Chemical Composition, Anti-bacterial Activity and Molecular Docking Studies of Essential Oil Isolated from Sa Sam Nam (Launaea sarmentosa).

Launaea sarmentosa, also known as Sa Sam Nam, is a widely used remedy in Vietnamese traditional medicine and cuisine. However, the chemical composition and bioactivity of its essential oil have not been elucidated yet. In this study, we identified 40 compounds (98.6% of total peak area) in the essential oil via GC-MS analysis at the first time. Among them, five main compounds including Thymohydroquinone dimethyl ether (52.4%), (E)-α-Atlantone (9.0%), Neryl isovalerate (6.6%), Davanol D2 (isomer 2) (3.9%), and trans-Sesquisabinene hydrate (3.9%) have accounted for 75.8% of total peak area. The anti-bacterial activity of the essential oil against 4 microorganisms including Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa has also investigated via agar well diffusion assay. The results showed that the essential oil exhibited a strong antibacterial activity against Bacillus subtilis with the inhibition zones ranging from 8.2 to 18.7 mm. To elucidate the anti-bacterial effect mechanism of the essential oil, docking study of five main compounds of the essential oil (Thymohydroquinone dimethyl ether, (E)-α-Atlantone, Neryl isovalerate, Davanol D2 (isomer 2), and trans-Sesquisabinene hydrate) against some key proteins for bacterial growth such as DNA gyrase B, penicillin binding protein 2A, tyrosyl-tRNA synthetase, and dihydrofolate reductase were performed. The results showed that the main constituents of essential oil were highly bound with penicillin binding protein 2A with the free energies ranging -27.7 to -44.8 kcal/mol, which suggests the relationship between the antibacterial effect of essential oil and the affinity of main compounds with penicillin binding protein. In addition, the free energies of main compounds of the essential oil with human cyclooxygenase 1, cyclooxygenase 2, and phospholipase A2, the crucial proteins related with inflammatory response were less than diclofenac, a non-steroidal antiinflammatory drug. These findings propose the essential oil as a novel and promising anti-bacterial and anti-inflammatory medicine or cosmetic products.

Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

Efficacy of artemether-lumefantrine and dihydroartemisinin-piperaquine and prevalence of molecular markers of anti-malarial drug resistance in children in Togo in 2021.

BACKGROUND: Artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) are the currently recommended first- and second-line therapies for uncomplicated Plasmodium falciparum infections in Togo. This study assessed the efficacy of these combinations, the proportion of Day3-positive patients (D3 +), the proportion of molecular markers associated with P. falciparum resistance to anti-malarial drugs, and the variable performance of HRP2-based malaria rapid diagnostic tests (RDTs). METHODS: A single arm prospective study evaluating the efficacy of AL and DP was conducted at two sites (Kouvé and Anié) from September 2021 to January 2022. Eligible children were enrolled, randomly assigned to treatment at each site and followed up for 42 days after treatment initiation. The primary endpoint was polymerase chain reaction (PCR) adjusted adequate clinical and parasitological response (ACPR). At day 0, samples were analysed for mutations in the Pfkelch13, Pfcrt, Pfmdr-1, dhfr, dhps, and deletions in the hrp2/hrp3 genes. RESULTS: A total of 179 and 178 children were included in the AL and DP groups, respectively. After PCR correction, cure rates of patients treated with AL were 97.5% (91.4-99.7) at day 28 in Kouvé and 98.6% (92.4-100) in Anié, whereas 96.4% (CI 95%: 89.1-98.8) and 97.3% (CI 95%: 89.5-99.3) were observed at day 42 in Kouvé and Anié, respectively. The cure rates of patients treated with DP at day 42 were 98.9% (CI 95%: 92.1-99.8) in Kouvé and 100% in Anié. The proportion of patients with parasites on day 3 (D3 +) was 8.5% in AL and 2.6% in DP groups in Anié and 4.3% in AL and 2.1% DP groups in Kouvé. Of the 357 day 0 samples, 99.2% carried the Pfkelch13 wild-type allele. Two isolates carried nonsynonymous mutations not known to be associated with artemisinin partial resistance (ART-R) (A578S and A557S). Most samples carried the Pfcrt wild-type allele (97.2%). The most common Pfmdr-1 allele was the single mutant 184F (75.6%). Among dhfr/dhps mutations, the quintuple mutant haplotype N51I/C59R/S108N + 437G/540E, which is responsible for SP treatment failure in adults and children, was not detected. Single deletions in hrp2 and hrp3 genes were detected in 1/357 (0.3%) and 1/357 (0.3%), respectively. Dual hrp2/hrp3 deletions, which could affect the performances of HRP2-based RDTs, were not observed. CONCLUSION: The results of this study confirm that the AL and DP treatments are highly effective. The absence of the validated Pfkelch13 mutants in the study areas suggests the absence of ART -R, although a significant proportion of D3 + cases were found. The absence of dhfr/dhps quintuple or sextuple mutants (quintuple + 581G) supports the continued use of SP for IPTp during pregnancy and in combination with amodiaquine for seasonal malaria chemoprevention. TRIAL REGISTRATION: ACTRN12623000344695.

