Dihydrofolate Reductase Activity Research Articles

Design, synthesis, antimicrobial evaluation, and molecular modeling of new sulfamethoxazole and trimethoprim analogs as potential DHPS/DHFR inhibitors

Trimethoprim (TMP) and sulfonamides and are typically utilized to hinder the activities of dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes, respectively. In this context, we aimed to enhance the understanding of the inhibitory effects on dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes by synthesizing and evaluating two series of compounds. These compounds, designed as derivatives of p-aminobenzoic acid (related to sulfamethoxazole, SUL) and trimethoprim analogs, were rigorously tested for their in vitro antimicrobial efficacy and their ability to hinder the activities of DHFR and DHPS enzymes.Among the synthesized compounds, compounds 6 and 10 demonstrated exceptional antimicrobial efficacy against a diverse spectrum of bacterial and fungal strains. The assessment of these compounds also included their impact on biofilm formation, evaluated through crystal violet staining. Our in vitro enzyme assays revealed that compound 10 emerged as the most potent inhibitor of DHPS, with an IC50 value of 224.91 μg/mL, surpassing SUL by 1.6 times. Simultaneously, Compound 6 exhibited particularly potent DHFR-inhibitory action, with an IC50 value of 3.731 μg/mL, surpassing TMP by a factor of three.Molecular modeling studies further elucidated the mode of action, illustrating that compound 10 effectively occupied the DHPS pterin-binding site alongside p-aminobenzoic acid. In contrast, compound 6 was adeptly positioned within the DHFR-binding pocket.In summary, our findings underscore the promising prospects of compounds 6 and 10 in the management of bacterial infections. The results not only demonstrate their remarkable antimicrobial efficacy but also position them as potentially effective in a synergistic combination targeting the DHPS/DHFR enzymatic cascade.

Using Integrated Multi-Omics to Explore the Differences in the Three Developmental Stages of Thelephora ganbajun Zang

Thelephora ganbajun Zang, a rare wild macrofungus, has significant culinary and medicinal value. However, it also has a high cost attributed to its inability to achieve artificial cultivation and its strict environmental requirements. To reveal the intricacies of its development, we conducted a comprehensive analysis of the proteome and metabolome in three pivotal developmental stages: the mycelium, the primordium, and the fruiting body. In our investigation, genes exhibiting various expression levels across multi-omics analyses were identified as potential candidates implicated in growth, development, or metabolic regulation. The aim of this study was to provide a clearer direction for understanding the fundamental metabolic activities and growth stages of this species. Label-free proteomic sequencing revealed a critical juncture in ectomycorrhiza formation, particularly during the transition from the mycelium to the primordium. Secreted proteins, signaling proteins, membrane proteins, and proteins with unidentified functions were rapidly synthesized, with certain amino acids contributing to the synthesis of proteins involved in signaling pathways or hormone precursor substances. In the metabolomics analysis, the classification of secondary metabolites revealed a noteworthy increase in lipid substances and organic acids, contributing to cell activity. The early mycelial development stage exhibited vigorous cell metabolism, contrasting with a decline in cell division activity during fruiting body formation. In our findings, the integration of metabolomic and transcriptomic data highlighted the potential key role of folate biosynthesis in regulating early ectomycorrhiza development. Notably, the expression of alkaline phosphatase and dihydrofolate synthase genes within this pathway was significantly up-regulated in the mycelium and fruiting body stages but down-regulated in the primordium stage. This regulation primarily influences dihydrofolate reductase activity and B vitamin synthesis.

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 invivo 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.

Isolation of Lactobacillus and Enzymatic Activity of Dihydrofolate Reductase (DHFR) in Musa spp.

