Unveiling the Therapeutic Potential of Folate-Dependent One-Carbon Metabolism in Cancer and Neurodegeneration.

Cellular metabolism is crucial for various physiological processes, with folate-dependent one-carbon (1C) metabolism playing a pivotal role. Folate, a B vitamin, is a key cofactor in this pathway, supporting DNA synthesis, methylation processes, and antioxidant defenses. In dividing cells, folate facilitates nucleotide biosynthesis, ensuring genomic stability and preventing carcinogenesis. Additionally, in neurodevelopment, folate is essential for neural tube closure and central nervous system formation. Thus, dysregulation of folate metabolism can contribute to pathologies such as cancer, severe birth defects, and neurodegenerative diseases. Epidemiological evidence highlights folate's impact on disease risk and its potential as a therapeutic target. In cancer, antifolate drugs that inhibit key enzymes of folate-dependent 1C metabolism and strategies targeting folate receptors are current therapeutic options. However, folate's impact on cancer risk is complex, varying among cancer types and dietary contexts. In neurodegenerative conditions, including Alzheimer's and Parkinson's diseases, folate deficiency exacerbates cognitive decline through elevated homocysteine levels, contributing to neuronal damage. Clinical trials of folic acid supplementation show mixed outcomes, underscoring the complexities of its neuroprotective effects. This review integrates current knowledge on folate metabolism in cancer and neurodegeneration, exploring molecular mechanisms, clinical implications, and therapeutic strategies, which can provide crucial information for advancing treatments.

Anti-virulence potential of iclaprim, a novel folic acid synthesis inhibitor, against Staphylococcus aureus.

Infections caused by Staphylococcus aureus pose a significant global public problem. Therefore, new antibiotics and therapeutic strategies are needed to combat this pathogen. This investigation delves into the effects of iclaprim, a newly discovered inhibitor of folic acid synthesis, on S. aureus virulence. The phenotypic and genotypic effects of iclaprim were thoroughly examined in relation to virulence factors, biofilm formation, and dispersal, as well as partial virulence-encoding genes associated with exoproteins, adherence, and regulation in S. aureus MW2, N315, and ATCC 25923. Then, the in vivo effectiveness of iclaprim on S. aureus pathogenicity was explored by a Galleria mellonella larvae infection model. The use of iclaprim at sub-inhibitory concentrations (sub-MICs) resulted in a reduction of α-hemolysin (Hla) production and a differential effect on the activity of coagulase in S. aureus strains. The results of biofilm formation and eradication assay showed that iclaprim was highly effective in depolymerizing the mature biofilm of S. aureus strains at concentrations of 1 MIC or greater, however, inhibited the biofilm-forming ability of only strains N315 and ATCC 25923 at sub-MICs. Interestingly, treatment of strains with sub-MICs of iclaprim resulted in significant stimulation or suppression of most virulence-encoding genes expression. Iclaprim did not affect the production of δ-hemolysin or staphylococcal protein A (SpA), nor did it impact the total activity of proteases, nucleases, and lipases. In vivo testing showed that sub-MICs of iclaprim significantly improves infected larvae survival. The present study offered valuable insights towards a better understating of the influence of iclaprim on different strains of S. aureus. The findings suggest that iclaprim may have potential as an anti-virulence and antibiofilm agent, thus potentially mitigating the pathogenicity of S. aureus and improving clinical outcomes associated with infections caused by this pathogen. KEY POINTS: • Iclaprim effectively inhibits α-hemolysin production and biofilm formation in a strain-dependent manner and was an excellent depolymerizing agent of mature biofilm • Iclaprim affected the mRNA expression of virulence-encoding genes associated with exoproteins, adherence, and regulation • In vivo study in G. mellonella larvae challenged with S. aureus exhibited that iclaprim improves larvae survival.

In vitro efficacy of next-generation dihydrotriazines and biguanides against babesiosis and malaria parasites.

