. patsnap.com

Research has shown that this compound inhibits the activity of human DHFR in various cancer cell lines, including lung cancer cells. aacrjournals.org While both this compound and the well-known DHFR inhibitor methotrexate can inhibit the proliferation of cancer cells by targeting DHFR, this compound has also been observed to inhibit other cancer-promoting processes like epithelial-mesenchymal transition (EMT), metastasis, and invasion, suggesting it may have additional targets. aacrjournals.org The inhibitory effect of this compound on different tumor cell lines has been shown to be positively correlated with the expression level of DHFR. aacrjournals.org

Table 2: Effects of this compound on DHFR and Cancer Cell Proliferation

STAT3 Pathway Inhibition

The Signal Transducer and Activator of Transcription 3 (STAT3) pathway is a key regulator of many processes that promote tumor growth, including inflammation, proliferation, and survival. nih.govnih.gov Persistent activation of STAT3 is a common feature in many human cancers, making it a promising therapeutic target. nih.govnih.govoup.com this compound has been identified as a novel and specific inhibitor of STAT3 transcriptional activity. nih.govnih.gov

Interestingly, this compound does not inhibit the initial steps of STAT3 activation, such as its phosphorylation or movement into the nucleus. nih.gov Instead, it appears to work downstream, inhibiting the ability of STAT3 to activate the transcription of its target genes. nih.gov This inhibitory effect on STAT3 is a consequence of DHFR inhibition and the resulting disruption of folate metabolism. nih.gov The link between folate metabolism and STAT3 activity represents a previously unknown regulatory point in this critical cancer pathway. nih.govnih.gov In preclinical models of breast cancer, this compound has been shown to have both direct tumor-inhibiting and immune-stimulatory effects through its inhibition of STAT3. researchgate.netresearchgate.net

p38-NF-κB Axis Inhibition

In the context of prostate cancer, this compound has been shown to exert its antitumor effects by inhibiting the p38-NF-κB signaling pathway. nih.govresearchgate.netnih.govresearchgate.net Studies on metastatic prostate cancer cell lines (DU145 and PC3) have demonstrated that this compound inhibits cell proliferation, induces cell cycle arrest in the S phase, and promotes apoptosis. nih.govresearchgate.net It also suppressed tumor growth in animal models. nih.govresearchgate.net

The mechanism behind these effects involves the inhibition of the p38 mitogen-activated protein kinase (MAPK). nih.gov Specifically, this compound was found to inhibit the phosphorylation of p38 without affecting the total amount of the p38 protein. nih.gov This inhibition of p38, in turn, affects the nuclear factor kappa B (NF-κB) pathway, a key player in inflammation and cancer progression. nih.gov By suppressing the p38-NF-κB axis, this compound demonstrates a novel mechanism for its antiproliferative and proapoptotic effects in prostate cancer. nih.govresearchgate.netnih.gov

Role of Thymidine Phosphorylase in Cancer

Thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor (PD-ECGF), is an enzyme with a multifaceted and dual role in the landscape of oncology. uah.es It is a key enzyme in the pyrimidine salvage pathway, catalyzing the reversible conversion of thymidine to thymine and 2-deoxy-D-ribose-1-phosphate. nih.govresearchgate.net While this function is primarily catabolic, the significance of TP in cancer extends far beyond its metabolic duties. nih.gov

Upregulation of TP is a common feature in a wide array of solid tumors, including breast, colorectal, gastric, glioblastoma, and lung cancers. uah.esaacrjournals.org This overexpression is often associated with a more aggressive tumor phenotype and a poorer prognosis for patients. uah.esaacrjournals.org The elevated levels of TP in the tumor microenvironment can be triggered by various cellular stressors such as hypoxia, low pH, and exposure to radio- and chemotherapy. uah.esnih.gov

