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.

Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance.

BACKGROUND: Pralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and explored potential therapeutic strategies to overcome PDX resistance. METHODS: To investigate PDX resistance, we established two PDX-resistant T-lymphoblastic leukemia cell lines (CEM and MOLT4) through continuous exposure to increasing doses of PDX. The resistance mechanisms were evaluated by measuring PDX uptake, apoptosis induction and folate metabolism-related protein expression. We also applied gene expression analysis and methylation profiling to identify the mechanisms of resistance. We then explored rational drug combinations using a spheroid (3D)-culture assay. RESULTS: Compared with their parental cells, PDX-resistant cells exhibited a 30-fold increase in half-maximal inhibitory concentration values. Induction of apoptosis by PDX was significantly decreased in both PDX-resistant cell lines. Intracellular uptake of [14C]-PDX decreased in PDX-resistant CEM cells but not in PDX-resistant MOLT4 cells. There was no significant change in expression of dihydrofolate reductase (DHFR) or folylpolyglutamate synthetase (FPGS). Gene expression array analysis revealed that DNA-methyltransferase 3ß (DNMT3B) expression was significantly elevated in both cell lines. Gene set enrichment analysis revealed that adipogenesis and mTORC1 signaling pathways were commonly upregulated in both resistant cell lines. Moreover, CpG island hypermethylation was observed in both PDX resistant cells lines. In the 3D-culture assay, decitabine (DAC) plus PDX showed synergistic effects in PDX-resistant cell lines compared with parental lines. CONCLUSIONS: The resistance mechanisms of PDX were associated with reduced cellular uptake of PDX and/or overexpression of DNMT3B. Epigenetic alterations were also considered to play a role in the resistance mechanism. The combination of DAC and PDX exhibited synergistic activity, and thus, this approach might improve the clinical efficacy of PDX.

A novel virtual screening procedure identifies Pralatrexate as inhibitor of SARS-CoV-2 RdRp and it reduces viral replication in vitro.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus poses serious threats to the global public health and leads to worldwide crisis. No effective drug or vaccine is readily available. The viral RNA-dependent RNA polymerase (RdRp) is a promising therapeutic target. A hybrid drug screening procedure was proposed and applied to identify potential drug candidates targeting RdRp from 1906 approved drugs. Among the four selected market available drug candidates, Pralatrexate and Azithromycin were confirmed to effectively inhibit SARS-CoV-2 replication in vitro with EC50 values of 0.008µM and 9.453 µM, respectively. For the first time, our study discovered that Pralatrexate is able to potently inhibit SARS-CoV-2 replication with a stronger inhibitory activity than Remdesivir within the same experimental conditions. The paper demonstrates the feasibility of fast and accurate anti-viral drug screening for inhibitors of SARS-CoV-2 and provides potential therapeutic agents against COVID-19.

Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells.

In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine-aminopterin-thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876-887.

Cost-utility analysis of pralatrexate for relapsed or refractory peripheral T-cell lymphoma based on a case-matched historical control study along with single arm clinical trial.

BACKGROUND: Patients with relapsed or refractory peripheral T-cell lymphoma (R/R PTCL) treated with pralatrexate have previously shown superior overall survival (OS) compared to those who underwent conventional chemotherapy (CC, 15.4 vs. 4.07 months). We conducted an economic evaluation of pralatrexate from a societal perspective in Korea based on data from the PROPEL phase II study. METHODS: Using a Markov model with a weekly cycle, we simulated the experience of patients with R/R PTCL receiving pralatrexate or CC for 15 years. The model consists of five health states; initial treatment, treatment pause, subsequent treatment, stem cell transplantation (SCT) success, and death. Comparative effectiveness was based on PROPEL phase II single-arm study and its matched historical control analysis. Costs included drug, drug administration, monitoring, adverse event management, and SCT costs. RESULTS: The incremental cost-effectiveness ratio of the base case was $39,153 per quality-adjusted life-year (QALY) gained. The results of one-way sensitivity analysis ranged from $33,949 to $51,846 per QALY gained, which remained within an implicit willingness-to-pay (WTP) threshold of anticancer drugs in Korea. CONCLUSIONS: Pralatrexate is a cost-effective intervention with improved OS and incremental costs within the WTP limit. Pralatrexate could function as a new therapeutic option for patients suffering from life-threatening R/R PTCL.