Background: Bananas grown in the tropic and subtropic regions are among the important crops worldwide. Banana is a general term embracing a number of species or hybrids in the genus Musa, family Musceae. Objective: The present research discusses extensively the breakthrough in the utilization of Musa i.e. Banana from its unripe to ripening phase in order to conclude the presence of Lactobacilli and enzymatic activity of Dihydrofolate Reductase (DHFR). DHFR, a ubiquitous enzyme, is involved in the metabolism and stability of folate (vitamin B9), and causes the reduction of dihydrofolate into tetrahydrofolate (folate or vit B9), which is utilized in amino acids and purine production. It has been studied that the deficiency of DHFR or its inhibition can lead to Megaloblastic anemia, Neural tube defects, improper synthesis of DNA and RNA, colorectal cancer, etc. The presence of the DHFR enzyme in bananas can be utilized in the treatment of diseases related to deficiency of folate, which can become a cheaper and cost-effective way to keep a balance in the folate metabolism and keep the activity of the enzyme maximum. Methodology: The fresh unripe banana samples were collected from an urban fruit market in Karachi, serially diluted, and inoculated into the Nutrient agar, Mannitol salt agar, and De Man, Rogosa, Sharpe agar media to check for microbial growth. The enzymatic activity of DHFR and its association with Musa was also checked by using DHFR substrate. Results: The results concluded from our experiment are that the Musa contains Gram-positive bacilli that were further tested to characterize the microorganism into its respective genus, indicating the presence of Lactobacillus in the banana. The enzymatic activity of DHFR was observed at maximum on day 2 and minimum enzymatic activity was observed on day 4. Conclusion: Hence, people suffering from folic acid deficiency, DHFR enzyme and consumption of day 2 bananas can be prescribed. Not only this, the presence of Lactobacilli as probiotics in bananas restore consumer’s intestinal microbial dysbiosis and maintain healthy intestinal ecosystem.  Furthermore, herbal medicines can be made from banana and given to patients in order to restore the deficiency.

Dihydrofolate reductase activity controls neurogenic transitions in the developing neocortex.

One-carbon/folate (1C) metabolism supplies methyl groups required for DNA and histone methylation, and is involved in the maintenance of self-renewal in stem cells. Dihydrofolate reductase (DHFR), a key enzyme in 1C metabolism, is highly expressed in human and mouse neural progenitors at the early stages of neocortical development. Here, we have investigated the role of DHFR in the developing neocortex and report that reducing its activity in human neural organoids and mouse embryonic neocortex accelerates indirect neurogenesis, thereby affecting neuronal composition of the neocortex. Furthermore, we show that decreasing DHFR activity in neural progenitors leads to a reduction in one-carbon/folate metabolites and correlates with modifications of H3K4me3 levels. Our findings reveal an unanticipated role for DHFR in controlling specific steps of neocortex development and indicate that variations in 1C metabolic cues impact cell fate transitions.

Effects of Chronic Alcohol Consumption and Iron Deficiency on Maternal Folate Status and Reproductive Outcome in Mice

The influence of chronic alcohol consumption with or without iron deficiency on reproductive performance and folate status was studied. Female CBA/J mice were fed isocaloric liquid diets prior to and during pregnancy. A 2 × 2 factorial design was employed with ethanol-derived calories (EDC) and iron (Fe) as dietary variables. Groups received 0% EDC and 30 ppm Fe (CA); 0% EDC and 2 ppm Fe (CD); 20% EDC and 30 ppm Fe (EA); and 20% EDC and 2 ppm Fe (ED). Animals were killed on day 18 of gestation. Mean body weights were reduced in CD, EA and ED groups, while daily caloric intakes reduced only in CD and ED groups. Maternal values for hemoglobin, transferrin saturation and red cell folates decreased with iron deficiency and/or 20% EDC; hematocrit, serum iron and serum folates decreased only with iron deficiency. Blood ethanol levels were similar in EA and ED groups. Maternal liver total lipids and alcohol dehydrogenase activity increased only with 20% EDC, while dihydrofolate reductase activity decreased with iron deficiency and/or 20% EDC. Iron deficiency and/or 20% EDC adversely affected gestational performances in mice as indicated by increased resorptions and decreased percentages of live fetuses/litters and live fetal weights. Data indicate that iron deficiency and/or chronic ethanol consumption induce adverse effects on maternal reproductive performance of CBA/J mice possibly via alteration of folate metabolism.

