Inhibition Of Heme Synthesis Research Articles

Endogenous heme positively contributes to ALA-enhanced salt stress resistance in cucumber

In order to investigate the time-dependent rule of heme that is implicated in the salt resistance of cucumber seedlings, it is imperative to conduct a thorough investigation.Hence, this study endeavors subject cucumber (Cucumis sativus) plants to treatments involving exogenous heme synthesis inhibitors (DPD, 0.2 mmol∙L−1), heme scavengers (Hx, 0.4 μg∙L−1), and ALA (25 mg∙L−1) to investigate the response and regulatory mechanisms, photosynthetic capacity, and antioxidant capacity under salt stress (50 mmol∙L−1). The findings revealed that the concentration of heme in cucumber leaves increased by 55.8%, 107.8%, and 166.9%, respectively, on the 3rd, 5th, and 7th days under salt stress, in comparison to the level of control. The malondialdehyde (MDA) content in the leaves of seedlings under single salt stress increased by 16.3%, 27.6%, and 11.8%, respectively, and increased by 30.9%, 104.1%, and 89.4% in the roots. Proline content in leaves and roots of seedlings gradually accumulated under single salt stress. Furthermore, the CAT activity increased by 2.8% in comparison with the normal level on the 7th day of salt stresss. On the seventh day of treatment, the total chlorophyll content experienced a decrease of 36.8% in comparison to the control level. Correspondingly, the photosynthetic capacity of the leaves was also inhibited. On the 7th day of NaCl+DPD+Hx treatment, the concentrations of MDA in leaves and MDA in roots exhibited an increase by 39.6% and 17.3%, respectively, the concentrations of ChlT, Y(II), Pn, E, and gs were observed to decrease by 11.0%, 17.6%, 38.3%, 23.8%, and 44.0%, respectively, in comparison to a single salt treatment. Nonetheless, subsequent to the addition of exogenous ALA under stress, the concentrations of MDA and proline decreased, the activity of antioxidant enzymes remained stable, the concentrations of heme and chlorophyll increased, and the photosynthetic capacity augmented.In summary, heme is positively involved in its own defense mechanism and exogenous ALA-induced salt resistance enhancement in cucumber seedlings.

Roles for Heme Synthesis in the Maintenance of Hematopoietic Stem and Progenitor Cells

Heme is required not only in red blood cells for oxygen transport but also in non-erythroid cells for the proper function of hemoproteins. Cytochrome c is one of the hemoproteins and inevitable for mitochondrial oxidative phosphorylation, suggesting that heme possesses important roles for ATP synthesis and mitochondria maintenance. Heme also can skew the differentiation of hematopoietic stem and progenitor cells (HSPCs) by altering the functions of transcription factors. Recent CRISPR Cas9 dropout screens repeatedly showed that some of the heme synthases, such as CPOX, might be inevitable for the survival and/or proliferation of HSPCs. However, still little is known about the roles for heme synthesis in the maintenance (survival, proliferation and differentiation) of HSPCs. To reveal the roles for heme synthesis in HSPCs in vivo, we developed polyI:C inducible hematopoietic cell specific Cpox knock-out mice ( Cpox f/f; Mx1-Cre). PolyI:C treated Cpox f/f; Mx1-Cre mice (KO mice) showed significant increase in the numbers of hematopoietic stem cells (HSCs) and multipotent stem and progenitor cells (MPP2s and MPP3s) but decrease in the numbers of common myeloid progenitors (CMPs) and some of their progenies (i.e., Gr1 positive cells and platelets). In contrast, its effects on erythroid and lymphoid lineages were limited. This result suggests that heme synthesis inhibition in HSPCs (HSCs, MPP2 and MPP3s) can cause their differentiation arrest. On the other hand, competitive transplantation analysis of LSKs (lineage -sca1 +c-kit +) or HSCs revealed that myelopoiesis were markedly decreased compared to control, suggesting that heme synthesis in HSPCs is important for the proper reconstitution of hematopoiesis. These results indicate that cell intrinsic heme synthesis is inevitable for the maintenance of HSPCs and their lineage specific output in vivo. In depth, we revealed that mitochondrial respiratory activity of Cpox KO HSCs were increased compared to control HSCs, suggesting that alterations of mitochondrial status and/or functions by the inhibition of heme synthesis might contribute to the aberrance in Cpox KO HSPCs. Comprehensive gene expression analysis is now ongoing to support this notion. To further reveal the roles for heme synthesis in human HSPCs, we treated human leukemia cell lines (MV4-11, KG1a and U937) with heme synthesis inhibitors (SA or NMPP). Concentration-dependent cell growth inhibitory effect and cell death were observed with increased apoptosis factors (cleaved caspase 3 and cleaved PARP), suggesting that heme synthesis inhibition triggered apoptosis. We also revealed that CPOX inhibition under doxycycline inducible CRISPR Cas9 system caused cell growth inhibitory effect and cell death in these cells. Second mitochondria-derived activator of caspases (Smac) and cytochrome c were released to cytoplasm, and the inhibitor of apoptosis protein (XIAP) was decreased by heme synthesis inhibitions. Therefore, caspase-3 activation by accumulation of cytoplasmic cytochrome c and Smac might be the inducer of this apoptosis pathway. Comprehensive transcriptomic analysis (RNA-seq) revealed that heme synthesis inhibition induced the release of cytochrome c and Smac from mitochondria into cytoplasm by the transcriptomic upregulation of Oxidative Stress Induced Growth Inhibitor 1 (Osgin1) that is the known target of NRF2. Collectively, this study demonstrated the significance of heme synthesis in the maintenance of HSPCs especially in vivo (mouse) and in vitro (human). Dysfunctions of heme synthesis in HSPCs can cause their aberrance by the alterations in their mitochondrial status which can cause apoptosis and/or differentiation arrest. Considering the metabolic differences (i.e., demands for mitochondrial oxidative phosphorylation and subsequent ATP synthesis) between normal HSPCs and leukemia or unperturbed hematopoiesis and perturbed hematopoiesis (i.e., infections and hematopoietic stem cell transplantations), further analysis to reveal the roles for heme synthesis in the maintenance of HSPCs will pave the way to establish optical approaches in these clinical settings.