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.

'Rod and ring' formation from IMP dehydrogenase is regulated through the one-carbon metabolic pathway.

'Rods and rings' (RRs) are conserved, non-membrane-bound intracellular polymeric structures composed, in part, of inosine monophosphate dehydrogenase (IMPDH), a key enzyme leading to GMP and GTP biosynthesis. RR formation is induced by IMPDH inhibitors as well as glutamine deprivation. They also form upon treatment of cells with glutamine synthetase inhibitors. We now report that depriving cells of serine and glycine promotes RR formation, and we have traced these effects to dihydrofolate reductase (DHFR) and serine hydroxymethyltransferase-2 (SHMT2), pivotal enzymes in one-carbon metabolism and nucleotide biosynthesis. RR assembly is likewise induced upon DHFR inhibition by methotrexate or aminopterin as well as siRNA-mediated knockdown of DHFR or SHMT2. Because RR assembly occurs when guanine nucleotide biosynthesis is inhibited, and because RRs rapidly disassemble after the addition of guanine nucleotide precursors, RR formation might be an adaptive homeostatic mechanism, allowing IMPDH to sense changes in the one-carbon folate pathway.

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.

Adenosine blocks aminopterin-induced suppression of osteoclast differentiation.

To search cell surface molecules involved in the regulation of osteoclastogenesis, especially in fusion process, it is one powerful approach to obtain monoclonal antibodies bearing ability to block formation of multinucleated osteoclasts. Ideally, direct bio-assay of hybridoma supernatants is quite convenient to screen monoclonal antibodies of interest from numerous culture wells. However, addition of hybridoma supernatant containing hypoxanthine-aminopterin-thymidine (HAT), components of the selection medium, to whole bone marrow cultures strikingly suppressed osteoclastogenesis. Here we clarified aminopterin is the responsible component in HAT medium to inhibit osteoclastogenesis. Methotrexate (MTX), mono-methylated aminopterin, showed similar suppressive effect on osteoclastogenesis. When bone marrow cells were cultured in the presence of all nucleosides, aminopterin and MTX-induced suppression of osteoclastogenesis was abrogated. Among four nucleosides only adenosine canceled aminopterin-induced suppression of osteoclastogenesis. Direct bio-assay of hybridoma supernatant containing HAT selection medium is now available to screen monoclonal antibodies if adenosine-containing culture medium was utilized for evaluating osteoclastogenesis.

Clinical development of new formulations of cytotoxics in solid tumors.

PURPOSE OF REVIEW: To discuss the clinical development of new formulations of old cytotoxic agents and highlight the value of adopting this strategy. RECENT FINDINGS: Several drugs are currently in clinical development with high potential in improving clinical outcomes compared with their older counterparts. We emphasize on the results of four of these agents, each belonging to a known group of cytotoxics namely amrubicin, EndoTAG-1, pralatrexate and NKTR-102. Each has shown promising results that have the potential in addressing some limitations that have been observed with the 'earlier generation' agents. SUMMARY: Improvement in drug development strategies and the appreciation of the mechanisms of action and resistance of the cytotoxic agents currently available in the clinic open the door for developing agents that have the potential of improving clinical outcomes with better safety profiles. It is important to adopt innovative clinical trials designs integrating molecular markers in early clinical development in order to identify the subgroups of patients who would derive the maximal benefit of these novel agents.

Pralatrexate (Folotyn).