Babesia and Plasmodium pathogens, the causative agents of babesiosis and malaria, are vector-borne intraerythrocytic protozoan parasites, posing significant threats to both human and animal health. The widespread resistance exhibited by these pathogens to various classes of antiparasitic drugs underscores the need for the development of novel and more effective therapeutic strategies. Antifolates have long been recognized as attractive antiparasitic drugs as they target the folate pathway, which is essential for the biosynthesis of purines and pyrimidines, and thus is vital for the survival and proliferation of protozoan parasites. More efficacious and safer analogs within this class are needed to overcome challenges due to resistance to commonly used antifolates, such as pyrimethamine, and to address liabilities associated with the dihydrotriazines, WR99210 and JPC-2067. Here, we utilized an in vitro culture condition suitable for the continuous propagation of Babesia duncani, Babesia divergens, Babesia MO1, and Plasmodium falciparum in human erythrocytes to screen a library of 50 dihydrotriazines and 29 biguanides for their efficacy in vitro and compared their potency and therapeutic indices across different species and isolates. We identified nine analogs that inhibit the growth of all species, including the P. falciparum pyrimethamine-resistant strain HB3, with IC50 values below 10 nM, and display excellent in vitro therapeutic indices. These compounds hold substantial promise as lead antifolates for further development as broad-spectrum antiparasitic drugs.

folA thyA knockout E. coli as a suitable surrogate model for evaluation of antifolate sensitivity against PfDHFR-TS.

A new superior bacteria complementation model was achieved for testing antifolate compounds and investigating antifolate resistance in the dihydrofolate reductase (DHFR) enzyme of the malaria parasite. Earlier models depended on the addition of trimethoprim (TMP) to chemically suppress the host Escherichia coli (Ec) DHFR function. However, incomplete suppression of EcDHFR and potential interference of antibiotics needed to maintain plasmids for complementary gene expression can complicate the interpretations. To overcome such limitations, the folA (F) and thyA (T) genes were genetically knocked out (Δ) in E. coli BL21(DE3). The resulting EcΔFΔT cells were thymidine auxotroph where thymidine supplementation or functional complementation with heterologous DHFR-thymidylate synthase (TS) is needed to restore the loss of gene functions. When tested against pyrimethamine (PYR) and its analogs designed to target Plasmodium falciparum (Pf) DHFR-TS, the 50 % inhibitory concentration values obtained from EcΔFΔT surrogates expressing wildtype (PfTM4) or double mutant (PfK1) DHFR-TS showed strong correlations to the results obtained from the standard in vitro P. falciparum growth inhibition assay. Interestingly, while TMP had little effect on the susceptibility to PYR and analogs in EcΔFΔT expressing PfDHFR-TS, it hypersensitized the chemically knockdown E. coli BL21(DE3) expressing PfTM4 DHFR-TS but desensitized the one carrying PfK1 DHFR-TS. The low intrinsic expression level of PfTM4 in E. coli BL21(DE3) by western blot analysis may explain the hypersensitive to antifolates of chemical knockdown bacteria surrogate. These results demonstrated the usefulness of EcΔFΔT surrogate as a new tool for antifolate antimalarial screening with potential application for investigation of antifolate resistance mechanism.

Role of Mitochondrial and Cytosolic Folylpolyglutamate Synthetase in One-Carbon Metabolism and Antitumor Efficacy of Mitochondrial-Targeted Antifolates.