The Differential Translation Capabilities of the Human DHFR2 Gene Indicates a Developmental and Tissue-Specific Endogenous Protein of Low Abundance.

A functional role has been ascribed to the human dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homolog, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2-specific peptides as evidence of its translation. We show definitive evidence that the DHFR2 activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, are differentially associated with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2-specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level.

Investigating the anti-cancer potential of pyrimethamine analogues through a modern chemical biology lens.

Pharmacological inhibition of dihydrofolate reductase (DHFR) is an established approach for treating a variety of human diseases, including foreign infections and cancer. However, treatment with classic DHFR inhibitors, such as methotrexate (MTX), are associated with negative side-effects and resistance mechanisms that have prompted the search for alternatives. The DHFR inhibitor pyrimethamine (Pyr) has compelling anti-cancer activity in in vivo models, but lacks potency compared to MTX, thereby requiring higher concentrations to induce therapeutic responses. The purpose of this work was to investigate structural analogues of Pyr to improve its in vitro and cellular activity. A series of 36 Pyr analogues were synthesized and tested in a sequence of in vitro and cell-based assays to monitor their DHFR inhibitory activity, cellular target engagement, and impact on breast cancer cell viability. Ten top compounds were identified, two of which stood out as potential lead candidates, 32 and 34. These functionalized Pyr analogues potently engaged DHFR in cells, at concentrations as low as 1 nM and represent promising DHFR inhibitors that could be further explored as potential anti-cancer agents.

Methotrexate-based PROTACs as DHFR-specific chemical probes.

Methotrexate (MTX) is a tight-binding dihydrofolate reductase (DHFR) inhibitor, used as both an antineoplastic and immunosuppressant therapeutic. MTX, like folate undergoes folylpolyglutamate synthetase-mediated γ-glutamylation, which affects cellular retention and target specificity. Mechanisms of MTX resistance in cancers include a decrease in MTX poly-γ-glutamylation and an upregulation of DHFR. Here, we report a series of potent MTX-based proteolysis targeting chimeras (PROTACs) to investigate DHFR degradation pharmacology and one-carbon biochemistry. These on-target, cell-active PROTACs show proteasome- and E3 ligase-dependent activity, and selective degradation of DHFR in multiple cancer cell lines. By comparison, treatment with MTX increases cellular DHFR protein expression. Importantly, these PROTACs produced distinct, less-lethal phenotypes compared to MTX. The chemical probe set described here should complement conventional DHFR inhibitors and serve as useful tools for studying one-carbon biochemistry and dissecting complex polypharmacology of MTX and related drugs. Such compounds may also serve as leads for potential autoimmune and antineoplastic therapeutics.

A genetically encoded protein tag for control and quantitative imaging of CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has been successful for hematological malignancies. Still, a lack of efficacy and potential toxicities have slowed its application for other indications. Furthermore, CAR T cells undergo dynamic expansion and contraction in vivo that cannot be easily predicted or controlled. Therefore, the safety and utility of such therapies could be enhanced by engineered mechanisms that engender reversible control and quantitative monitoring. Here, we use a genetic tag based on the enzyme Escherichia coli dihydrofolate reductase (eDHFR), and derivatives of trimethoprim (TMP) to modulate and monitor CAR expression and T cell activity. We fused eDHFR to the CAR C terminus, allowing regulation with TMP-based proteolysis-targeting chimeric small molecules (PROTACs). Fusion of eDHFR to the CAR does not interfere with cell signaling or its cytotoxic function, and the addition of TMP-based PROTACs results in a reversible and dose-dependent inhibition of CAR activity via the proteosome. We show the regulation of CAR expression in vivo and demonstrate imaging of the cells with TMP radiotracers. In vitro immunogenicity assays using primary human immune cells and overlapping peptide fragments of eDHFR showed no memory immune repertoire for eDHFR. Overall, this translationally-orientied approach allows for temporal monitoring and image-guided control of cell-based therapies.