T-cell lymphoma accounts for 10% to 15% of all cases of non-Hodgkin lymphoma in the United States (approximately 5000 to 6000 cases a year). Peripheral T-cell lymphoma (PTCL) comprises a subgroup of rare and aggressive non-Hodgkin lymphomas that develop from T cells in different stages of maturity outside of the thymus. Cutaneous T-cell lymphoma is a subgroup that falls within the T-cell lymphoma population but is classified differently than other PTCLs. Most cases of CTCL are considered indolent and can often be treated with less aggressive therapies. Eight percent to 55% of CTCL cases undergo transformation, and once this transformation occurs, the disease acts similarly to other PTCLs and its classification changes to that of a PTCL. Transformed CTCL requires aggressive systemic therapy. Pralatrexate is the first Food and Drug Administration-approved drug for relapsed and refractory PTCL and has also gained compendia approval for treatment of CTCL. Pralatrexate is an antifolate chemotherapeutic inhibitor of dihydrofolatereductase. It has a high affinity for the one carbon-reduced folate carrier, which leads to better cellular internalization of the drug and has a greater antitumor effect than methotrexate. Several clinical trials have been conducted to evaluate the use of this drug in PTCL and other malignancies such as non-small cell lung cancer. This review offers focused information for dermatologists about pralatrexate and its use as a novel treatment for relapsed or refractory PTCL.

Pralatrexate Sustainably Released from Polypeptide Thermogel Is Effective for Chondrogenic Differentiation of Mesenchymal Stem Cells.

Folic acid was reported to significantly improve chondrogenic differentiation of mesenchymal stem cells. In a similar mechanism of action, we investigated clinically approved antifolates by the U.S. Food and Drug Administration as chondrogenic-promoting compounds for tonsil-derived mesenchymal stem cells. A poly(ethylene glycol)-poly(l-alanine) thermogelling system was used as a three-dimensional cell culture matrix, where stem cells and antifolates could be incorporated simultaneously during a heat-induced in situ sol-to-gel transition. The antifolates could be supplied over several days by the sustained release of the drug from the thermogel. Initially, seven antifolates were prescreened based on cell viability and expression of a typical chondrogenic biomarker of type II collagen (COL II) at the mRNA level. Then, dapsone, pralatrexate, and trimethoprim were selected as candidate compounds in the second round screening, and detailed studies were carried out on the mRNA and protein expression of various chondrogenic biomarkers including COL II, SRY box transcription factor 9, and aggrecan. Three-dimensional cultures of stem cells in the thermogel in the absence of a chondrogenic promoter compound and in the presence of kartogenin (KGN) were performed as a negative control and positive control, respectively. The chondrogenic biomarkers were significantly increased in the selected antifolate-incorporating systems compared to the negative control system, without an increase in type I collagen (an osteogenic biomarker) expression. Pralatrexate was the best compound for inducing chondrogenic differentiation of the stem cells, even better than the positive control (KGN). Nuclear translocation of the core-binding factor ß subunit (CBFß) and enhanced nuclear runt-related transcription factor 1 (RUNX1) by antifolate treatment suggested that the chondrogenesis-enhancing mechanism is mediated by CBFß and RUNX1. An in silico modeling study confirmed the mechanism by proving the high binding affinity of pralatrexate to a target protein of filamin A compared with other antifolate candidates. To conclude, pralatrexate was rediscovered as a lead compound, and the polypeptide thermogel incorporating pralatrexate and mesenchymal stem cells can be a very effective system in promoting chondrogenic differentiation of stem cells and might be used in injectable tissue engineering for cartilage repair.

Single agent and combination studies of pralatrexate and molecular correlates of sensitivity.

BACKGROUND: Pralatrexate is a dihydrofolate reductase (DHFR) inhibitor with high affinity for reduced folate carrier 1 (RFC-1) and folylpolyglutamate synthetase (FPGS), resulting in extensive internalization and accumulation in tumour cells. Pralatrexate is approved in the US for the treatment of relapsed or refractory peripheral T-cell lymphoma and is being investigated in various malignancies. Here, we evaluated molecular correlates of sensitivity to pralatrexate and explored combinations with a variety of anticancer agents. METHODS: Antiproliferative effects of pralatrexate were evaluated in 15 human-cancer cell lines using the MTT assay. Gene expression was evaluated using qRT-PCR. RESULTS: Pralatrexate and methotrexate had a similar pattern of cytotoxicity, pralatrexate being more potent. Pralatrexate potentiated the effects of platinum drugs, antimetabolites and EGFR inhibitors. Dose- and time-dependent cytotoxicity of pralatrexate correlated with high mRNA expression of FPGS. Acquired resistance to pralatrexate was associated with decreased RFC-1 expression, whereas methotrexate resistance correlated with increased DHFR expression, suggesting different mechanisms of acquired resistance. CONCLUSION: Pralatrexate was more potent than methotrexate in a panel of solid tumour lines. Our findings support the further clinical development of pralatrexate in combination with certain cytotoxics and targeted therapies, and suggest that RFC-1, FPGS and DHFR may be potential biomarkers of outcome.