Narirutin downregulates lipoxygenase-5 expression and induces G0/G1 arrest in triple-negative breast carcinoma cells

BackgroundTriple-negative breast cancer (TNBC) accounts for 20% of breast cancer that does not express HER2, progesterone and estrogen receptors. It is associated with a high mortality rate, morbidity, metastasis, recurrence, poor prognosis and resistance to chemotherapy. Lipoxygenase-5 (LOX-5), cyclooxygenase-2 (COX-2), cathepsin-D (CATD), ornithine decarboxylase (ODC) and dihydrofolate reductase (DHFR) are involved in breast cancer carcinogenesis; hence, there is a pressing need to identify novel chemicals that targets these enzymes. Narirutin, a flavanone glycoside abundantly present in citrus fruits, is reported to have immune-modulatory, anti-allergic and antioxidant potential. Still, the cancer chemopreventive mechanism against TNBC has not been explored. MethodsIn vitro experiments, enzyme activity, expression analysis, molecular docking and MD simulation were carried out. ResultsNarirutin suppressed the growth of MDA-MB-231 and MCF-7 in a dose-proportional manner. The pronounced effect with >50% inhibition was observed in SRB and MTT assays for MDAMB-231 cells. Unexpectedly, narirutin suppressed the proliferation of normal cells (24.51%) at 100 μM. Further, narirutin inhibits the activity of LOX-5 in cell-free (18.18 ± 3.93 μM) and cell-based (48.13 ± 7.04 μM) test systems while moderately affecting COX-2, CATD, ODC and DHFR activity. Moreover, narirutin revealed a down-regulation of LOX-5 expression with a fold change of 1.23. Besides, MD simulation experiments confirm that narirutin binding forms a stable complex with LOX-5 and improves the stability and compactness of LOX-5. In addition, the prediction analysis demonstrates that narirutin could not cross the blood-brain barrier and did not act as an inhibitor of different CYPs. Conclusions and significanceNarirutin could be a potent cancer chemopreventive lead for TNBC, further paving the way for synthesizing novel analogues.

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

Quantifying the evolvability of allostery at different protein surfaces.

Allosteric regulation requires the cooperative action of multiple amino acid residues to relay a signal from a distal surface site to the protein active site. It is an open problem to understand how new regulation evolves through a Darwinian process of single mutation and selection. Yet a better understanding of how allostery emerges is critical to our ability to engineer new custom biosensors. Are some protein surfaces more prone to evolve into allosteric sites than others? Or is the capacity to evolve allostery homogenously distributed across a protein's entire surface? Recent work has identified that allosteric regulation is more easily engineered at certain protein surfaces termed “allosteric hotspots.” These hotspots occur at surface positions that co-evolve with enzyme active sites. Allosteric hotspots have been used to engineer new allosteric regulation of dihydrofolate reductase (DHFR) by the light-oxygen-voltage-sensing domain (LOV2). When LOV2 is fused to allosteric hotspots on DHFR, DHFR activity is weakly regulated by light (1.3-fold). However, fusing LOV2 to non-hotspot positions does not result in allosteric control. The low level of regulation observed in DHFR/LOV2 fusions presents an opportunity to study how allostery can be optimized by evolution. In my work, I am using random mutagenesis combined with a high-throughput screen to evolve new allosteric regulation. I am performing forward evolution of allostery for a weakly allosteric, hotspot-connected fusion, and for a non-allosteric, non-hotspot-connected fusion. I am monitoring the distribution of mutational effects on allostery for each construct over rounds of selection. With this approach, I will quantify the evolvability of allosteric regulation at different protein surface sites. I hypothesize that allosteric hotspots will have a higher prevalence of allostery-tuning mutations, and that these mutations will result in higher levels of allosteric regulation.

A conserved SH3-like fold in diverse putative proteins tetramerizes into an oxidoreductase providing an antimicrobial resistance phenotype.

We present a potential mechanism for emergence of catalytic activity that is essential for survival, from a non-catalytic protein fold. The type B dihydrofolate reductase (DfrB) family of enzymes were first identified in pathogenic bacteria because their dihydrofolate reductase activity is sufficient to provide trimethoprim (TMP) resistance. DfrB enzymes are described as poorly evolved as a result of their unusual structural and kinetic features. No characterized protein shares sequence homology with DfrB enzymes; how they evolved to emerge in the modern resistome is unknown. In this work, we identify DfrB homologues from a database of putative and uncharacterized proteins. These proteins include an SH3-like fold homologous to the DfrB enzymes, embedded in a variety of additional structural domains. By means of functional, structural and biophysical characterization, we demonstrate that these distant homologues and their extracted SH3-like fold can display dihydrofolate reductase activity and confer TMP resistance. We provide evidence of tetrameric assembly and catalytic mechanism analogous to that of DfrB enzymes. These results contribute, to our knowledge, the first insights into a potential evolutionary path taken by this SH3-like fold to emerge in the modern resistome following introduction of TMP. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Discovery of new STAT3 inhibitors as anticancer agents using ligand-receptor contact fingerprints and docking-augmented machine learning.