A critical role for heme synthesis and succinate in the regulation of pluripotent states transitions.

Using embryonic stem cells (ESCs) in regenerative medicine or in disease modeling requires a complete understanding of these cells. Two main distinct developmental states of ESCs have been stabilized in vitro, a naïve pre-implantation stage and a primed post-implantation stage. Based on two recently published CRISPR-Cas9 knockout functional screens, we show here that the exit of the naïve state is impaired upon heme biosynthesis pathway blockade, linked in mESCs to the incapacity to activate MAPK- and TGFβ-dependent signaling pathways after succinate accumulation. In addition, heme synthesis inhibition promotes the acquisition of 2 cell-like cells in a heme-independent manner caused by a mitochondrial succinate accumulation and leakage out of the cell. We further demonstrate that extracellular succinate acts as a paracrine/autocrine signal, able to trigger the 2C-like reprogramming through the activation of its plasma membrane receptor, SUCNR1. Overall, this study unveils a new mechanism underlying the maintenance of pluripotency under the control of heme synthesis.

Abstract 2681: Combination therapy of heme inhibitory protein (HeSP2) and Cyclopamine tartrate (CycT) with chemotherapeutic drugs is an effective strategy for treatment of TNBC

Abstract Breast cancer (BC) is the second leading cause of cancer death in women. Among the four main types of BC, triple-negative breast cancer (TNBC) accounts for 10 to 15% of BC. Although most TNBC patients have a poor prognosis, there are subsets of patients with tumors that respond well to chemotherapy. However, the outcome of chemotherapy in TNBC worsens over time due to the high expression of the oxidative phosphorylation (OXPHOS) complex. Likewise, drug resistant TNBC cells depend on OXPHOS; targeting oxidative metabolism and mitochondrial respiration can potentially overcome their drug resistance. Our lab recently engineered a heme-sequestering protein 2 (HeSP2) based on HasA hemophore. HeSP2 can effectively inhibit heme uptake and decrease the OXPHOS levels in cancer cells due to its high affinity for heme. Another agent, cyclopamine tartrate (CycT), previously known as a modulator of hedgehog signaling and mitochondrial respiration, can inhibit heme synthesis and reduce the OXPHOS levels. Therefore, using these heme-targeting agents in combination with chemotherapy could be an effective strategy to increase the therapeutic efficacy in TNBC. Cisplatin and etoposide are two chemotherapeutic drugs that have recently been used for treating TNBC. For this purpose, we examine the combination therapy of our OXPHOS inhibitory agents, HeSP2 and CycT, with cisplatin and etoposide in TNBC. In vitro experiment using three different TNBC cell lines, 4T1-Fluc-Neo, EMT6_Fluc_Puro, and MDA-MBA-231 confirmed the inhibitory effect of both HeSP2 and CycT in cell proliferation and colony formation in combination with or without chemo drugs. Results indicate that combination therapy significantly reduced the proliferation and inhibited colony formation in TNBC cells. As HeSP2 and CycT are therapeutic agents that reduce hypoxia and OXPHOS, they may also delay the emergence of drug resistance in cancer cells treated with chemotherapy. Our results indicate that HeSP2 and CycT in combination with chemotherapy drugs could serve as a promising therapy to diminish the tumorigenic function in TNBC and overcome resistance to chemotherapy. Citation Format: Parinaz Sadat Alemi, Maria del Carmen Chacon Castro, Eranda Berisha, Zakia Akter, Li Zhang. Combination therapy of heme inhibitory protein (HeSP2) and Cyclopamine tartrate (CycT) with chemotherapeutic drugs is an effective strategy for treatment of TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2681.