Pralatrexate-induced tumor cell apoptosis in the epidermis of a patient with HTLV-1 adult T-cell lymphoma/leukemia causing skin erosions.

Pralatrexate is a novel antifolate, which shows increased antitumor activity in human tumor xenograft studies in mice compared with methotrexate. We investigated the effects of pralatrexate in a patient with adult T-cell lymphoma/leukemia with significant skin involvement. Atypical lymphocytes in epidermal Pautrier microabscesses were positive for HTLV-1. After the patient presented with leukemic conversion and with worsening of an erythematous generalized papular rash, he received one dose of pralatrexate. Within one week, his skin developed innumerable small erosions limited to the areas of the papular rash, sparing unaffected skin. Here we present in vivo evidence that pralatrexate-induced erosions in skin affected by adult T-cell lymphoma/leukemia are a manifestation of apoptosis of tumor cells infiltrating the epidermis and are not the result of cytotoxicity by pralatrexate on keratinocytes. This distinction is critical and may profoundly influence the clinical decision to continue pralatrexate treatment. Pralatrexate-induced skin erosions may indicate response to treatment.

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.

Generation of functional human T cell hybrids.

Human T cell hybrids were generated by fusing lectin-activated normal and leukemic human T cells with an aminopterin-sensitive human T cell line. This mutant cell line, designated CEM-T15, was derived from the human T cell line CEM after chemical mutagenesis with ethane methylsulfonate and subsequent culture in medium containing 6-thioguanine. After polyethylene glycol-induced fusion, the cells were cultured in hypoxanthine-aminopterin-thymidine selective medium. More than 5 wk after fusion, evidence for successful hybridization was obtained by three independent criteria: (a) The majority of the cultures contained cells expressing the OKT3 surface antigen: this antigen is expressed on normal T cells but not on CEM-T15 cells. (b) Most of the cultures contained polyploid cells. (c) Some of the cultures provided helper activity in the generation of antibody-forming cells. This functional activity is absent from the CEM-T15 parental cell line. Evidence for functional stability of the hybrids greater than 20 wk after fusion was provided by several clones that not only continue growing exponentially but also maintain expression of OKT3 surface antigen and high levels of helper function. These T cell hybrids constructed using antigen-specific human T cells should be of considerable importance in further studies of the immunobiology of human T cells.

Pralatrexate in Combination with Oxaliplatin in Advanced Esophagogastric Cancer: A Phase II Trial with Predictive Molecular Correlates.

The purpose of our study was to evaluate the efficacy of a combination of pralatrexate plus oxaliplatin in advanced esophagogastric cancer (EGC), analyze the impact of polymorphisms in folate metabolism pathway genes on toxicity and efficacy of pralatrexate, and to evaluate microRNA profile of tumor epithelium as a predictive biomarker. This was a two-stage trial with a safety lead in cohort and a primary endpoint of overall response rate (ORR). Patients received biweekly intravenous oxaliplatin (85 mg/m2) and pralatrexate (Dose level 1 [D1], 120 mg/m2; dose level-1 [D-1] 100 mg/m2). Single-nucleotide polymorphisms (SNP) in genes encoding proteins involved in pralatrexate metabolism were evaluated in germline DNA. microRNA profiling of the tumor epithelium was performed. ORR was 26%. Dose-limiting toxicities were observed in 2 of 4 patients at D1 and none at D-1. The T>C polymorphism in DHFR rs11951910 was significantly associated with lower progression-free survival (PFS; P ≤ 0.01), whereas the presence of the SLC19A1 rs2838957 G>A polymorphism was associated with improved PFS (P = 0.02). Presence of the GGH rs3780130 A>T and SLC19A1 rs1051266 G>A polymorphisms were significantly associated with better overall survival (OS; P = 0.01), whereas GGH rs7010484 T>C polymorphism was associated significantly with reduced OS (P = 0.04). There was no correlation between epithelial microRNA expression profile with disease progression or response. We conclude that the combination of oxaliplatin and pralatrexate is safe, is well tolerated, and has modest efficacy in advanced EGC. Pharmacogenomic analysis may be relevant to the use of pralatrexate in combination with platinum agents.