Regulation of eDHFR-tagged proteins with trimethoprim PROTACs.

Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, control of engineered proteins could be used in therapeutic applications. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a small-molecule-driven strategy to achieve rapid, post-translational regulation of protein abundance via recruitment of an E3 ligase to the target protein of interest. Here, we develop several PROTAC molecules by covalently linking the antibiotic trimethoprim (TMP) to pomalidomide, a ligand for the E3 ligase, Cereblon. These molecules induce degradation of proteins of interest (POIs) genetically fused to a small protein domain, E. coli dihydrofolate reductase (eDHFR), the molecular target of TMP. We show that various eDHFR-tagged proteins can be robustly degraded to 95% of maximum expression with PROTAC molecule 7c. Moreover, TMP-based PROTACs minimally affect the expression of immunomodulatory imide drug (IMiD)-sensitive neosubstrates using proteomic and biochemical assays. Finally, we show multiplexed regulation with another known degron-PROTAC pair, as well as reversible protein regulation in a rodent model of metastatic cancer, demonstrating the formidable strength of this system. Altogether, TMP PROTACs are a robust approach for selective and reversible degradation of eDHFR-tagged proteins in vitro and in vivo.

Elongation Factor P Modulates the Incorporation of Structurally Diverse Noncanonical Amino Acids into Escherichia coli Dihydrofolate Reductase.

The introduction of noncanonical amino acids into proteins and peptides has been of great interest for many years and has facilitated the detailed study of peptide/protein structure and mechanism. In addition to numerous nonproteinogenic α-l-amino acids, bacterial ribosome modification has provided the wherewithal to enable the synthesis of peptides and proteins with a much greater range of structural diversity, as has the use of endogenous bacterial proteins in reconstituted protein synthesizing systems. In a recent report, elongation factor P (EF-P), putatively essential for enabling the incorporation of contiguous proline residues into proteins, was shown to facilitate the introduction of an N-methylated amino acid in addition to proline. This finding prompted us to investigate the properties of this protein factor with a broad variety of structurally diverse amino acid analogues using an optimized suppressor tRNAPro that we designed. While these analogues can generally be incorporated into proteins only in systems containing modified ribosomes specifically selected for their incorporation, we found that EF-P could significantly enhance their incorporation into model protein dihydrofolate reductase using wild-type ribosomes. Plausibly, the increased yields observed in the presence of structurally diverse amino acid analogues may result from the formation of a stabilized ribosomal complex in the presence of EF-P that provides more favorable conditions for peptide bond formation. This finding should enable the facile incorporation of a much broader structural variety of amino acid analogues into proteins and peptides using native ribosomes.

Exploration and Biological Evaluation of 1,3-Diamino-7H-pyrrol[3,2-f]quinazoline Derivatives as Dihydrofolate Reductase Inhibitors.

Dihydrofolate reductase (DHFR), a core enzyme of folate metabolism, plays a crucial role in the biosynthesis of purines and thymidylate for cell proliferation and growth in both prokaryotic and eukaryotic cells. However, the development of new DHFR inhibitors is challenging due to the limited number of scaffolds available for drug development. Hence, we designed and synthesized a new class of DHFR inhibitors with a 1,3-diamino-7H-pyrrol[3,2-f]quinazoline derivative (PQD) structure bearing condensed rings. Compound 6r exhibited therapeutic effects on mouse models of systemic infection and thigh infection caused by methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. Moreover, methyl-modified PQD compound 8a showed a strong efficacy in a murine model of breast cancer, which was better than the effects of taxol. The findings showcased in this study highlight the promising capabilities of novel DHFR inhibitors in addressing bacterial infections as well as breast cancer.