Functional Characterization of the Co2+ Transporter AitP in Sinorhizobium meliloti: A New Player in Fe2+ Homeostasis.

Co2+ induces the increase of the labile-Fe pool (LIP) by Fe-S cluster damage, heme synthesis inhibition, and "free" iron import, which affects cell viability. The N2-fixing bacteria, Sinorhizobium meliloti, is a suitable model to determine the roles of Co2+-transporting cation diffusion facilitator exporters (Co-eCDF) in Fe2+ homeostasis because it has a putative member of this subfamily, AitP, and two specific Fe2+-export systems. An insertional mutant of AitP showed Co2+ sensitivity and accumulation, Fe accumulation and hydrogen peroxide sensitivity, but not Fe2+ sensitivity, despite AitP being a bona fide low affinity Fe2+ exporter as demonstrated by the kinetic analyses of Fe2+ uptake into everted membrane vesicles. Suggesting concomitant Fe2+-dependent induced stress, Co2+ sensitivity was increased in strains carrying mutations in AitP and Fe2+ exporters which did not correlate with the Co2+ accumulation. Growth in the presence of sublethal Fe2+ and Co2+ concentrations suggested that free Fe-import might contribute to Co2+ toxicity. Supporting this, Co2+ induced transcription of Fe-import system and genes associated with Fe homeostasis. Analyses of total protoporphyrin content indicates Fe-S cluster attack as the major source for LIP. AitP-mediated Fe2+-export is likely counterbalanced via a nonfutile Fe2+-import pathway. Two lines of evidence support this: (i) an increased hemin uptake in the presence of Co2+ was observed in wild-type (WT) versus AitP mutant, and (ii) hemin reversed the Co2+ sensitivity in the AitP mutant. Thus, the simultaneous detoxification mediated by AitP aids cells to orchestrate an Fe-S cluster salvage response, avoiding the increase in the LIP caused by the disassembly of Fe-S clusters or free iron uptake. IMPORTANCE Cross-talk between iron and cobalt has been long recognized in biological systems. This is due to the capacity of cobalt to interfere with proper iron utilization. Cells can detoxify cobalt by exporting mechanisms involving membrane proteins known as exporters. Highlighting the cross-talk, the capacity of several cobalt exporters to also export iron is emerging. Although biologically less important than Fe2+, Co2+ induces toxicity by promoting intracellular Fe release, which ultimately causes additional toxic effects. In this work, we describe how the rhizobia cells solve this perturbation by clearing Fe through a Co2+ exporter, in order to reestablish intracellular Fe levels by importing nonfree Fe, heme. This piggyback-ride type of transport may aid bacterial cells to survive in free-living conditions where high anthropogenic Co2+ content may be encountered.

Is heme biosynthesis influenced the mitochondrial function and cell proliferation in cancer?

Heme is a compound consisting of an iron (Fe) atom bound to a pyrrole ring forming protoporphyrin IX (PPIX). Protoporphyrin combines with a protein-forming hemoprotein compound that plays an essential role in oxygen-binding and transport as well as in the process of energy production in the mitochondria. Some cancer cells have more heme biosynthesis than normal cells, which is thought to be linked to more cancer cell growth. Inhibition of heme biosynthesis in some cancer cells leads to decreased cell proliferation. This review article discusses the synthesis of heme, the role of heme in energy metabolism, which is needed for cell proliferation, the inhibition of heme synthesis and its effect on cancer cell proliferation, and the possibility of the inhibition of heme biosynthesis as an approach in therapy of cancer in the future.
 
 
 

SOSIALISASI PEMAKAIAN DAN PEMBAGIAN SARUNG TANGAN PADA MEKANIK BENGKEL ANTISIPASI TERPAPAR CEMARAN LOGAM DI DESA LALANG KABUPATEN DELI SERDANG

Personal protective equipment (PPE) is very necessary when working, one of which is a workplace that requires gloves, namely workshop mechanics. One of the PPE needed by workshop mechanics is gloves. Even though it's not like masks are always required to be used to avoid exposure to heavy metals, gloves are considered to be able to protect themselves from exposure to heavy metals. Pb (Lead) is a heavy metal. Exposure to Lead (Pb) is closely related to its accumulation in body tissues which causes disruption of physiological processes both directly and indirectly at the molecular level. Lead can cause chronic effects due to lead entering through the respiratory tract, nails, skin which enters the body and digestive tract and then spreads into the blood and will inhibit heme synthesis. As a result, Pb can reduce blood Hb production which can result in the emergence of other health problems. Therefore hand protection needs to be used by people who need more protection such as tire patchers who handle motorbike repairs. Gloves have many benefits for protecting mechanics working in workshops. The main purpose of giving gloves is to protect the hands of the tire patcher when working in a workshop where the nails have the potential to be exposed to heavy metals. After distributing these gloves, it is hoped that they will be able to suppress or minimize the exposure of workshop mechanics to heavy metals, in addition to educating mechanics and workshop entrepreneurs about the importance of maintaining and protecting themselves while doing work.