Protein folding causes an arrest of preprotein translocation into mitochondria in vivo.

With vital yeast cells, a hybrid protein consisting of the amino-terminal third of the precursor to cytochrome b2 and of the entire dihydrofolate reductase was arrested on the import pathway into mitochondria. Accumulation of the protein in the mitochondrial membranes was achieved by inducing a stable tertiary structure of the dihydrofolate reductase domain. Thereby, three salient features of mitochondrial protein uptake in vivo were demonstrated: its posttranslational character; the requirement for unfolding of precursors; and import through translocation contact sites. The permanent occupation of translocation sites by the fusion protein inhibited the import of other precursors; it did, however, not lead to leakage of mitochondrial ions, implying the existence of a channel that is sealed around the membrane spanning polypeptide segment.

Mitochondrial protein synthesis in HeLa cells.

HeLa cell mitochondrial proteins have been shown to be the products of two separate protein-synthesizing systems; one, the general cellular mechanism, sensitive to inhibition by cycloheximide, the other, a specific mitochondrial system subject to inhibition by low concentrations of chloramphenicol (Galper, J. B., and J. E. Darnell. 1971. J. Mol. Biol 57:363). Preliminary data have suggested that a mitochondrial N-formyl-methionyl-tRNA (f-Met-tRNA) might be the initiator tRNA in the latter (Galper, J. B., and J. E. Darnell. 1969. Biochem. Biophys. Res. Commun. 34:205; 1971. J. Mol. Biol. 57:363). It is demonstrated here that the synthesis of these endogenous mitochondrial proteins is also subject to inhibition by ethidium bromide and decays with a half-life of 1(1/2)-2 h in cultures incubated with low concentrations of this dye. The role of formylated f-Met-tRNA as the initiator tRNA in the synthesis of mitochondrial proteins is supported by data from several experiments. The rates of ethidium bromide inhibition of both the charging of f-Met-tRNA and of the synthesis of mitochondrial proteins are strikingly similar. Inhibition by aminopterin of the formylation of f-Met-tRNA greatly depresses the rate of mitochondrial-specific protein synthesis. In the absence of the synthesis of these proteins, respiration, the levels of cytochromes a-a(3) and b, and the number of mitochondrial cristae are decreased. The implications of these findings as they relate to mitochondrial biogenesis are discussed.

Import of a DHFR hybrid protein into glycosomes in vivo is not inhibited by the folate-analogue aminopterin.

Dihydrofolate reductase fusion proteins have been widely used to study conformational properties of polypeptides translocated across membranes. We have studied the import of dihydrofolate reductase fusion proteins into glycosomes and mitochondria of Trypanosoma brucei. As signal sequences we used the last 22 carboxy-terminal amino acids of glycosomal phosphoglycerate kinase for glycosomes, and the cleavable presequences of yeast cytochrome b2 or cytochrome oxidase subunit IV for mitochondria. Upon addition of aminopterin, a folate analogue that stabilizes the dihydrofolate reductase moiety, import of the fusion protein targeted to glycosomes was not inhibited, although the results of protease protection assays showed that the fusion protein could bind the drug. Under the same conditions, import of a DHFR fusion protein targeted to mitochondria was inhibited by aminopterin. When DHFR fusion proteins targeted simultaneously to both glycosomes and mitochondria were expressed, import into mitochondria was inhibited by aminopterin, whereas uptake of the same proteins into glycosomes was either unaffected or slightly increased. These findings suggest that the glycosomes possess either a strong unfolding activity or an unusually large or flexible translocation channel.