Folate-dependent one-carbon (C1) metabolism encompasses distinct cytosolic and mitochondrial pathways connected by an interchange among serine, glycine, and formate. In both the cytosol and mitochondria, folates exist as polyglutamates, with polyglutamylation catalyzed by folylpolyglutamate synthetase (FPGS), including cytosolic and mitochondrial isoforms. Serine is metabolized by serine hydroxymethyltransferase (SHMT)2 in the mitochondria and generates glycine and C1 units for cellular biosynthesis in the cytosol. AGF347 is a novel pyrrolo[3,2-day]pyrimidine antifolate that targets SHMT2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. FPGS is expressed in primary pancreatic cancer specimens, and FPGS levels correlate with in vitro efficacies of AGF347 toward human pancreatic cancer cells. MIA PaCa-2 pancreatic cancer cells with CRISPR knockout of FPGS were engineered to express doxycycline-inducible FPGS exclusively in the cytosol (cFPGS) or in both the cytosol and mitochondria (mFPGS). Folate and AGF347 accumulations increased in both the cytosol and mitochondria with increased mFPGS but were restricted to the cytosol with cFPGS. AGF347-Glu5 inhibited SHMT2 ∼19-fold greater than AGF347 By metabolomics analysis, mFPGS stimulated the C1 flux from serine in the mitochondria and de novo purine and dTTP synthesis far greater than cFPGS. mFPGS enhanced in vitro inhibition of MIA PaCa-2 cell proliferation by AGF347 (∼30-fold) more than cFPGS (∼4.9-fold). Similar results were seen with other pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320); however, elevated mFPGS adversely impacted inhibition by the nonclassical SHMT2/SHMT1 inhibitor SHIN1. These results suggest a critical role of mFPGS levels in determining antitumor efficacies of mitochondrial-targeted pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer. SIGNIFICANCE STATEMENT: AGF347 is a novel pyrrolo[3,2-d]pyrimidine antifolate that targets serine hydroxymethyltransferase (SHMT)2 in the mitochondria and SHMT1 and de novo purine biosynthesis in the cytosol. AGF347 accumulation increases with folylpolyglutamate synthetase (FPGS) levels in both the cytosol and mitochondria. Increased mitochondrial FPGS stimulated one-carbon metabolic fluxes in the cytosol and mitochondria and substantially enhanced in vitro inhibition of pancreatic cancer cells by AGF347. Mitochondrial FPGS levels play important roles in determining the antitumor efficacies of pyrrolo[3,2-d]pyrimidine antifolates for pancreatic cancer.

Identification of Innovative Folate Inhibitors Leveraging the Amino Dihydrotriazine Motif from Cycloguanil for Their Potential as Anti-Trypanosoma brucei Agents.

Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (Ki = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.

Synthesis, molecular modeling simulations and anticancer activity of some new Imidazo[2,1-b]thiazole analogues as EGFR/HER2 and DHFR inhibitors.

New imidazo[2,1-b]thiazole analogs were designed, synthesized, and biologically evaluated as anticancer agents. In vitro biological evaluation of the anticancer properties of the compounds was performed against different cancer cell lines. Compounds 23 and 39 showed remarkable broad -spectrum cytotoxic potency on most of the tested cell lines. Compounds 23 and 39 exhibited potent activity against the MCF-7 breast cancer cell line, with IC50 values of 1.81 and 4.95 µM, respectively, compared to DOX and SOR (IC50 values of 4.17 and 7.26 µM, respectively). An enzyme inhibition assay was carried out to clarify the possible mode of action of the tested compounds. Compounds 23 and 39 were identified as possible EGFR, HER-2, and DHFR inhibitors. Cell cycle arrest results indicated that compound 23 caused cell cycle arrest at the G0/G1 phase in the MCF-7 cells and at the G2/M phase in the Hep G2 cells. Compound 39 induced cell cycle arrest at the G2/M phase in Hela cells. In vivo testing of the anticancer activity of the two most promising molecules in this study was conducted, and the results indicated that they possess considerable in vivo anticancer activity in mice. Data obtained from the molecular modeling simulation study were consistent with the biological evaluation results.

Synthesis, molecular docking study and biological evaluation of new pyrrole scaffolds as potential antitubercular agents for dual targeting of enoyl ACP reductase and dihydrofolate reductase.