STAT3 belongs to a family of seven vital transcription factors. High levels of STAT3 are detected in several types of cancer. Hence, STAT3 inhibition is considered a promising therapeutic anti-cancer strategy. In this work, we used multiple docked poses of STAT3 inhibitors to augment training data for machine learning QSAR modeling. Ligand-Receptor Contact Fingerprints and scoring values were implemented as descriptor variables. Escalating docking-scoring consensus levels were scanned against orthogonal machine learners, and the best learners (Random Forests and XGBoost) were coupled with genetic algorithm and Shapley additive explanations (SHAP) to identify critical descriptors that determine anti-STAT3 bioactivity to be translated into pharmacophore model(s). Two successful pharmacophores were deduced and subsequently used for in silico screening against the National Cancer Institute (NCI) database. A total of 26 hits were evaluated in vitro for their anti-STAT3 bioactivities. Out of which, three hits of novel chemotypes, showed cytotoxic IC50 values in the nanomolar range (35 nM to 6.7 μM). However, two are potent dihydrofolate reductase (DHFR) inhibitors and therefore should have significant indirect STAT3 inhibitory effects. The third hit (cytotoxic IC50 = 0.44 μM) is purely direct STAT3 inhibitor (devoid of DHFR activity) and caused, at its cytotoxic IC50, more than two-fold reduction in the expression of STAT3 downstream genes (c-Myc and Bcl-xL). The presented work indicates that the concept of data augmentation using multiple docked poses is a promising strategy for generating valid machine learning models capable of discriminating active from inactive compounds.

Discovery of Highly Trimethoprim-Resistant DfrB Dihydrofolate Reductases in Diverse Environmental Settings Suggests an Evolutionary Advantage Unrelated to Antibiotic Resistance.

Type B dihydrofolate reductases (DfrB) are intrinsically highly resistant to the widely used antibiotic trimethoprim, posing a threat to global public health. The ten known DfrB family members have been strongly associated with genetic material related to the application of antibiotics. Several dfrB genes were associated with multidrug resistance contexts and mobile genetic elements, integrated both in chromosomes and plasmids. However, little is known regarding their presence in other environments. Here, we investigated the presence of dfrB beyond the traditional areas of enquiry by conducting metagenomic database searches from environmental settings where antibiotics are not prevalent. Thirty putative DfrB homologues that share 62 to 95% identity with characterized DfrB were identified. Expression of ten representative homologues verified trimethoprim resistance in all and dihydrofolate reductase activity in most. Contrary to samples associated with the use of antibiotics, the newly identified dfrB were rarely associated with mobile genetic elements or antibiotic resistance genes. Instead, association with metabolic enzymes was observed, suggesting an evolutionary advantage unrelated to antibiotic resistance. Our results are consistent with the hypothesis that multiple dfrB exist in diverse environments from which dfrB were mobilized into the clinically relevant resistome. Our observations reinforce the need to closely monitor their progression.

Methotrexate-induced acute cardiotoxicity requiring veno-arterial extracorporeal membrane oxygenation support: a case report

BackgroundMethotrexate is an antifolate antimetabolite that inhibits the activity of dihydrofolate reductase by acting as a false substrate, which leads to defects of DNA synthesis, specifically the inhibition of purine and pyrimidine synthesis. Thus, methotrexate is a powerful agent for treating autoimmune diseases and cancer. In general, methotrexate is thought to be cardioprotective and reports of methotrexate-induced cardiomyopathy are rare. We present a case of methotrexate-induced severe cardiotoxicity diagnosed by exclusion of all other potential causes.Case presentationThe patient was a 54-year-old Caucasian man presenting to an outside hospital with a chief complaint of abdominal pain and bloating who reported taking methotrexate up to 20 mg per week for systemic sclerosis. After a transthoracic echocardiogram found a left ventricular ejection fraction of 10% and coronary catheterization demonstrated no significant disease, he was transferred to our hospital for advanced heart failure therapies. His condition deteriorated, and he was eventually placed on veno-arterial extracorporeal membrane oxygenation. Owing to a lack of an identifiable etiology of cardiac failure, toxicology consultation recommended 24 hours of intravenous leucovorin therapy to overcome any residual and potentially cardiotoxic methotrexate still in his system. Over the next 5 days, his cardiac function improved daily, such that on day 5 of extracorporeal membrane oxygenation, he had a left ventricular ejection fraction of 40% and was able to be decannulated. Two days later, his ejection fraction improved to 60% and normal right ventricular function. Initially, his renal function improved while on extracorporeal membrane oxygenation, but over the next week deteriorated such that he required intermittent hemodialysis until hospital discharge.ConclusionsAfter a process of elimination, the most likely cause of this patient’s acute decline and rapid recovery of bi-ventricular function was methotrexate toxicity. Leucovorin may have aided the reversal of methotrexate toxicity.