A novel antifolate suppresses growth of FPGS-deficient cells and overcomes methotrexate resistance.

Cancer cells make extensive use of the folate cycle to sustain increased anabolic metabolism. Multiple chemotherapeutic drugs interfere with the folate cycle, including methotrexate and 5-fluorouracil that are commonly applied for the treatment of leukemia and colorectal cancer (CRC), respectively. Despite high success rates, therapy-induced resistance causes relapse at later disease stages. Depletion of folylpolyglutamate synthetase (FPGS), which normally promotes intracellular accumulation and activity of natural folates and methotrexate, is linked to methotrexate and 5-fluorouracil resistance and its association with relapse illustrates the need for improved intervention strategies. Here, we describe a novel antifolate (C1) that, like methotrexate, potently inhibits dihydrofolate reductase and downstream one-carbon metabolism. Contrary to methotrexate, C1 displays optimal efficacy in FPGS-deficient contexts, due to decreased competition with intracellular folates for interaction with dihydrofolate reductase. We show that FPGS-deficient patient-derived CRC organoids display enhanced sensitivity to C1, whereas FPGS-high CRC organoids are more sensitive to methotrexate. Our results argue that polyglutamylation-independent antifolates can be applied to exert selective pressure on FPGS-deficient cells during chemotherapy, using a vulnerability created by polyglutamylation deficiency.

Plasmodium falciparum drug resistance-associated mutations in isolates from children living in endemic areas of Burkina Faso.

BACKGROUND: Artemisinin-based combinations therapy (ACT) is the current frontline curative therapy for uncomplicated malaria in Burkina Faso. Sulfadoxine-pyrimethamine (SP) is used for the preventive treatment of pregnant women (IPTp), while SP plus amodiaquine (SP-AQ) is recommended for children under five in seasonal malaria chemoprevention (SMC). This study aimed to assess the proportions of mutations in the P. falciparum multidrug-resistance 1 (Pfmdr1), P. falciparum chloroquine resistance transporter (Pfcrt), P. falciparum dihydrofolate reductase (pfdhfr), and P. falciparum dihydropteroate synthase (pfdhps), genes from isolates collected during household surveys in Burkina Faso. METHODS: Dried blood spots from Plasmodium falciparum-positive cases at three sites (Orodara, Gaoua, and Banfora) collected during the peak of transmission were analysed for mutations in Pfcrt (codons 72-76, 93, 97, 145, 218, 343, 350 and 353), Pfmdr-1 (codons 86, 184, 1034, 1042 and 1246) dhfr (codons 51, 59, 108, 164) and dhps (at codons 431, 436, 437, 540, 581, 613) genes using deep sequencing of multiplexed Polymerase chaine reaction (PCR) amplicons. RESULTS: Of the 377 samples analysed, 346 (91.7%), 369 (97.9%), 368 (97.6%), and 374 (99.2%) were successfully sequenced for Pfcrt, Pfmdr-1, dhfr, and dhps, respectively. Most of the samples had a Pfcrt wild-type allele (89.3%). The 76T mutation was below 10%. The most frequent Pfmdr-1 mutation was detected at codon 184 (Y > F, 30.9%). The single mutant genotype (NFSND) predominated (66.7%), followed by the wild-type genotype (NYSND, 30.4%). The highest dhfr mutations were observed at codon 59R (69.8%), followed by codons 51I (66.6%) and 108 N (14.7%). The double mutant genotype (ACIRSI) predominated (52.4%). For mutation in the dhps gene, the highest frequency was observed at codon 437 K (89.3%), followed by codons 436 A (61.2%), and 613 S (14.4%). The double mutant genotype (IAKKAA) and the single mutant genotype (ISKKAA) were predominant (37.7% and 37.2%, respectively). The most frequent dhfr/dhps haplotypes were the triple mutant ACIRSI/IAKKAA (23%), the wild-type ACNCSI/ISKKAA (19%) and the double mutant ACIRSI/ISKKAA (14%). A septuple mutant ACIRNI/VAKKGA was observed in 2 isolates from Gaoua (0.5%). CONCLUSION: The efficacy of ACT partner drugs and drugs used in IPTp and SMC does not appear to be affected by the low proportion of highly resistant mutants observed in this study. Continued monitoring, including molecular surveillance, is critical for decision-making on effective treatment policy in Burkina Faso.