The pro-tumorigenic effects of TP are largely attributed to its angiogenic and anti-apoptotic properties. uah.esnih.gov TP stimulates the growth of new blood vessels, a process crucial for tumor expansion and metastasis, by promoting the migration of endothelial cells. uah.es One of its metabolites, 2-deoxy-D-ribose, acts as a chemoattractant for endothelial cells. nih.gov Furthermore, TP can protect cancer cells from apoptosis induced by various stimuli, including hypoxia and DNA-damaging agents. uah.es

Conversely, TP plays a critical role in the activation of certain chemotherapeutic agents, most notably the 5-fluorouracil (5-FU) prodrug capecitabine. uah.esresearchgate.netingentaconnect.com TP converts capecitabine into its active, cytotoxic form, 5-FU, within the tumor tissue. This has led to therapeutic strategies that aim to induce TP expression to enhance the efficacy of capecitabine-based chemotherapy. uah.es

Recent research has identified TP as a direct target of the antimalarial drug this compound. aacrjournals.orgnih.gov While this compound is known to inhibit dihydrofolate reductase (DHFR), its ability to also target TP contributes to its dual role in inhibiting both cancer cell proliferation and metastasis. aacrjournals.orgnih.gov This finding is particularly relevant as data from The Cancer Genome Atlas (TCGA) database reveals a correlation between TP overexpression and poor prognosis in lung cancer patients. aacrjournals.orgnih.gov

Ubiquitin-Mediated Degradation of AIMP2-DX2

Aminoacyl-tRNA synthetase-interacting multifunctional protein 2 (AIMP2) is recognized as a tumor suppressor. However, a splice variant of this protein, AIMP2-DX2, which lacks exon 2, exhibits oncogenic properties. nih.govmdpi.com High expression levels of AIMP2-DX2 are found in various human cancers, particularly lung cancer, where the ratio of AIMP2-DX2 to the full-length AIMP2 increases with tumor progression. nih.gov This elevated AIMP2-DX2 level is associated with poorer patient survival.

The pro-cancer activity of AIMP2-DX2 stems from its ability to interfere with the tumor-suppressive functions of the wild-type AIMP2. For instance, AIMP2-DX2 can competitively inhibit the binding of AIMP2 to proteins like p53, thereby preventing the AIMP2-mediated stabilization and pro-apoptotic activity of p53.

A key regulatory mechanism for protein levels in cells is the ubiquitin-proteasome system, which tags proteins for degradation. It has been discovered that this compound can induce the ubiquitin-mediated degradation of AIMP2-DX2. nih.govmdpi.com In a study involving lung cancer cell lines, this compound was shown to decrease the levels of AIMP2-DX2 in a dose-dependent manner, without affecting the levels of the full-length AIMP2 protein. nih.govmdpi.com Further experiments confirmed that this reduction in AIMP2-DX2 was due to its ubiquitination and subsequent degradation. nih.govmdpi.com

This targeted degradation of the oncogenic AIMP2-DX2 by this compound has shown significant antitumor effects. In a panel of lung cancer cell lines, this compound most potently suppressed the growth of H460 cells, which have high endogenous levels of AIMP2-DX2. nih.govmdpi.com Moreover, in a mouse xenograft model using H460 cells, the administration of this compound led to a significant reduction in tumor size and weight, an effect comparable to the standard chemotherapy drug taxol. nih.govmdpi.com Analysis of the tumor tissue from these mice revealed decreased levels of AIMP2-DX2, confirming the drug's mechanism of action in vivo. mdpi.com These findings highlight the potential of repurposing this compound for the treatment of cancers with high AIMP2-DX2 expression. nih.govmdpi.com

NRF2 Suppression and its Implications

Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays a central role in the cellular response to oxidative stress. nih.govnih.gov While its protective functions are crucial for normal cell homeostasis, aberrant and persistent activation of NRF2 is a common feature in several cancers, including lung and upper aerodigestive cancers. nih.govnih.gov This hyperactivation of NRF2 can promote tumor initiation and progression, and importantly, confers resistance to various cancer therapies, including chemotherapy, radiation therapy, and immune checkpoint inhibitors. nih.govnih.gov