Heme is involved in the exogenous ALA-promoted growth and antioxidant defense system of cucumber seedlings under salt stress

A biosynthetic precursor of tetrapyrrol, 5-aminolevulinic acid (ALA), is widely used in agricultural production, as an exogenous regulatory substance that effectively regulates plant growth. Previous studies have shown that heme and chlorophyll accumulate in plants under salt stress, when treated with exogenous ALA. In this study, we explored the regulatory role of heme in plants, by spraying 25 mg L–1 ALA onto the leaves of cucumber seedlings treated with heme synthesis inhibitor (2,2'-dipyridyl, DPD) and heme scavenger (hemopexin, Hx), under 50 mmol L–1 NaCl stress. The results showed that NaCl alone and DPD + Hx treatments to cucumber seedlings subjected to salt stress adversely affected their growth, by decreasing biomass accumulation, root activity, and root morphology. In addition, these treatments induced an increase in membrane lipid oxidation, as well as enhancement of anti-oxidase activities, proline content, and glutamate betaine. However, exogenous ALA application increased the plant growth and root architecture indices under NaCl stress, owing to a lack of heme in the seedlings. In addition, cucumber seedlings treated with DPD and Hx showed inhibition of growth under salt stress, but exogenous ALA effectively improved cucumber seedling growth as well as the physiological characteristics; moreover, the regulation of ALA in plants was weakened when heme synthesis was inhibited. Heme biosynthesis and metabolism genes, HEMH and HO1, which are involved in the ALA metabolic pathway, were upregulated under salinity conditions, when ferrochelatase activity was inhibited. Application of exogenous ALA increased the heme content in the leaves. Thus, exogenous ALA may supplement the substrates for heme synthesis. These results indicated that heme plays a vital role in the response of plants to salinity stress. In conclusion, heme is involved in ALA-mediated alleviation of damage caused to cucumber seedlings and acts as a positive regulator of plant adaption.

ISCA2 deficiency leads to heme synthesis defects and impaired erythroid differentiation in K562 cells by indirect ROS-mediated IRP1 activation

Iron‑sulfur (Fe-S) clusters have been shown to play important roles in various cellular physiological process. Iron‑sulfur cluster assembly 2 (ISCA2) is a vital component of the [4Fe-4S] cluster assembly machine. Several studies have shown that ISCA2 is highly expressed during erythroid differentiation. However, the role and specific regulatory mechanisms of ISCA2 in erythroid differentiation and erythroid cell growth remain unclear. RNA interference was used to deplete ISCA2 expression in human erythroid leukemia K562 cells. The proliferation, apoptosis, and erythroid differentiation ability of the cells were assessed. We show that knockdown of ISCA2 has profound effects on [4Fe-4S] cluster formation, diminishing mitochondrial respiratory chain complexes, leading to reactive oxygen species (ROS) accumulation and mitochondrial damage, inhibiting cell proliferation. Excessive ROS can inhibit the activity of cytoplasmic aconitase (ACO1) and promote ACO1, a bifunctional protein, to perform its iron-regulating protein 1(IRP1) function, thus inhibiting the expression of 5′-aminolevulinate synthase 2 (ALAS2), which is a key enzyme in heme synthesis. Deficiency of ISCA2 results in the accumulation of iron divalent. In addition, the combination of excessive ferrous iron and ROS may lead to damage of the ACO1 cluster and higher IRP1 function. In brief, ISCA2 deficiency inhibits heme synthesis and erythroid differentiation by double indirect downregulation of ALAS2 expression. We conclude that ISCA2 is essential for normal functioning of mitochondria, and is necessary for erythroid differentiation and cell proliferation.