Biological Activity of NHC-Gold-Alkynyl Complexes Derived from 3-Hydroxyflavones.

In this paper we describe the synthesis of new N-heterocyclic carbene (NHC) gold(I) derivatives with flavone-derived ligands with a propargyl ether group. The compounds were screened for their antimicrobial and anticancer activities, showing greater activity against bacteria than against colon cancer cells (Caco-2). Complexes [Au(L2b)(IMe)] (1b) and [Au(L2b)(IPr)] (2b) were found to be active against both Gram-positive and Gram-negative strains. The mechanism of action of 1b was evaluated by measurement of thioredoxin reductase (TrxR) and dihydrofolate reductase (DHFR) activity, besides scanning electron microscopy (SEM). Inhibition of the enzyme thioredoxin reductase is not observed in either Escherichia Coli or Caco-2 cells; however, DHFR activity is compromised after incubation of E. coli cells with complex 1b. Moreover, loss of structural integrity and change in bacterial shape is observed in the images obtained from scanning electron microscopy (SEM) after treatment E. coli cells with complex 1b.

Joseph R. Bertino: In Memoriam (1930–2021)

Few individuals define the physician scientist as well as Joseph R. Bertino, MD, FAACR, who died October 11 surrounded by family and friends. He was 91.His commitment to patients can best be summed up by his own words, “When I went to medical school, I took an elective in hematology. At the same time, my brother's child came down with acute leukemia. This beautiful kid, 3 years old, died of leukemia. That made me think about a career in cancer research, hematologic malignancies, and it was a stimulus for me to continue in that area.”Joe's legacy and career journey are inextricably linked to the history of cancer chemotherapy and the training of medical oncologists. When Milton Winternitz became Yale's fifth dean in 1920, little could he imagine what would follow. Enlisted in the U.S. Armed Forces during World War II, he established a center for the study of toxic war gases. The attack on allied ships in the Italian port of Barre on December 2, 1943, spilled mustard chemicals into the oily water covering the sailors who dove for safety; many died. An investigation by Lieutenant Colonel Stewart Francis Alexander showing that these chemicals were not only skin vesicants but also had destructive effects on the lymphoid and hematopoietic systems caught the attention of Colonel Cornelius P. “Dusty” Rhoads, who served as head of New York's Memorial Hospital for the Treatment of Cancer and Allied Diseases and believed that mustard derivatives might be used to treat cancer. In 1945, he persuaded General Motors' Alfred P. Sloan and Charles F. Kettering to fund the Sloan Kettering Institute for Cancer Research to develop medicines to treat patients with cancer.Winternitz shared Alexander's observations with two Yale assistant professors, Alfred Gilman and Louis Goodman, who began top secret studies on the effects of nitrogen mustard, a more stable derivative of sulfur mustard, on murine lymphomas and demonstrated the first signs of activity in patients. In 1949, Mustargen (mechlorethamine) became the first chemotherapeutic drug approved by the FDA.From this early work on chemotherapy arose the need for physicians trained in both pharmacology and medicine. The Yale School of Medicine formed a division of Oncology and Chemotherapy, the first of what is today known as Medical Oncology. Initially led by Paul Calabresi, Bertino was appointed in 1966 and served for 20 years as the chief and became the first director of the Yale Cancer Center in 1973. With his colleagues in the departments of Medicine and Pharmacology, he developed today's concept of translational research, that is, applying laboratory discoveries to clinical medicine. Joe left Yale in 1986 and served until 2002 as chairman of the Molecular Pharmacology and Therapeutics Program at the Sloan Kettering Institute for Cancer Research, the institute originally conceived of by Dusty Rhoads.I, a former Bertino lab fellow at Yale, recruited Joe along with several of his former Yale trainees and associates in 2002 to help build the Rutgers Cancer Institute of New Jersey, the state's first and only NCI designated comprehensive cancer center, where he served as University Professor of Medicine and Pharmacology and Chief Scientific Officer until his death.Bertino's major contributions are best appreciated in the context of today's vernacular, including cancer metabolism, gene editing, molecular mechanisms of drug sensitivity and resistance, clinical and translational research, and drug discovery.The Bertino lab developed and implemented the tools that elucidated the critical roles that folates play in cancer metabolism, leading to major advances in the pharmacology of antifolates in humans. These insights into folate metabolism in cancer versus normal tissues produced his pioneering clinical use of leucovorin rescue following high-dose methotrexate for patients with osteosarcoma and acute lymphocytic leukemia. His lab identified NAD kinase, encoded by NADK, as a target for anticancer drug development that protects tumor cells from reactive oxygens species and mediates macromolecular biosynthesis. Together with the Scotto lab, Bertino discovered an activating mutation in NADK that enhanced tumor cell proliferation. The finding that NAD kinase mRNA is