Crystal structure of dihydrofolate reductase from the filarial nematode W. bancrofti in complex with NADPH and folate.

Lymphatic filariasis is a debilitating illness with an estimated 50 million cases as of 2018. The majority of cases are caused by the parasitic worm W. bancrofti and additional cases by the worms B. malayi and B. timori. Dihydrofolate reductase (DHFR) is an established target in the treatment of cancer, bacterial, and protozoal infections and may be a potential target for drugs targeting parasitic worm infections, including filariasis. Recent studies have shown that known antifolate compounds, including methotrexate, inhibit the activity of W. bancrofti DHFR (WbDHFR). However, the absence of structural information for filarial DHFRs has limited the study of more in-depth structure-function relationships. We report the structure of WbDHFR complexed with NADPH and folate using X-ray diffraction data measured to 2.47 Å resolution. The structure of WbDHFR reveals the usual DHFR fold and is currently only the second nematode DHFR structure in the Protein Data Bank. The equilibrium dissociation constants for NADPH (90 ± 29 nM) and folate (23 ± 4 nM) were determined by equilibrium titrations. The interactions of known antifolates with WbDHFR were analyzed using molecular docking programs and molecular dynamics simulations. Antifolates with a hydrophobic core and extended linker formed favorable interactions with WbDHFR. These combined data should now facilitate the rational design of filarial DHFR inhibitors, which in turn can be used to determine whether DHFR is a viable drug target for filariasis and whether existing antifolates may be repurposed for its treatment.

In silico screening of inhibitors against human dihydrofolate reductase to identify potential anticancer compounds.

In all species, dihydrofolate reductase (DHFR) is an essential enzyme that regulates the cellular amount of tetrahydrofolate. Human DHFR (hDHFR) activity inhibition results in tetrahydrofolate depletion and cell death. This property has made hDHFR a therapeutic target for cancer. Methotrexate is a well-known hDHFR inhibitor, but its administration has shown some light to severe adverse effects. Therefore, we aimed to find new potential hDHFR inhibitors using structure-based virtual screening, ADMET prediction, molecular docking, and molecular dynamics simulations. Here, we used the PubChem database to find all compounds with at least 90% structural similarity to known natural DHFR inhibitors. To explore their interaction pattern and estimate their binding affinities, the screened compounds (2023) were subjected to structure-based molecular docking against hDHFR. The fifteen compounds that showed higher binding affinity to the hDHFR than the reference compound (methotrexate) displayed important molecular orientation and interactions with key residues in the enzyme's active site. These compounds were subjected to Lipinski and ADMET prediction. PubChem CIDs: 46886812 and 638190 were identified as putative inhibitors. In addition, molecular dynamics simulations revealed that the binding of compounds (CIDs: 46886812 and 63819) stabilized the hDHFR structure and caused minor conformational changes. Our findings suggest that two compounds (CIDs: 46886812 and 63819) could be promising potential inhibitors of hDHFR in cancer therapy.Communicated by Ramaswamy H. Sarma.

Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing.

Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α' isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR.

Unveiling the Efficacy of Sesquiterpenes from Marine Sponge Dactylospongia elegans in Inhibiting Dihydrofolate Reductase Using Docking and Molecular Dynamic Studies.

Dihydrofolate reductase (DHFR) is a crucial enzyme that maintains the levels of 5,6,7,8-tetrahydrofolate (THF) required for the biological synthesis of the building blocks of DNA, RNA, and proteins. Over-activation of DHFR results in the progression of multiple pathological conditions such as cancer, bacterial infection, and inflammation. Therefore, DHFR inhibition plays a major role in treating these illnesses. Sesquiterpenes of various types are prime metabolites derived from the marine sponge Dactylospongia elegans and have demonstrated antitumor, anti-inflammation, and antibacterial capacities. Here, we investigated the in silico potential inhibitory effects of 87 D. elegans metabolites on DHFR and predicted their ADMET properties. Compounds were prepared computationally for molecular docking into the selected crystal structure of DHFR (PDB: 1KMV). The docking scores of metabolites 34, 28, and 44 were the highest among this series (gscore values of -12.431, -11.502, and -10.62 kcal/mol, respectively), even above the co-crystallized inhibitor SRI-9662 score (-10.432 kcal/mol). The binding affinity and protein stability of these top three scored compounds were further estimated using molecular dynamic simulation. Compounds 34, 28, and 44 revealed high binding affinity to the enzyme and could be possible leads for DHFR inhibitors; however, further in vitro and in vivo investigations are required to validate their potential.