Identification and characterization of a heme exporter from the MRP family in Drosophila melanogaster

BackgroundThe heme group constitutes a major functional form of iron, which plays vital roles in various biological processes including oxygen transport and mitochondrial respiration. Heme is an essential nutrient, but its pro-oxidant nature may have toxic cellular effects if present at high levels, and its synthesis is therefore tightly regulated. Deficiency and excess of heme both lead to pathological processes; however, our current understanding of metazoan heme transport is largely limited to work in mammals and the worm Caenorhabditis elegans, while functional analyses of heme transport in the genetically amenable Drosophila melanogaster and other arthropods have not been explored.ResultsWe implemented a functional screening in Schneider 2 (S2) cells to identify putative heme transporters of D. melanogaster. A few multidrug resistance-associated protein (MRP) members were found to be induced by hemin and/or involved in heme export. Between the two plasma membrane-resident heme exporters CG4562 and CG7627, the former is responsible for heme transit across the intestinal epithelium. CG4562 knockdown resulted in heme accumulation in the intestine and lethality that could be alleviated by heme synthesis inhibition, human MRP5 (hMRP5) expression, heme oxygenase (HO) expression, or zinc supplement. CG4562 is mainly expressed in the gastric caeca and the anterior part of the midgut, suggesting this is the major site of heme absorption. It thus appears that CG4562 is the functional counterpart of mammalian MRP5. Mutation analyses in the transmembrane and nucleotide binding domains of CG4562 characterized some potential binding sites and conservative ATP binding pockets for the heme transport process. Furthermore, some homologs in Aedes aegypti, including that of CG4562, have also been characterized as heme exporters.ConclusionsTogether, our findings suggest a conserved heme homeostasis mechanism within insects, and between insects and mammals. We propose the fly model may be a good complement to the existing platforms of heme studies.

Inhibition of Heme Export and/or Heme Synthesis Potentiates Metformin Anti-Proliferative Effect on Cancer Cell Lines.

Simple SummaryTumor initiation and progression are sustained by the ability of the cancer cell to reshape its metabolism in a way that favors cell proliferation and survival. Recently, it was shown that heme metabolism contributes to metabolic adaptation of tumor cell and that interfering with heme homeostasis reduces tumor cell growth. Here, we show that the alteration of heme metabolism, either by RNA-interference or pharmacological approaches, increases the sensitivity of tumor cell lines to the antitumor agent metformin. These findings strengthen the concept of targeting heme metabolism to counteract tumor progression.Cancer is one of the leading causes of mortality worldwide. Beyond standard therapeutic options, whose effectiveness is often reduced by drug resistance, repurposing of the antidiabetic drug metformin appears promising. Heme metabolism plays a pivotal role in the control of metabolic adaptations that sustain cancer cell proliferation. Recently, we demonstrated the existence of a functional axis between the heme synthetic enzyme ALAS1 and the heme exporter FLVCR1a exploited by cancer cells to down-modulate oxidative metabolism. In colorectal cancer cell lines, the inhibition of heme synthesis-export system was associated with reduced proliferation and survival. Here, we aim to assess whether the inhibition of the heme synthesis-export system affects the sensitivity of colorectal cancer cells to metformin. Our data demonstrate that the inhibition of this system, either by blocking heme efflux with a FLVCR1a specific shRNA or by inhibiting heme synthesis with 5-aminolevulinic acid, improves metformin anti-proliferative effect on colorectal cancer cell lines. In addition, we demonstrated that the same effect can be obtained in other kinds of cancer cell lines. Our study provides an in vitro proof of concept of the possibility to target heme metabolism in association with metformin to counteract cancer cell growth.

Lack of human relevance for rat developmental toxicity of flumioxazin is revealed by comparative heme synthesis assay using embryonic erythroid cells derived from human and rat pluripotent stem cells.

Fetal rat anemia from flumioxazin, an N-phenylimide herbicide, is caused by suppression of heme synthesis resulting from inhibition of protoporphyrinogen oxidase (PPO). A series of studies to investigate the effects of flumioxazin have revealed that developmental toxicity is caused in rats but not in rabbits, and the adverse effects are not likely to occur in humans. In this study, as a final weight-of-evidence approach for assessing the human safety of flumioxazin, we compared the toxic potential of inhibition of heme synthesis leading to anemia between human and rat embryonic erythroid cells, which were degenerated as the target of flumioxazin in the rat developmental toxicity. To obtain embryonic erythroid cells, we established respective differentiation methods for embryonic erythroid cells from both human and rat pluripotent stem cells. Derived human and rat embryonic erythroid cells were treated with flumioxazin or dihydroartemisinin (DHA), an anti-malarial drug that causes reduction of embryonic erythroid cells and leads to anemia without species differences. In the human embryonic erythroid cells, DHA inhibited cell proliferation and heme synthesis, whereas there were no effects on heme content or cell proliferation with flumioxazin. In the rat embryonic erythroid cells, however, a dose-related reduction in heme synthesis occurred with treatment of flumioxazin and of DHA. These results confirmed that flumioxazin has no effect on heme synthesis in human embryonic erythroid cells. The present data were in accordance with the results of previous studies and demonstrated that there are no concerns in humans regarding the developmental toxicity of flumioxazin observed in rats.