amplified in some tumor cells prompted efforts to discover inhibitors.Bertino helped define the mechanisms of action and resistance to methotrexate, historically one of the most important cancer chemotherapeutic agents. While on sabbatical with Robert Schimke at Stanford, Bertino, Rod Kellems, and Fred Alt reported that increased dihydrofolate reductase (DHFR) activity in methotrexate-resistant cells was due to amplification of the DHFR gene, providing the first example of gene amplification as a mechanism of drug resistance. This benchmark discovery led to the appreciation that gene amplification is an important mechanism of resistance to tight binding inhibitors. He translated these insights to studies of drug resistance in patients and reported that DHFR amplification and impaired methotrexate uptake were major mechanisms of resistance in blast cells from patients with acute leukemia. He later described a novel mechanism of resistance to methotrexate due to mutations in the 3′ untranslated region of DHFR that abrogated binding of miRNA 34a, leading to increased expression of DHFR mRNA and protein.With colleagues at the Rutgers Cancer Institute of New Jersey, he identified new biomarkers for methotrexate and pemetrexed sensitivity. Highly proliferative breast cancer and high-grade lymphoma cells have increased mitochondrial folate enzymes and are sensitive to treatment with methotrexate. He identified mitochondrial methylenetetrahydrofolate dehydrogenase 2 as an exciting drug target as it is expressed in embryos and not in adult tissues but is overexpressed in rapidly proliferating tumors.Using early genomic tools, his laboratory created DHFR and thymidylate synthase (TYMS) genetic variants and demonstrated that these edited genes could render hematopoietic stem cells resistant to the toxic effects of methotrexate, 5-fluorouracil, and pemetrexed and protect mice from long-term toxicity.Unravelling much of the pharmacology of methotrexate and his important work on cytosine arabinoside (AraC) allowed Bertino to design and implement one of the first curative regimens for diffuse large cell lymphoma (“COMLA,” cyclophosphamide, vincristine, methotrexate with leucovorin rescue and AraC). With Seth Rudnick and Robert Capizzi, he pioneered the use of high-dose AraC in the treatment of acute leukemia. His work on the pharmacologic basis for the timing of combining methotrexate and fluorouracil led to a collaboration with Bernard Fisher and the National Surgical Adjuvant Breast and Bowel Project (NSABP) to investigate “sequential methotrexate→ fluorouracil” as one of the first alkylating agent–free adjuvant chemotherapy regimens for premenopausal women with breast cancer. Bertino's work on 5-fluorouracil included the seminal discovery that this drug produced cytotoxicity by different mechanisms of action depending on the schedule of drug administration. These findings were translated into current clinical regimens to treat colorectal cancer that use both pulse and infusional fluorouracil.By defining the mechanisms by which folates rescue cancer cells from antimetabolites and the mechanism by which antifolates are transported across cell membranes, Bertino drove the development of trimetrexate, a lipid-soluble folate analog that does not utilize the reduced folate carrier and is not cross-resistant with methotrexate. This work was translated to the clinic by his former postdoctoral fellow, Bruce Chabner, who demonstrated the activity of trimetrexate with leucovorin rescue for the treatment of toxoplasmosis. Bertino's lab also isolated and characterized carboxypeptidase G1, an enzyme that metabolizes methotrexate, that was approved by the FDA for treating patients at risk of kidney damage following high-dose methotrexate.Joe received numerous prestigious awards and honors. He served both as President of the American Association for Cancer Research (AACR) and President of the American Association of Clinical Oncology (ASCO) and was the inaugural editor of ASCO's Journal of Clinical Oncology, highlighting the esteem in which he was held in the basic, translational, and clinical research communities. A few of his many awards include, American Cancer Society Research Professorship; AACR Richard and Hinda Rosenthal Award; Memorial Sloan Kettering Cancer Center Chester C. Stock Award; ASCO Karnofsky Award; American Cancer Society Medal of Honor; ASCO Statesman Award; AACR Joseph H. Burchenal Memorial Award for Outstanding Achievement in Clinical Research; Int'l Congress on Hematologic Malignancies Outstanding Medical Research Scientist Award; American College of Clinical Pharmacology Distinguished Investigator Award; AACR Lifetime Achievement Award; and inaugural Fellow of the AACR Academy.Joe published more than 450 peer-reviewed articles and would be quick to credit his accomplishments to his collaborators and the over 50 postdoctoral fellows whom he trained from around the world.“Dr. B” was a beloved physician, husband, and father. His grateful patients would shower him each year with Christmas gifts. He would leave the clinic looking like a big teddy bear of a Christmas tree. The world of medical oncology has lost a giant.I would like to thank Kathleen Scotto, Mary Todd, Bruce Haffty, and Roger Strair for reviewing the article and apologize to the many fellows, faculty and other colleagues whom I failed to acknowledge.