Given the role of NRF2 in promoting cancer cell survival and therapeutic resistance, there is a significant effort to develop inhibitors of this pathway. nih.gov this compound has been identified as an inhibitor of NRF2. nih.govnih.gov Studies have shown that this compound can suppress both NRF2 mRNA and protein levels. nih.gov

The mechanism by which this compound inhibits NRF2 is linked to its known function as an inhibitor of dihydrofolate reductase (DHFR). nih.govwustl.edu Inhibition of DHFR disrupts one-carbon metabolism, which appears to be essential for maintaining the NRF2 signaling pathway. nih.gov In fact, research has demonstrated that the inactivation of DHFR is a prerequisite for the suppression of NRF2 by this compound. nih.govnih.gov Proteomic analyses have revealed that the effects of this compound on cellular protein expression overlap with those of methotrexate, a classical DHFR inhibitor, and include the suppression of the NRF2 oxidative stress response. nih.govnih.gov

Furthermore, some studies suggest that this compound may also promote the ubiquitination and subsequent degradation of the NRF2 protein, thereby reducing its half-life in cancer cells. researchgate.netbiorxiv.orgaacrjournals.org This dual mechanism of inhibiting NRF2 expression and promoting its degradation makes this compound a compelling candidate for targeting NRF2-driven cancers. The suppression of NRF2 by this compound has been shown to inhibit the growth of NRF2-hyperactive esophageal squamous cell carcinoma cells in preclinical models. researchgate.netaacrjournals.org

Table 1: Effects of this compound on AIMP2-DX2 and NRF2

Sensitization to Temozolomide

Temozolomide (TMZ) is a standard-of-care alkylating agent used in the treatment of glioblastoma and metastatic melanoma. nih.govoaepublish.com However, both intrinsic and acquired resistance to TMZ pose significant clinical challenges. oaepublish.com One of the key mechanisms of resistance involves the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which removes the cytotoxic lesions induced by TMZ. jbuon.com

Research has shown that this compound can enhance the cytotoxic effects of temozolomide in cancer cells, effectively sensitizing them to the drug. nih.govnih.gov In a screening of 2,000 compounds, this compound was identified as a molecule that, while having little intrinsic cytotoxicity on its own, significantly enhanced the growth-inhibitory effects of TMZ in melanoma cell lines. nih.gov The combination of this compound and TMZ resulted in a synergistic inhibition of cell proliferation. nih.gov

This sensitization is attributed to the antifolate activity of this compound. nih.gov The combination treatment leads to an increase in DNA double-strand breaks and apoptosis, along with cell cycle arrest. nih.gov Notably, the enhanced cell death induced by the combination can be rescued by the administration of leucovorin, a reduced folate, which confirms the role of folate antagonism in this synergistic interaction. nih.gov An important finding is that the sensitizing effect of this compound to TMZ appears to be independent of the MGMT status of the cancer cells, making it a potentially valuable strategy for both MGMT-expressing and MGMT-negative tumors. jbuon.com

The potential of this combination has also been explored in glioblastoma. In vitro studies using glioblastoma stem-like cells and primary glioblastoma cells have demonstrated that this compound enhances the cytotoxic effect of TMZ and sensitizes these cells to radiation therapy. jbuon.comnih.gov Given that this compound is an orally available drug that can cross the blood-brain barrier, its combination with TMZ holds promise for improving the treatment of brain tumors like glioblastoma. nih.gov

Combination with Standard Chemotherapy Drugs

The potential of this compound as an adjunct to standard chemotherapy extends beyond its combination with temozolomide. Its multifaceted mechanisms of action, including the inhibition of DHFR, STAT3, and thymidine phosphorylase, make it a candidate for combination therapies in various cancers. mdpi.comoncoscience.usnih.gov