Prevalence of Plasmodium falciparum haplotypes associated with resistance to sulfadoxine-pyrimethamine and amodiaquine before and after upscaling of seasonal malaria chemoprevention in seven African countries: a genomic surveillance study.

BACKGROUND: Seasonal malaria chemoprevention is used in 13 countries in the Sahel region of Africa to prevent malaria in children younger than 5 years. Resistance of Plasmodium falciparum to seasonal malaria chemoprevention drugs across the region is a potential threat to this intervention. METHODS: Between December, 2015, and March, 2016, and between December, 2017, and March, 2018, immediately following the 2015 and 2017 malaria transmission seasons, community surveys were done among children younger than 5 years and individuals aged 10-30 years in districts implementing seasonal malaria chemoprevention with sulfadoxine-pyrimethamine and amodiaquine in Burkina Faso, Chad, Guinea, Mali, Nigeria, Niger and The Gambia. Dried blood samples were collected and tested for P falciparum DNA by PCR. Resistance-associated haplotypes of the P falciparum genes crt, mdr1, dhfr, and dhps were identified by quantitative PCR and sequencing of isolates from the collected samples, and survey-weighted prevalence and prevalence ratio between the first and second surveys were estimated for each variant. FINDINGS: 5130 (17·5%) of 29 274 samples from 2016 and 2176 (7·6%) of 28 546 samples from 2018 were positive for P falciparum on quantitative PCR. Among children younger than 5 years, parasite carriage decreased from 2844 of 14 345 samples (19·8% [95% CI 19·2-20·5]) in 2016 to 801 of 14 019 samples (5·7% [5·3-6·1]) in 2018 (prevalence ratio 0·27 [95% CI 0·24-0·31], p<0·0001). Genotyping found no consistent evidence of increasing prevalence of amodiaquine resistance-associated variants of crt and mdr1 between 2016 and 2018. The dhfr haplotype IRN (consisting of 51Ile-59Arg-108Asn) was common at both survey timepoints, but the dhps haplotype ISGEAA (431Ile-436Ser-437Gly-540Glu-581Ala-613Ala), crucial for resistance to sulfadoxine-pyrimethamine, was always rare. Parasites carrying amodiaquine resistance-associated variants of both crt and mdr1 together with dhfr IRN and dhps ISGEAA occurred in 0·05% of isolates. The emerging dhps haplotype VAGKGS (431Val-436Ala-437Gly-540Lys-581Gly-613Ser) was present in four countries. INTERPRETATION: In seven African countries, evidence of a significant reduction in parasite carriage among children receiving seasonal malaria chemoprevention was found 2 years after intervention scale-up. Combined resistance-associated haplotypes remained rare, and seasonal malaria chemoprevention with sulfadoxine-pyrimethamine and amodiaquine is expected to retain effectiveness. The threat of future erosion of effectiveness due to dhps variant haplotypes requires further monitoring. FUNDING: Unitaid.

Design, synthesis and antitumor evaluation of novel pyrazolo[3,4-d]pyrimidines incorporating different amino acid conjugates as potential DHFR inhibitors.