Effects of lead exposure on blood electrical impedance spectroscopy of mice

BackgroundLead is a nonessential heavy metal, which can inhibit heme synthesis and has significant cytotoxic effects. Nevertheless, its effect on the electrical properties of red blood cells (RBCs) remains unclear. Consequently, this study aimed to investigate the electrical properties and the electrophysiological mechanism of lead exposure in mouse blood using Electrical Impedance Spectroscopy (EIS) in 0.01–100 MHz frequency range. Data characteristic of the impedance spectrum, Bodes plot, Nyquist plot and Nichols plot, and Constant Phase Element (CPE) equivalent circuit model were used to explicitly analyze the differences in amplitude–frequency, phase–frequency, and the frequency characteristics of blood in electrical impedance properties.ResultsCompared with the healthy blood in control mice, the changes in blood exposed to lead were as follows: (i) the hematocrit decreased; (ii) the amplitude–frequency and phase–frequency characteristics of electrical impedance decreased; (iii) the characteristic frequencies (f0) were significantly increased; (iv) the electrical impedance of plasma, erythrocyte membrane, and hemoglobin decreased, while the conductivity increased. (v) The pseudo-capacitance of cell membrane (CPE_Tm) and the intracellular pseudo-capacitance (CPE-Ti) were decreased.ConclusionsTherefore, EIS can be used as an effective method to monitor blood and RBC abnormalities caused by lead exposure. The electrical properties of the cells can be applied as an important observation in the evaluation of the toxic effects of heavy metals.

New Insights into the Pivotal Role of Iron/Heme Metabolism in TLR4/NF-κB Signaling-Mediated Inflammatory Responses in Human Monocytes.

Iron metabolism and heme biosynthesis are essential processes in cells during the energy cycle. Alteration in these processes could create an inflammatory condition, which results in tumorigenesis. Studies are conducted on the exact role of iron/heme metabolism in induced inflammatory conditions. This study used lipopolysaccharide (LPS)- or high-glucose-induced inflammation conditions in THP-1 cells to study how iron/heme metabolism participates in inflammatory responses. Here, we used iron and heme assays for measuring total iron and heme. We also used flow cytometry and Western blotting to analyze molecular responses. Our results demonstrated that adding LPS or high-glucose induced iron formation and heme synthesis and elevated the expression levels of proteins responsible for iron metabolism and heme synthesis. We then found that further addition of heme or 5-aminolevulinic acid (ALA) increased heme biosynthesis and promoted inflammatory responses by upregulating TLR4/NF-κB and inflammatory cytokine expressions. We also demonstrated the inhibition of heme synthesis using succinylacetone (SA). Moreover, N-MMP inhibited LPS- or high-glucose-induced inflammatory responses by inhibiting TLR4/NF-κB signaling. Hence, iron/heme metabolism checkpoints could be considered a target for treating inflammatory conditions.

The immunometabolite itaconate inhibits heme synthesis and remodels cellular metabolism in erythroid precursors

AbstractAs part of the inflammatory response by macrophages, Irg1 is induced, resulting in millimolar quantities of itaconate being produced. This immunometabolite remodels the macrophage metabolome and acts as an antimicrobial agent when excreted. Itaconate is not synthesized within the erythron but instead may be acquired from central macrophages within the erythroid island. Previously, we reported that itaconate inhibits hemoglobinization of developing erythroid cells. Herein we show that this action is accomplished by inhibition of tetrapyrrole synthesis. In differentiating erythroid precursors, cellular heme and protoporphyrin IX synthesis are reduced by itaconate at an early step in the pathway. In addition, itaconate causes global alterations in cellular metabolite pools, resulting in elevated levels of succinate, 2-hydroxyglutarate, pyruvate, glyoxylate, and intermediates of glycolytic shunts. Itaconate taken up by the developing erythron can be converted to itaconyl–coenzyme A (CoA) by the enzyme succinyl-CoA:glutarate-CoA transferase. Propionyl-CoA, propionyl-carnitine, methylmalonic acid, heptadecanoic acid, and nonanoic acid, as well as the aliphatic amino acids threonine, valine, methionine, and isoleucine, are increased, likely due to the impact of endogenous itaconyl-CoA synthesis. We further show that itaconyl-CoA is a competitive inhibitor of the erythroid-specific 5-aminolevulinate synthase (ALAS2), the first and rate-limiting step in heme synthesis. These findings strongly support our hypothesis that the inhibition of heme synthesis observed in chronic inflammation is mediated not only by iron limitation but also by limitation of tetrapyrrole synthesis at the point of ALAS2 catalysis by itaconate. Thus, we propose that macrophage-derived itaconate promotes anemia during an inflammatory response in the erythroid compartment.

