Efficacy and tolerability of folate-aminopterin therapy in a rat focal model of multiple sclerosis.

BACKGROUND: Activated macrophages in the experimental model of multiple sclerosis (MS) express folate receptor-ß (FR-ß), representing a promising target for the treatment of MS. Here, we both evaluated the efficacy of a novel folate-aminopterin construct (EC2319) in a rat focal model of multiple sclerosis (MS) and investigated the utility of 68Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid-conjugated folate (68Ga-FOL) for assessing inflammatory lesions. In addition, we investigated whether FR-ß is expressed in the brain of patients with MS. METHODS: Focal delayed-type hypersensitivity experimental autoimmune encephalomyelitis (fDTH-EAE) was induced in 40 Lewis rats; 20 healthy Lewis rats were used as controls. Rats were divided into six groups according to the duration of disease (control, acute, or chronic) and intervention (vehicle versus EC2319). 68Ga-FOL analyses, histology, and immunofluorescence of the brain were performed to evaluate the efficacy of subcutaneously administered EC2319 on lesion development. Immunofluorescence was used to assess FR-ß expression in postmortem brain samples from 5 patients with MS and 5 healthy controls. RESULTS: Immunofluorescence and histological analyses revealed significant reductions in FR-ß expression (P < 0.05) and lesion size (P < 0.01), as well as improved inducible nitric oxide synthase/mannose receptor C type 1 ratios (P < 0.01) in macrophages and microglia during the chronic but not acute phase of fDTH-EAE in EC2319-treated rats. The uptake of IV-injected 68Ga-FOL in the brain was low and did not differ between the groups, but the in vitro binding of 68Ga-FOL was significantly lower in EC2319-treated rats (P < 0.01). FR-ß positivity was observed in chronically active lesions and in normal-appearing white matter in MS brain samples. CONCLUSIONS: EC2319 was well tolerated and attenuated inflammation and lesion development in a rat model of a chronic progressive form of MS. Human MS patients have FR-ß-positive cells in chronically active plaques, which suggests that these results may have translational relevance.

Auxiliary in vitro and in vivo biological evaluation of hydrogen peroxide sensitive prodrugs of methotrexate and aminopterin for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes severe joints damage and other extra-articular alterations. Despite the efficacy of low-dose methotrexate (LD-MTX) in RA treatment, adverse effects are the predominant reasons for discontinuation of therapy. As a therapeutic targeting strategy, the presence of increased concentrations of reactive oxygen species (ROS) in the inflammatory environment can serve as the stimulus for prodrug activation in site-selective drug delivery systems. Our group has previously reported novel ROS sensitive prodrugs (1-3) of MTX and aminopterin (AMT) for site-selective delivery to inflammatory tissue associated with RA, with the aim of reducing side effects in RA therapy. Herein, we investigate the effect and toxicity of the same prodrugs in a rat CIA (collagen-induced arthritis) model of RA. We find that prodrug 1, an arylboronic acid ROS-sensitive MTX-prodrug, displays similar in vivo efficacy as MTX at an equimolar dose, while avoiding adverse effects known to restrict MTX treatment. To further characterize prodrug 1 and its ROS mediated activation, we synthesized compound 4, a negative control lacking the boronic acid moiety. We then investigated the effect of molecules on cell proliferation and cytotoxicity in the presence of the ROS scavenger pyruvate, as well as their stability in buffer and cell media, demonstrating a direct correlation between ROS concentration and the prodrug activity. Moreover, the in vitro ADME properties were investigated, including permeability, rat plasma and microsomal stability.

Folate receptor-targeted aminopterin therapy is highly effective and specific in experimental models of autoimmune uveitis and autoimmune encephalomyelitis.