In this study, new series of N'-(2-(substitutedphenoxy)acetyl)-4-(1H-pyrrol-1-yl)benzohydrazides (3a-j) 4-(2,5-dimethyl-1H-pyrrol-1-yl)-N'-(2-(substitutedphenoxy)acetyl)benzohydrazides (5a-j) were synthesized, characterized and assessed as inhibitors of enoyl ACP reductase and DHFR. Most of the compounds exhibited dual inhibition against the enzymes enoyl ACP reductase and DHFR. Several synthesized substances also demonstrated significant antibacterial and antitubercular properties. A molecular docking analysis was conducted in order to determine the potential mechanism of action of the synthesized compounds. The results indicated that there were binding interactions seen with the active sites of dihydrofolate reductase and enoyl ACP reductase. Additionally, important structural details were identified that play a critical role in sustaining the dual inhibitory activity. These findings were useful for the development of future dual inhibitors. Therefore, this study provided strong evidence that several synthesized molecules could exert their antitubercular properties at the cellular level through multi-target inhibition. By shedding light on the mechanisms through which these compounds exert their inhibitory effects, this research opens up promising avenues for the future development of dual inhibitors with enhanced antibacterial and antitubercular properties. The study's findings underscore the importance of multi-target approaches in drug design, providing a strong foundation for the design and optimization of novel compounds that can effectively target bacterial infections at the cellular level.

Synthesis and in vitro antitumor evaluation of new thieno[2,3-d]pyrimidine derivatives as EGFR and DHFR inhibitors.

New thienopyrimidine derivatives 2-16 have been synthesized and their in vitro cytotoxicity was evaluated against five different human cancer cell lines HCT-116, Hela, MDA-MB-231, MCF7 and PC3. Compounds 6e, 7a, 7b, 7d, 10c and 10e displayed the highest antitumor activity against all tested cell lines compared to Doxorubicin. Enzyme inhibition assay revealed that compounds 6e and 10e showed high inhibitory activity against EGFR-TK, with IC50 values of 0.133 and 0.151 µM, compared to Olmutinib (IC50 = 0.028 µM); while the highest DHFR inhibitory activity was shown by compounds 7d and 10e with IC50 values of 0.462 and 0.541 µM, compared to Methotrexate (IC50 = 0.117 µM). Cell cycle analysis following a flow cytometric study using colorectal HCT-116 cancer cell line proved that compound 6e induced cell cycle arrest in G0-G1 phase, while compound 10e arrested the cell cycle at both G0-G1 and S phases. Additionally, both compounds (6e and 10e) were potently able to induce apoptosis in HCT-116 cell line. Docking results of compounds 6e and 10e into the pocket of EGFR active site showed their similar main binding features with Olmutinib, while compounds 7d and 10e showed only moderate fitting into DHFR compared to methotrexate. In silico studies revealed that most of the tested compounds obeyed Lipinski's RO5 and showed positive drug likeness scores.

Cymoxanil disrupts RNA synthesis through inhibiting the activity of dihydrofolate reductase.

The agricultural fungicide cymoxanil (CMX) is commonly used in the treatment of plant pathogens, such as Phytophthora infestans. Although the use of CMX is widespread throughout the agricultural industry and internationally, the exact mechanism of action behind this fungicide remains unclear. Therefore, we sought to elucidate the biocidal mechanism underlying CMX. This was accomplished by first performing a large-scale chemical-genomic screen comprising the 4000 haploid non-essential gene deletion array of the yeast Saccharomyces cerevisiae. We found that gene families related to de novo purine biosynthesis and ribonucleoside synthesis were enriched in the presence of CMX. These results were confirmed through additional spot-test and colony counting assays. We next examined whether CMX affects RNA biosynthesis. Using qRT-PCR and expression assays, we found that CMX appears to target RNA biosynthesis possibly through the yeast dihydrofolate reductase (DHFR) enzyme Dfr1. To determine whether DHFR is a target of CMX, we performed an in-silico molecular docking assay between CMX and yeast, human, and P. infestans DHFR. The results suggest that CMX directly interacts with the active site of all tested forms of DHFR using conserved residues. Using an in vitro DHFR activity assay we observed that CMX inhibits DHFR activity in a dose-dependent relationship.