Recent Trends in the Development of Benzimidazole Hybrid Derivatives and Their Antimalarial Activities

A parasite of the Plasmodium species initiates malaria. The parasite is transmitted to communities through the bite of an infected mosquito. Malarial resistance towards the commonly used antimalarial agents is a genuine human health problem. Benzimidazole derivatives exhibit a wide range of antimalarial activities against Plasmodium falciparum (P. falciparum) strain. The present review has summarized the antimalarial activity of benzimidazole hybrid derivatives and described its structural activity relationship (SAR) and quantitative structural-activity relationship (QSAR) model. A total of 14 papers were systematically reviewed. The literature survey has revealed that novel benzimidazole hybrid derivatives diminished the P. falciparum activity in the liver and gametocyte stages and inhibited heme synthesis and β-hematin formation. The QSAR models explain imminent antimalarial agent's growth through multiple linear regression (MLR) and artificial neural networks (ANN).

The Role of TCA Cycle Compounds in Erythroid Heme Synthesis and Its Regulation During an Inflammatory Response

One early hallmark event of terminal erythroid differentiation is the induction of heme and globin synthesis. Onset of heme synthesis occurs when the first pathway enzyme, erythroid-specific 5-aminolevulinate synthase (ALAS2), is induced. The steps of heme biosynthesis are identical between non-erythroid (housekeeping) and differentiating erythroid cells, but two genes encode the first enzyme, 5-aminolevulinate synthase (ALAS). The housekeeping gene (ALAS1) is expressed in non-erythroid cells while the erythroid-specific gene (ALAS2) is expressed only in the erythron. Heme synthesis in metazoan cells requires iron, glycine, and succinyl CoA. The supply of iron required for heme synthesis has been well studied and a reasonable view of the components involved in this process exists. Glycine is abundant in plasma (~250 µM) and studies of glycine transporters are consistent with glycine being supplied from extracellular sources. For succinyl CoA the general assumption is that it is produced by the TCA cycle. This may be possible for housekeeping heme synthesis where only modest levels of heme are made at a given time, but this mechanism would clearly place cells under strain during erythroid differentiation when cells make ~109 molecules of heme in a span of 2-3 days.ALAS2, but not ALAS1, physically interacts with the succinyl CoA synthetase (SCS) ATP-dependent subunit SUCLA2. Since ALAS utilizes succinyl CoA to synthesize aminolevulinic acid (ALA), and because SUCLA2 as a subunit of SCS may be involved in the ATP-dependent reverse reaction of SCS to generate succinyl CoA from succinate, one popular hypothesis is that the ALAS2-SUCLA2 interaction exists to provide succinyl CoA for ALAS2. While the evidence for an association between ALAS2 and SUCLA2 is strong, there is no experimental data showing that SUCLA2 has an impact on ALAS2 activity or that there is a role for this interaction in the supply of succinyl CoA for ALAS2.In the current work we have sought to determine if succinyl CoA for heme synthesis during erythropoiesis in MEL cells originates from the TCA cycle or more directly from metabolism of glutamine via transaminases and α-ketoglutarate dehydrogenase (α-KGDH). Employing 13C-labeled glucose, diethyl succinate, or glutamine we determined that succinate is a poor source of carbons for erythroid heme synthesis, providing a maximum of 5 carbons out of a possible 26 (by isotopomer analysis) and labeling only 2.5% of carbons of isolated heme. Glucose contributes up to 18 carbons and a total of 8.3%, and glutamine contributes to 23 carbons and a total of 23%. Enzyme assays of TCA cycle enzymes revealed that during erythropoiesis only aconitase, isocitrate dehydrogenase, and α-KGDH are induced, which is consistent with glutamine, and not succinate, being the major source of carbon for heme.We also examined the impact of itaconate, a compound produced from the TCA cycle in LPS-induced macrophages, on erythroid differentiation. Itaconate is known to inhibit succinate dehydrogenase, and so might be expected to induce heme synthesis if succinyl CoA is being supplied from accumulating succinate via ATP-dependent SCS. However, dimethyl itaconate (a cell permeable form of itaconate) inhibits heme synthesis in differentiating erythroid K562 cells and this inhibition is reversed by ALA. It has long been assumed that SCS has broad specificity and is capable of making itaconyl CoA (Adler J. et al. 1957, JBC 229:865). However, we found that purified human and porcine SCS do not synthesize itaconyl CoA, but that the enzyme succinyl CoA:glutarate CoA transferase (SUGCT) generates itaconyl CoA from itaconate and succinyl CoA. Additionally we demonstrated that itaconyl CoA inhibits purified ALAS2 at µM concentrations. Previously published data along with our studies on K562 cells in the presence of LPS-induced RAW cells suggest that during the inflammatory response macrophages resident in erythroblastic islands produce itaconate as well as cytokines that induce proximal developing red cells to express a plasmalemmal transporter (possibly SLC13A5) required for itaconate import. Intracellular itaconate is then converted to itaconyl CoA via SUGCT, resulting in ALAS2 inhibition and a concomitant decrease in heme synthesis. This process restricts the amount of protoporphyrin synthesized during infection and, thereby, complements mechanisms that restrict the availability of iron to microbial pathogens. DisclosuresNo relevant conflicts of interest to declare.