EC0746 is a rationally designed anti-inflammatory drug conjugate consisting of a modified folic acid-based ligand linked to a γ-hydrazide analog of aminopterin. In this report, EC0746's effectiveness was evaluated against experimental retinal S-antigen (PDSAg) induced autoimmune uveitis (EAU) and myelin-basic-protein induced autoimmune encephalomyelitis (EAE). In both models, functional FR-ß was detected on activated macrophages in local (retinal or central-nervous-system, respectively) and systemic (peritoneal cavity) sites of inflammation. In myelin-rich regions of EAE rats, an increased uptake of (99m)Tc-EC20 (etarfolatide; a FR-specific radioimaging agent) was also observed. EC0746 treatment at disease onset suppressed the clinical severity of both EAU and EAE, and it strongly attenuated progressive histopathological changes in the affected organs. In all parameters assessed, EC0746 activity was completely blocked by a benign folate competitor, suggesting that these therapeutic outcomes were specifically FR-ß mediated. EC0746 may emerge as a useful macrophage-modulating agent for treating inflammatory episodes of organ-specific autoimmunity.

Shedding light into the biological activity of aminopterin, via molecular structural, docking, and molecular dynamics analyses.

In this study, the structural and anticancer properties of aminopterin, as well as its antiviral characteristics, were elucidated. The preferred conformations of the title molecule were investigated with semiempirical AM1 method, and the obtained the lowest energy conformer was then optimized by using density functional (DFT/B3LYP) method with 6-311++G(d,p) as basis set. The vibrational frequencies of the optimized structure were calculated by the same level of theory and were compared with the experimental values. The vibrational assignments were performed based on the computed potential energy distribution (PED) of the vibrational modes. The molecular electrostatic potential (MEP) and frontier molecular orbitals (HOMO, LUMO) analyses were carried out for the optimized structure and the chemical reactivity has been scrutinized. To enlighten the biological activity of aminopterin as anticancer and anti-COVID-19 agents, aminopterin was docked into DNA, αIIBß3 and α5ß1integrins, human dihydrofolate reductase, main protease (Mpro) of SARS-CoV-2 and SARS-CoV-2/ACE2 complex receptor. The binding mechanisms of aminopterin with the receptors were clarified. The molecular docking results revealed the strong interaction of the aminopterin with DNA (-8.2 kcal/mol), αIIBß3 and α5ß1 integrins (-9.0 and -10.8 kcal/mol, respectively), human dihydrofolate reductase (-9.7 kcal/mol), Mpro of SARS-CoV-2 (-6.7 kcal/mol), and SARS-CoV-2/ACE2 complex receptor (-8.1 kcal/mol). Moreover, after molecular docking calculations, top-scoring ligand-receptor complexes of the aminopterin with SARS-CoV-2 enzymes (6M03 and 6M0J) were subjected to 50 ns all-atom MD simulations to investigate the ligand-receptor interactions in more detail, and to determine the binding free energies accurately. The predicted results indicate that the aminopterin may significantly inhibit SARS-CoV-2 infection. Thus, in this study, as both anticancer and anti-COVID-19 agents, the versatility of the biological activity of aminopterin was shown.Communicated by Ramaswamy H. Sarma.

A Novel Treatment for Glomerular Disease: Targeting the Activated Macrophage Folate Receptor with a Trojan Horse Therapy in Rats.

Since activated macrophages express a functional folate receptor ß (FRß), targeting this macrophage population with folate-linked drugs could increase selectivity to treat inflammatory diseases. Using a macrophage-mediated anti-glomerular basement membrane (anti-GBM) glomerulonephritis (GN) in WKY rats, we investigated the effect of a novel folic acid-aminopterin (AMT) conjugate (EC2319) designed to intracellularly deliver AMT via the FR. We found that treatment with EC2319 significantly attenuated kidney injury and preserved renal function. Kidney protection with EC2319 was blocked by a folate competitor, indicating that its mechanism of action was specifically FRß-mediated. Notably, treatment with methotrexate (MTX), another folic acid antagonist related to AMT, did not protect from kidney damage. EC2319 reduced glomerular and interstitial macrophage infiltration and decreased M1 macrophage recruitment but not M2 macrophages. The expression of CCL2 and the pro-fibrotic cytokine TGF-ß were also reduced in nephritic glomeruli with EC2319 treatment. In EC2319-treated rats, there was a significant decrease in the deposition of collagens. In nephritic kidneys, FRß was expressed on periglomerular macrophages and macrophages present in the crescents, but its expression was not observed in normal kidneys. These data indicate that selectively targeting the activated macrophage population could represent a novel means for treating anti-GBM GN and other acute crescentic glomerulonephritis.