Next Generation Chemiluminescent Probes for Antimalarial Drug Discovery.

Malaria is caused by parasites of the Plasmodium genus and remains one of the most pressing human health problems. The spread of parasites resistant to or partially resistant to single or multiple drugs, including frontline antimalarial artemisinin and its derivatives, poses a serious threat to current and future malaria control efforts. In vitro drug assays are important for identifying new antimalarial compounds and monitoring drug resistance. Due to its robustness and ease of use, the [3H]-hypoxanthine incorporation assay is still considered a gold standard and is widely applied, despite limited sensitivity and the dependence on radioactive material. Here, we present a first-of-its-kind chemiluminescence-based antimalarial drug screening assay. The effect of compounds on P. falciparum is monitored by using a dioxetane-based substrate (AquaSpark ß-D-galactoside) that emits high-intensity luminescence upon removal of a protective group (ß-D-galactoside) by a transgenic ß-galactosidase reporter enzyme. This biosensor enables highly sensitive, robust, and cost-effective detection of asexual, intraerythrocytic P. falciparum parasites without the need for parasite enrichment, washing, or purification steps. We are convinced that the ultralow detection limit of less than 100 parasites of the presented biosensor system will become instrumental in malaria research, including but not limited to drug screening.

A folate inhibitor exploits metabolic differences in Pseudomonas aeruginosa for narrow-spectrum targeting.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections for which the development of antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks enzymes required to scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa is to disrupt thymidine synthesis while providing exogenous thymine. However, known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa.Here we characterize fluorofolin, a dihydrofolate reductase (DHFR) inhibitor derived from Irresistin-16, that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. Fluorofolin is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics. Metabolomics and in vitro assays using purified folA confirm that fluorofolin inhibits P. aeruginosa DHFR. Importantly, in the presence of thymine supplementation, fluorofolin activity is selective for P. aeruginosa. Resistance to fluorofolin can emerge through overexpression of the efflux pumps MexCD-OprJ and MexEF-OprN, but these mutants also decrease pathogenesis. Our findings demonstrate how understanding species-specific genetic differences can enable selective targeting of important pathogens while revealing trade-offs between resistance and pathogenesis.

Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate.

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

Direct radiolabeling of methotrexate and methotrexate micelles with Tc-99m using QbD approach.

The aim of the work presented in this manuscript was to radiolabel methotrexate and prepare radiolabeled methotrexate micelles, an antifolate drug with Tc-99m using QbD approach. The radiolabeling was executed using the experimental design and the radiolabeled drug was further encapsulated in micelles. The authors are of the view that the radiolabeled MTX could be used to target the folate receptor overexpressing cancers such as the kidney, colorectal, breast, brain etc thereby opening newer possibilities to the theranostic applications of the formed conjugate.

De Novo Purine Metabolism is a Metabolic Vulnerability of Cancers with Low p16 Expression.

p16 is a tumor suppressor encoded by the CDKN2A gene whose expression is lost in approximately 50% of all human cancers. In its canonical role, p16 inhibits the G1-S-phase cell cycle progression through suppression of cyclin-dependent kinases. Interestingly, p16 also has roles in metabolic reprogramming, and we previously published that loss of p16 promotes nucleotide synthesis via the pentose phosphate pathway. However, the broader impact of p16/CDKN2A loss on other nucleotide metabolic pathways and potential therapeutic targets remains unexplored. Using CRISPR knockout libraries in isogenic human and mouse melanoma cell lines, we determined several nucleotide metabolism genes essential for the survival of cells with loss of p16/CDKN2A. Consistently, many of these genes are upregulated in melanoma cells with p16 knockdown or endogenously low CDKN2A expression. We determined that cells with low p16/CDKN2A expression are sensitive to multiple inhibitors of de novo purine synthesis, including antifolates. Finally, tumors with p16 knockdown were more sensitive to the antifolate methotrexate in vivo than control tumors. Together, our data provide evidence to reevaluate the utility of these drugs in patients with p16/CDKN2Alow tumors as loss of p16/CDKN2A may provide a therapeutic window for these agents. SIGNIFICANCE: Antimetabolites were the first chemotherapies, yet many have failed in the clinic due to toxicity and poor patient selection. Our data suggest that p16 loss provides a therapeutic window to kill cancer cells with widely-used antifolates with relatively little toxicity.