The dihydrofolate reductase 19-bp deletion modifies the beneficial effect of B-vitamin therapy in mild cognitive impairment: pooled study of two randomized placebo-controlled trials.

Background: Higher serum homocysteine is associated with cognitive decline in older people. But homocysteine-lowering trials including folic acid (FA) show inconsistent results on cognitive decline. The reduction of FA to dihydrofolate by dihydrofolate reductase (DHFR) is slow in humans. Objective: We examined the effects of the DHFR 19-bp deletion/insertion (del/ins) polymorphism on FA-containing treatment on cognitive decline and brain atrophy in older people with mild cognitive impairment (MCI). Methods: This study used pooled data from two randomized B-vitamin trials on 545 MCI subjects who received either FA-containing B vitamins or placebo for 24 months. Subjects were typed for the DHFR genotype. Primary outcome was the Clinical Dementia Rating scale-global score (CDR-global). Secondary outcomes were CDR-sum of boxes score (CDR-SOB), memory and executive Z-scores and whole brain atrophy rate by serial MRI. Results: The proportions of subjects with del/del, del/ins and ins/ins genotype were 29.5, 44.3 and 26.1%, respectively. DHFR genotypes modified the effects of B vitamins on CDR-global, CDR-SOB and executive function Z-score (Pinteraction = 0.017, 0.014 and 0.052, respectively), with significant benefits being observed only in those with ins/ins genotype (Beta = −1.367, −0.614 and 0.315, P = 0.004, 0.014 and 0.012, respectively). The interaction was not significant for memory Z-score and whole brain atrophy rate. Notably, the supplements only slowed brain atrophy in members of the ‘ins/ins’ group who were not using aspirin. Conclusions: Our data indicate that the beneficial effects of B vitamins including FA on cognitive function are only apparent in those with ins/ins genotype, i.e. relatively better preserved DHFR activity.

Switching an active site helix in dihydrofolate reductase reveals limits to subdomain modularity

To what degree are individual structural elements within proteins modular such that similar structures from unrelated proteins can be interchanged? We study subdomain modularity by creating 20 chimeras of an enzyme, Escherichia coli dihydrofolate reductase (DHFR), in which a catalytically important, 10-residue α-helical sequence is replaced by α-helical sequences from a diverse set of proteins. The chimeras stably fold but have a range of diminished thermal stabilities and catalytic activities. Evolutionary coupling analysis indicates that the residues of this α-helix are under selection pressure to maintain catalytic activity in DHFR. Reversion to phenylalanine at key position 31 was found to partially restore catalytic activity, which could be explained by evolutionary coupling values. We performed molecular dynamics simulations using replica exchange with solute tempering. Chimeras with low catalytic activity exhibit nonhelical conformations that block the binding site and disrupt the positioning of the catalytically essential residue D27. Simulation observables and in vitro measurements of thermal stability and substrate-binding affinity are strongly correlated. Several E. coli strains with chromosomally integrated chimeric DHFRs can grow, with growth rates that follow predictions from a kinetic flux model that depends on the intracellular abundance and catalytic activity of DHFR. Our findings show that although α-helices are not universally substitutable, the molecular and fitness effects of modular segments can be predicted by the biophysical compatibility of the replacement segment.