Drug Screening of Potential Multiple Target Inhibitors for Estrogen Receptor-α-positive Breast Cancer.

BACKGROUND/AIM: Estrogen receptor α (ERα) antagonist is the most common treatment for ERα-positive breast cancer. However, compensatory signaling contributes to resistance to ERα antagonists. Thus, to explore the potential agents for targeting compensatory signaling, we screened multiple target inhibitors for breast cancer treatment. MATERIALS AND METHODS: We attempted to build a structure-based virtual screening model that can find potential compounds and assay the anticancer ability of these drugs by overall cell survival assay. The downstream compensatory phosphorylated signaling was measured by immunoblotting. RESULTS: Hamamelitannin and glucocheirolin were hits for ERα, phosphoinositide 3-kinase (PI3K), and KRAS proto-oncogene, GTPase (KRAS), which were active against estrogen and epidermal growth factor-triggered proliferation. Additionally, we select aminopterin as a hit for ERα, PI3K, KRAS, and SRC proto-oncogene, non-receptor tyrosine kinase (SRC) with inhibitory activities toward AKT serine/threonine kinase 1 (AKT) and mitogen-activated protein kinase kinase (MEK) signaling. CONCLUSION: Our structure-based virtual screening model selected hamamelitannin, glucocheirolin, aminopterin, and pemetrexed as compounds that may act as potential inhibitors for improving endocrine therapies for breast cancer.

Targeting folate receptor beta on monocytes/macrophages renders rapid inflammation resolution independent of root causes.

Provoked by sterile/nonsterile insults, prolonged monocyte mobilization and uncontrolled monocyte/macrophage activation can pose imminent or impending harm to the affected organs. Curiously, folate receptor beta (FRß), with subnanomolar affinity for the vitamin folic acid (FA), is upregulated during immune activation in hematopoietic cells of the myeloid lineage. This phenomenon has inspired a strong interest in exploring FRß-directed diagnostics/therapeutics. Previously, we have reported that FA-targeted aminopterin (AMT) therapy can modulate macrophage function and effectively treat animal models of inflammation. Our current investigation of a lead compound (EC2319) leads to discovery of a highly FR-specific mechanism of action independent of the root causes against inflammatory monocytes. We further show that EC2319 suppresses interleukin-6/interleukin-1ß release by FRß+ monocytes in a triple co-culture leukemic model of cytokine release syndrome with anti-CD19 chimeric antigen receptor T cells. Because of its chemical stability and metabolically activated linker, EC2319 demonstrates favorable pharmacokinetic characteristics and cross-species translatability to support future pre-clinical and clinical development.

Recent developments in the medicinal chemistry of single boron atom-containing compounds.

Various boron-containing drugs have been approved for clinical use over the past two decades, and more are currently in clinical trials. The increasing interest in boron-containing compounds is due to their unique binding properties to biological targets; for example, boron substitution can be used to modulate biological activity, pharmacokinetic properties, and drug resistance. In this perspective, we aim to comprehensively review the current status of boron compounds in drug discovery, focusing especially on progress from 2015 to December 2020. We classify these compounds into groups showing anticancer, antibacterial, antiviral, antiparasitic and other activities, and discuss the biological targets associated with each activity, as well as potential future developments.

Evolution of modern treatment of childhood acute leukemia and cancer: adventures and battles in the 1970s and 1980s.

This article summarizes the adventures and explorations in the 1970s and 1980s in the treatment of children with leukemia and cancer that paved the way for the current success in childhood cancers. Indeed, these were adventures and bold steps into unchartered waters. Because childhood leukemia the most common of the childhood cancers, success in childhood leukemia was pivotal in the push toward cure of all childhood cancers. The success in childhood leukemia illustrates how treatment programs were designed using clinical- and biology-based risk factors seen in the patients.

Stable maintenance of de novo assembled human artificial chromosomes in embryonic stem cells and their differentiated progeny in mice.