In silico drug repurposing for filarial infection predicts nilotinib and paritaprevir as potential inhibitors of the Wolbachia 5\u2032-aminolevulinic acid synthase

Filarial infections affect millions of individuals and are responsible for some notorious disabilities. Current treatment options involve repeated mass drug administrations, which have been met with several challenges despite some successes. Administration of doxycycline, an anti-Wolbachia agent, has shown clinical effectiveness but has several limitations, including long treatment durations and contraindications. We describe the use of an in silico drug repurposing approach to screening a library of over 3200 FDA-approved medications against the filarial endosymbiont, Wolbachia. We target the enzyme which catalyzes the first step of heme biosynthesis in the Wolbachia. This presents an opportunity to inhibit heme synthesis, which leads to depriving the filarial worm of heme, resulting in a subsequent macrofilaricidal effect. High throughput virtual screening, molecular docking and molecular simulations with binding energy calculations led to the identification of paritaprevir and nilotinib as potential anti-Wolbachia agents. Having higher binding affinities to the catalytic pocket than the natural substrate, these drugs have the structural potential to bind and engage active site residues of the wolbachia 5′-Aminolevulinic Acid Synthase. We hereby propose paritaprevir and nilotinib for experimental validations as anti-Wolbachia agents.

Cell‐Based Identification of New IDO1 Modulator Chemotypes

AbstractThe immunoregulatory enzyme indoleamine‐2,3‐dioxygenase (IDO1) strengthens cancer immune escape, and inhibition of IDO1 by means of new chemotypes and mechanisms of action is considered a promising opportunity for IDO1 inhibitor discovery. IDO1 is a cofactor‐binding, redox‐sensitive protein, which calls for monitoring of IDO1 activity in its native cellular environment. We developed a new, robust fluorescence‐based assay amenable to high throughput, which detects kynurenine in cells. Screening of a ca. 150 000‐member compound library discovered unprecedented, potent IDO1 modulators with different mechanisms of action, including direct IDO1 inhibitors, regulators of IDO1 expression, and inhibitors of heme synthesis. Three IDO1‐modulator chemotypes were identified that bind to apo‐IDO1 and compete with the heme cofactor. Our new cell‐based technology opens up novel opportunities for medicinal chemistry programs in immuno‐oncology.

Oxygen-Enhanced Optoacoustic Tomography Reveals the Effectiveness of Targeting Heme and Oxidative Phosphorylation at Normalizing Tumor Vascular Oxygenation.

Multispectral optoacoustic tomography (MSOT) is an emerging noninvasive imaging modality that can detect real-time dynamic information about the tumor microenvironment in humans and animals. Oxygen enhanced (OE)-MSOT can monitor tumor vasculature and oxygenation during disease development or therapy. Here, we used MSOT and OE-MSOT to examine in mice the response of human non-small cell lung cancer (NSCLC) xenografts to a new class of antitumor drugs, heme-targeting agents heme-sequestering peptide 2 (HSP2) and cyclopamine tartrate (CycT). HSP2 inhibits heme uptake, while CycT inhibits heme synthesis in NSCLC cells, where heme is essential for ATP generation via oxidative phosphorylation. HSP2 and CycT can inhibit ATP generation and thereby suppress NSCLC cell tumorigenic functions. MSOT showed that treatment of NSCLC tumors with HSP2 or CycT reduced total hemoglobin, increased oxygen saturation, and enhanced the amplitude of response to oxygen gas breathing challenge. HSP2 and CycT normalized tumor vasculature and improved tumor oxygenation, where levels of several hypoxia markers in NSCLC tumors were reduced by treatment with HSP2 or CycT. Furthermore, treatment with HSP2 or CycT reduced levels of angiogenic factor VEGFA, its receptor VEGFR1, and vascular marker CD34. Together, our data show that heme-targeting drugs HSP2 and CycT elicit multiple tumor-suppressing functions, such as inhibiting angiogenic function, normalizing tumor vasculature, alleviating tumor hypoxia, and inhibiting oxygen consumption and ATP generation. SIGNIFICANCE: Heme-targeting agents HSP2 and CycT effectively normalize tumor vasculature and alleviate tumor hypoxia, raising the possibility of their combination with chemo-, radio-, and immunotherapies to improve antitumor efficacy.See related commentary by Tomaszewski, p. 3461.