A pH Fingerprint Assay to Identify Inhibitors of Multiple Validated and Potential Antimalarial Drug Targets.

New drugs with novel modes of action are needed to safeguard malaria treatment. In recent years, millions of compounds have been tested for their ability to inhibit the growth of asexual blood-stage Plasmodium falciparum parasites, resulting in the identification of thousands of compounds with antiplasmodial activity. Determining the mechanisms of action of antiplasmodial compounds informs their further development, but remains challenging. A relatively high proportion of compounds identified as killing asexual blood-stage parasites show evidence of targeting the parasite's plasma membrane Na+-extruding, H+-importing pump, PfATP4. Inhibitors of PfATP4 give rise to characteristic changes in the parasite's internal [Na+] and pH. Here, we designed a "pH fingerprint" assay that robustly identifies PfATP4 inhibitors while simultaneously allowing the detection of (and discrimination between) inhibitors of the lactate:H+ transporter PfFNT, which is a validated antimalarial drug target, and the V-type H+ ATPase, which was suggested as a possible target of the clinical candidate ZY19489. In our pH fingerprint assays and subsequent secondary assays, ZY19489 did not show evidence for the inhibition of pH regulation by the V-type H+ ATPase, suggesting that it has a different mode of action in the parasite. The pH fingerprint assay also has the potential to identify protonophores, inhibitors of the acid-loading Cl- transporter(s) (for which the molecular identity(ies) remain elusive), and compounds that act through inhibition of either the glucose transporter PfHT or glycolysis. The pH fingerprint assay therefore provides an efficient starting point to match a proportion of antiplasmodial compounds with their mechanisms of action.

Phase II study of the novel antifolate agent pralatrexate in combination with the histone deacetylase inhibitor romidepsin for the treatment of patients with mature T-cell lymphoma.

Previously, we conducted a Phase I study of the combination of pralatrexate and romidepsin in patients with relapsed/refractory (R/R) lymphomas and subsequently conducted a multicenter Phase II study in patients with untreated or R/R mature T cell lymphomas (MTCL). Patients received pralatrexate 25 mg/m2 and romidepsin 12 mg/m2 every 2 weeks. Fourteen patients were evaluable for efficacy. Overall response rate was 35.7% with CR in 14.3% and disease control in 50%. The mDOR was 8.2 months, mPFS was 3.6 months, and mOS was 20.2 months. Gastrointestinal side effects were most common in up to 33%; there was only one hematologic toxicity of grade 3 anemia. Combining results of MTCL patients from the Phase I and II studies (N = 28), the ORR was 53.5% with CR in 21.4%, disease control in67.8%, and DOR of 7.2 months. The combination was safe however does not out-perform other combination strategies.Trial Registration: www.clinicaltrials.gov (NCT01947140).

Quassinoids from Eurycoma longifolia as Potential Dihydrofolate Reductase Inhibitors: A Computational Study.