De novo assembled alphoid(tetO)-type human artificial chromosomes (HACs) represent a novel promising generation of high capacity episomal vectors. Their function and persistence, and any adverse effects, in various cell types in live animals, have not, however, been explored. In this study we transferred the alphoid(tetO)-HAC into mouse ES cells and assessed whether the presence of this extra chromosome affects their pluripotent properties. Alphoid(tetO)-HAC-bearing ES cells were indistinguishable from their wild-type counterparts: they retained self-renewal potential and full capacity for multilineage differentiation during mouse development, whereas the HAC itself was mitotically and transcriptionally stable during this process. Our data provide the first example of fully synthetic DNA behaving like a normal chromosome in cells of living animals. It also opens a new perspective into functional genetic studies in laboratory animals as well as stem cell-based regenerative medicine.

Waclaw Szybalski's contribution to immunotherapy: HGPRT mutation & HAT selection as first steps to gene therapy and hybrid techniques in mammalian cells.

In this report we describe Waclaw Szybalski's fundamental contribution to gene therapy and immunotherapy. His 1962 PNAS paper (Szybalska and Szybalski, 1962) documented the first successful gene repair in mammalian cells. Furthermore, this was also the first report on the HAT selection method used later in many applications. Most importantly, somatic cell fusion and HAT selection were subsequently used to develop monoclonal antibody technology, which contributed significantly to the progress of today's medicine.

Genotoxicity of quinocetone, cyadox and olaquindox in vitro and in vivo.

Quinocetone (QCT) and Cyadox (CYA) are important derivative of heterocyclic N-oxide quinoxaline (QdNO), used actively as antimicrobial feed additives in China. Here, we tested and compared the genotoxic potential of QCT and CYA with olaquindox (OLA) in Ames test, HGPRT gene mutation (HGM) test in V79 cells, unscheduled DNA synthesis (UDS) assay in human peripheral lymphocytes, chromosome aberration (CA) test, and micronucleus (MN) test in mice bone marrow. OLA was found genotoxic in all 5 assays. In Ames test, QCT produced His(+) mutants at 6.9 µg/plate in Salmonella typhimurium TA 97, at 18.2 µg/plate in TA 100, TA 1535, TA 1537, and at 50 µg/plate in TA 98. CYA produced His(+) mutants at 18.2 µg/plate in TA 97, TA 1535, and at 50 µg/plate in TA 98, TA 100 and TA 1537. QCT was found positive in HGM and UDS assay at concentrations ≥10 µg/ml while negative results were reported in CA test and MN test. Collectively, we found that OLA was more genotoxic than QCT and CYA. Genotoxicity of QCT was found at higher concentration levels in Ames test, HGM and UDS assays while CYA showed weak mutagenic potential to bacterial cells in Ames test.

Nonstructural proteins of Torque teno sus virus 2 from O2AUG: prediction to experimental validation.

The expression profiles of nonstructural proteins (NSPs) in Torque teno sus virus 2 (TTSuV2) have not yet been characterized. Here, we determined the coding sequences of the TTSuV2 NSPs ORF2, ORF2/2, and ORF2/2/3 by overlapping polymerase chain reaction (PCR) and subsequent expression in bacterial and mammalian cells. We generated two monoclonal antibodies (mAbs), 2E5 and 6F8, from mice immunized with mixed Escherichia coli expressing His-tagged ORF2 and ORF2/2. Enzyme-linked immunosorbent assay (ELISA) and western blot analysis revealed that, 2E5 mAbs bound to the consensus sequences of ORF2, ORF2/2, and ORF2/2/3, while 6F8 recognized the common sequences of ORF2/2 and ORF2/2/3. Immunofluorescence assay (IFA) revealed that ORF2 was localized in the cytoplasm, ORF2/2, in the nucleus but not the nucleolus, and ORF2/2/3, in the peri-nuclear region. To identify the expression profiles of TTSuV NSPs, a circular TTSuV2_ZJ (GenBank: KF660540) genomic DNA clone was constructed and transfected into HEK293T and HeLa cells. Splicing mRNAs and the expression and localization of ORF2/2 and ORF2/2/3 were identified by RT-PCR, western blot analysis, and IFA, respectively. However, ORF2 was not detected either at the RNA or protein level. Our study is the first to provide experimental evidence of the existence of ORF2/2 and ORF2/2/3 at the protein level. Moreover, the mAbs have potential applications in future research on TTSuV2 viral protein function and diagnosis of related diseases.