In the context of glioblastoma, the combination of this compound with the repurposed antifungal drug itraconazole and temozolomide has been investigated. oup.comugent.be In vitro studies using primary glioblastoma patient-derived stem cell lines showed that both itraconazole and this compound, at clinically relevant concentrations, reduced cell viability. oup.comugent.be The combination of these two repurposed drugs with low-dose temozolomide resulted in a significant decrease in cell viability compared to temozolomide monotherapy. oup.comugent.be Synergy analysis indicated mainly additive effects between itraconazole and this compound. oup.com

Another preclinical study investigated the combination of this compound with sorafenib, a multi-kinase inhibitor used in the treatment of various cancers. In a mouse model, the combination of this compound and sorafenib demonstrated a clear synergistic effect in reducing tumor size compared to either drug alone or the control group. nih.gov

The rationale for these combinations often lies in targeting multiple, often complementary, cancer-driving pathways. For instance, in a proposed multidrug adjunctive cancer treatment regimen, this compound is included alongside drugs like celecoxib, disulfiram, and itraconazole to interfere with common growth-driving elements in cancers such as cholangiocarcinoma, colon adenocarcinoma, and non-small-cell lung cancer. mdpi.com

Table 2: Preclinical Combinatorial Studies with this compound

Drug Interactions Affecting this compound Pharmacokinetics (e.g., co-administration with Azithromycin)

Correlation between Pharmacokinetic Parameters and Clinical Outcomes (e.g., treatment failure, resistance development)

The clinical efficacy of this compound is intrinsically linked to its pharmacokinetic (PK) profile. Research has established a significant correlation between specific PK parameters, such as plasma drug concentration and elimination half-life, and clinical outcomes, including treatment success, failure, and the emergence of drug-resistant parasite strains. The interplay between drug exposure and parasite susceptibility is a critical determinant in the therapeutic success of this compound-containing regimens.

Detailed Research Findings

Studies investigating the use of sulfadoxine-pyrimethamine (SP) for treating Plasmodium falciparum malaria have provided direct evidence linking drug concentrations to clinical results. A key factor in treatment failure is the presence of parasites with genetic mutations that confer resistance. researchgate.netasm.org However, even in the face of resistant genotypes, adequate drug exposure can play a decisive role in clearing the infection.

Conversely, suboptimal drug concentrations are a risk factor for treatment failure. researchgate.netresearchgate.net In one multivariate analysis, a low day 3 blood concentration of this compound was identified as a risk factor for late treatment failure. nih.gov Physiological conditions that alter pharmacokinetics can also impact outcomes. For instance, pregnancy is associated with a significantly lower area under the concentration-time curve (AUC) for this compound, which could compromise both curative efficacy and the prophylactic effect of the drug. asm.org

The long elimination half-life of this compound is another critical pharmacokinetic parameter that influences the development of resistance. asm.orgnih.govnih.gov While beneficial for maintaining therapeutic concentrations, a long half-life creates a prolonged period where sub-therapeutic drug levels persist in the body. This extended window provides a strong selective pressure for existing parasites with resistance-conferring mutations, allowing them to survive and propagate. asm.orgnih.govnih.gov Modeling studies have shown that this selection is the first stage in the development of widespread clinical resistance. asm.orgnih.gov

However, the relationship is not always direct. A study in Tanzania found uniform efficacy of SP even with reduced doses, but this was in a context where all tested parasite isolates showed full sensitivity in vitro. oup.com In another case, treatment failure in an individual was not attributable to parasite resistance or inadequate plasma drug levels, but rather to an undefined host factor, demonstrating the complexity of the host-parasite-drug interaction. sci-hub.se

Interactive Data Table: this compound Concentration and Clinical Outcome in Malaria Treatment

The following table summarizes findings from studies correlating this compound plasma concentrations with treatment outcomes.

Exploration of Structure-Activity Relationships (SAR)

Repurposing this compound for Emerging Disease Treatment