The present study aimed to investigate the antitumor effect of simultaneous inhibition of dihydrofolate reductase (DHFR) enzyme. We designed some novel pyrazolo[3,4-d]pyrimidines bearing different amino acid conjugates as efficient antifolate agents attributable to their structural similarity with methotrexate (MTX) and MTX-related antifolates. All compounds were tested to screen their enzymatic inhibition against DHFR compared with the reference drug MTX and for their in vitro antitumor cytotoxicity against six MTX-resistant cancer cell lines. The flow cytometry indicated that the most potent compound 7f arrested MCF-7 cells in the S-phase and induced apoptosis. Western blot for visualisation proved the ability of compound 7f to induce the expression of proapoptotic caspases and Bax proteins in MCF-7 breast cancer cell line beside its ability to diminish the expression of antiapoptotic Bcl-2 protein. Molecular modelling studies concluded that compound 7f displayed better binding energy than that of the normal ligand MTX. HIGHLIGHTSNew pyrazolo[3,4-d]pyrimidine derivatives 7a-m which are structurally similar to the classical methotrexate (MTX) and MTX-related antifolates were synthesised as antitumor agents.Novel N-acyl amino acid compound 7f exhibited marked DHFR inhibition activity that are parralel to both the molecular docking results and cytotoxic activity.Compound 7f could induce the expression of proapoptotic caspases and Bax proteins in MCF-7 breast cancer cell line beside its ability to diminish the expression of antiapoptotic Bcl-2 protein.All prepared compounds obey Lipinski rule of five except compound 7f.

New quinazolinone-based derivatives as DHFR/EGFR-TK inhibitors: Synthesis, molecular modeling simulations, and anticancer activity.

New 2-mercapto-quinazolin-4-one analogs were synthesized and tested for their in vitro anticancer activity, dihydrofolate reductase (DHFR) inhibition, and epidermal growth factor tyrosine kinase (EGFR-TK) inhibition activities. Compound 24, which is characterized by a 2-benzyl-thio function, showed broad-spectrum anticancer activity with high safety profile and selectivity index. The concentrations of 24 causing 50% growth inhibition (GI50 ) and total cell growth inhibition (TGI) and its lethal concentration 50 (LC50 ) were 15.1, 52.5, and 91.2 µM, respectively, using 5-fluorouracil as a positive control. Also, it showed EGFR-TK inhibitory activity with IC50 = 13.40 nM compared to gefitinib (IC50 = 18.14 nM) and DHFR inhibitory potency with 0.30 µM compared to methotrexate (MTX; IC50 = 0.08 µM). In addition, compound 24 caused cell cycle arrest and apoptosis on COLO-205 colon cancer cells. Compounds 37, 21, and 54 showed remarkable DHFR inhibitory activity with IC50 values of 0.03, 0.08, and 0.08 µM, respectively. The inhibitory properties of these compounds are due to an electron-withdrawing group on the quinazolinone ring, except for compound 54. In a molecular modeling study, compound 24 showed the same binding mode as gefitinib as it interacted with the amino acid Lys745 via π-π interaction. Compound 37 showed a similar binding mode as MTX through the binding interaction with Lys68, Asn64 via hydrogen bond acceptor, and Phe31 via arene-arene interaction. The obtained model and substitution pattern could be used for further development.

Anti-folate quintuple mutations in Plasmodium falciparum asymptomatic infections in Yaoundé, Cameroon.

A major challenge in the fight to effectively control malaria is the emergence of resistant parasite to drugs used in therapy as well as for chemoprevention. In this study, single nucleotide polymorphisms (SNPs) associated with Plasmodium falciparum resistance to sulfadoxine-pyrimethamine (SP), one of the partner drugs in artemisinin-based therapies (ACTs) were studied in asymptomatic P. falciparum isolates from Cameroon. Dried Blood spots were collected from children with asymptomatic malaria enrolled during a household survey. The P. falciparum dihydrofolate reductase (Pfdhfr), dihydropteroate synthase (Pfdhps) and Kelch 13 genes were amplified and point mutations in these gene sequences were analyzed by sequencing. Among a total of 234 samples collected, 51 showed parasitaemia after microscopic examination of which 47 were P. falciparum mono-infections. Molecular analysis revealed 97.3% of mutant alleles at codons 51I, 59R and 108 N in Pfdhfr gene. In Pfdhps gene the most common mutation was 437G (83.3%); followed by 436A (47.6%) and 436F (28.6%). The association of mutations in the two genes (dhfr + dhps) showed 11 different haplotypes including three sextuple mutants (IRNI + AGKGA, IRNI + AAKGS, IRNI + AGKAS) and one septuple mutant (IRNI + AGKGS). For K13 gene no SNPs were seen in the studied asymptomatic malaria samples. The findings revealed presence of SP-resistant alleles in asymptomatic infected individuals with presence of sextuples and septuple SNPs. This emphasizes that regular profiling of antimalarial drugs resistance markers in such population is essential for malaria control and elimination programmes.