Targeted Mitochondrial Fluorescence Imaging-Guided Tumor Antimetabolic Therapy with the Imprinted Polymer Nanomedicine Capable of Specifically Recognizing Dihydrofolate Reductase.

As we all know, inhibiting the activity of dihydrofolate reductase (DHFR) has always been an effective strategy for folate antimetabolites to treat tumors. In the past, it mainly relied on chemical drugs. Here, we propose a new strategy, (3-propanecarboxyl)triphenylphosphonium bromide (CTPB)-modified molecularly imprinted polymer nanomedicine (MIP-CTPB). MIP-CTPB prepared by imprinting the active center of DHFR can specifically bind to the active center to block the catalytic activity of DHFR, thereby inhibiting the synthesis of DNA and ultimately inhibiting the tumor growth. The modification of CTPB allows the nanomedicine to be targeted and enriched in mitochondria, where DHFR is abundant. The confocal laser imaging results show that MIP-CTPB can target mitochondria. Cytotoxicity experiments show that MIP-CTPB inhibits HeLa cell proliferation by 42.2%. In vivo experiments show that the tumor volume of the MIP-CTPB-treated group is only one-sixth of that of the untreated group. The fluorescent and paramagnetic properties of the nanomedicine enable targeted fluorescence imaging of mitochondria and T2-weighted magnetic resonance imaging of tumors. This research not only opens up a new direction for the application of molecular imprinting, but also provides a new idea for tumor antimetabolic therapy guided by targeted mitochondrial imaging.

Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype.

The relationship between sequence variation and phenotype is poorly understood. Here, we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in dihydrofolate reductase (DHFR). We find that partial loss of DHFR activity causes reversible filamentation despite SOS response indicative of DNA damage, in contrast to thymineless death (TLD) achieved by complete inhibition of DHFR activity by high concentrations of antibiotic trimethoprim. This phenotype is triggered by a disproportionate drop in intracellular dTTP, which could not be explained by drop in dTMP based on the Michaelis–Menten‐like in vitro activity curve of thymidylate kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation rescues filamentation and restores in vivo Tmk kinetics to Michaelis–Menten. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system‐level implications for bacterial phenotype.

Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype

The relationship between sequence variation and phenotype is poorly understood. This is largely due to its complexity, as mutations are pleiotropic and affect several layers of cellular hierarchy, from molecular properties to systems level to the phenotype. Though mapping of phenotype to molecular, cellular and certain systems level properties has been elucidated in some systems, a precise mechanistic understanding of how metabolic perturbations arising out of mutations affect phenotype is lacking. Here we use metabolomic analysis to elucidate the molecular mechanism underlying the filamentous phenotype of E. coli strains that carry destabilizing mutations in the Dihydrofolate Reductase (DHFR). We find that partial loss of DHFR activity causes SOS response indicative of DNA damage and cell filamentation. This phenotype is triggered by an imbalance in deoxy nucleotide levels, most prominently a disproportionate drop in the intracellular dTTP. However, dTTP levels could not be explained by drop in dTMP based on the Michaelis-Menten like in vitro activity curve of Thymidylate Kinase (Tmk), an enzyme downstream of DHFR that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation in the media rescues filamentation and restores in vivo Tmk kinetics to almost perfect Michaelis-Menten. The cooperative enzyme activity is best explained by the fractal nature of Tmk activity in vivo due to diffusion-limitation of substrate dTMP, possibly due to substrate channeling and metabolon formation. Overall, this study highlights the important role of cellular environment in sculpting enzyme kinetics. It also demonstrates the use of a systems level property of the cell – metabolome – as a stepping-stone to illustrate precise biophysical and biochemical mechanisms to bridge the multi-scale genotype-phenotype relationship.