BACKGROUND: Quassinoids are degraded triterpene compounds that can be obtained from various species of the Simaroubaceae plant family, including Eurycoma longifolia. Quassinoids are the major compounds in E. longifolia, and they are known to have various medicinal potentials, such as anticancer and antimalarial properties. Dihydrofolate reductase (DHFR) was reported to be one of the important targets for certain anticancer and antimalarial drugs. Twelve quassinoids from E. longifolia were identified to have anticancer effects based on their IC50 values. This study aimed to evaluate the interactions of these twelve quassinoids with DHFR via Autodock 4.2 software and Biovia Discovery Studio Visualiser. METHODS: Twelve quassinoids from E. longifolia and their interactions with DHFR were evaluated via Autodock 4.2 software and Biovia Discovery Studio Visualiser. Their drug-likeness and pharmacokinetic properties were also assessed using the ADMETlab 2.0 program. RESULTS: The molecular docking results showed that eleven quassinoids showed better docking scores than methotrexate, in which the binding energy (BE) of these quassinoids ranged from - 7.87 to -9.58 kcal/mol. Their inhibition constant (Ki) ranged from 0.095 to 1.71 µM. At the same time, the BE and Ki values for methotrexate were -7.80 kcal/mol and 1.64 µM, respectively. CONCLUSION: From the analysis, 6-dehydrolongilactone and eurycomalide B are among the twelve compounds that showed great potential as hit-to-lead compounds based on the docking score on DHFR, drug-likeness, and ADMET properties. These results suggest a great potential to pursue validation studies via in vitro and in vivo models.

Evaluation of the antimalarial activity of SAM13-2HCl with morpholine amide (SKM13 derivative) against antimalarial drug-resistant Plasmodium falciparum and Plasmodium berghei infected ICR mice.

Antimalarial drugs are an urgently need and crucial tool in the campaign against malaria, which can threaten public health. In this study, we examined the cytotoxicity of the 9 antimalarial compounds chemically synthesized using SKM13-2HCl. Except for SKM13-2HCl, the 5 newly synthesized compounds had a 50% cytotoxic concentration (CC50) > 100 µM, indicating that they would be less cytotoxic than SKM13-2HCl. Among the 5 compounds, only SAM13-2HCl outperformed SKM13-2HCl for antimalarial activity, showing a 3- and 1.3-fold greater selective index (SI) (CC50/IC50) than SKM13-2HCl in vitro against both chloroquine-sensitive (3D7) and chloroquine -resistant (K1) Plasmodium falciparum strains, respectively. Thus, the presence of morpholine amide may help to effectively suppress human-infectious P. falciparum parasites. However, the antimalarial activity of SAM13-2HCl was inferior to that of the SKM13-2HCl template compound in the P. berghei NK65-infected mouse model, possibly because SAM13-2HCl had a lower polarity and less efficient pharmacokinetics than SKM13-2HCl. SAM13-2HCl was more toxic in the rodent model. Consequently, SAM13-2HCl containing morpholine was selected from screening a combination of pharmacologically significant structures as being the most effective in vitro against human-infectious P. falciparum but was less efficient in vivo in a P. berghei-infected animal model when compared with SKM13-2HCl. Therefore, SAM13-2HCl containing morpholine could be considered a promising compound to treat chloroquine-resistant P. falciparum infections, although further optimization is crucial to maintain antimalarial activity while reducing toxicity in animals.

How can we optimize the use of methotrexate to treat pediatric patients with inflammatory skin diseases?

INTRODUCTION: Methotrexate (MTX) is a folic acid antagonist used in clinical practice in oncology and rheumatology, as well as in the treatment of inflammatory skin conditions in children. The low-doses of MTX are commonly used in children for the treatment of many inflammatory and autoimmune conditions, including inflammatory skin diseases, due to its anti-inflammatory and immunomodulatory effects. AREAS COVERED: This review discusses the possibilities for optimizing the use of methotrexate in the treatment of pediatric patients with inflammatory skin diseases. A thorough search through PubMed and Embase databases was performed to identify relevant literature. EXPERT OPINION: Clinical observations confirm the high efficacy and safety of low-dose MTX in children with inflammatory skin diseases. Unfortunately, to date there are few studies providing guidelines on the optimal dosage of MTX in children with inflammatory skin diseases; routes of administration; principles of monitoring; and the safety of long-term use of this medication in children. There is still a need for specific recommendations on the safest and most effective dosing and monitoring regimen for children treated with methotrexate for inflammatory skin diseases.