Resolving DJ-1 Glyoxalase Catalysis Using Mix-and-Inject Serial Crystallography at a Synchrotron.

DJ-1 (PARK7) is an intensively studied protein whose cytoprotective activities are dysregulated in multiple diseases. DJ-1 has been reported as having two distinct enzymatic activities in defense against reactive carbonyl species that are difficult to distinguish in conventional biochemical experiments. Here, we establish the mechanism of DJ-1 using a synchrotron-compatible version of mix-and-inject-serial crystallography (MISC), which was previously performed only at XFELs, to directly observe DJ-1 catalysis. We designed and used new diffusive mixers to collect time-resolved Laue diffraction data of DJ-1 catalysis at a pink beam synchrotron beamline. Analysis of structurally similar methylglyoxal-derived intermediates formed through the DJ-1 catalytic cycle shows that the enzyme catalyzes nearly two turnovers in the crystal and defines key aspects of its glyoxalase mechanism. In addition, DJ-1 shows allosteric communication between a distal site at the dimer interface and the active site that changes during catalysis. Our results rule out the widely cited deglycase mechanism for DJ-1 action and provide an explanation for how DJ-1 produces L-lactate with high chiral purity.

Methylglyoxal Adducts Are Prognostic Biomarkers for Diabetic Kidney Disease in Patients With Type 1 Diabetes.

More than 30% of patients with type 1 diabetes develop diabetic kidney disease (DKD), which significantly increases mortality risk. The Diabetes Control and Complications Trial (DCCT) and follow-up study, Epidemiology of Diabetes Interventions and Complications (EDIC), established that glycemic control measured by HbA1c predicts DKD risk. However, the continued high incidence of DKD reinforces the urgent need for additional biomarkers to supplement HbA1c. Here, we assessed biomarkers induced by methylglyoxal (MG), a metabolic by-product that forms covalent adducts on DNA, RNA, and proteins, called MG adducts. Urinary MG adducts were measured in samples from patients with type 1 diabetes enrolled in DCCT/EDIC who did (case patients; n = 90) or did not (control patients; n = 117) develop DKD. Univariate and multivariable analyses revealed that measurements of MG adducts independently predict DKD before established DKD biomarkers such as glomerular filtration rate and albumin excretion rate. Elevated levels of MG adducts bestowed the greatest risk of developing DKD in a multivariable model that included HbA1c and other clinical covariates. Our work establishes a novel class of biomarkers to predict DKD risk and suggests that inclusion of MG adducts may be a valuable tool to improve existing predictors of complications like DKD prior to overt disease, and to aid in identifying at-risk individuals and personalized risk management.

Methylglyoxal products in pre-symptomatic type 1 diabetes.

Introduction: Progression to type 1 diabetes has emerged as a complex process with metabolic alterations proposed to be a significant driver of disease. Monitoring products of altered metabolism is a promising tool for determining the risk of type 1 diabetes progression and to supplement existing predictive biomarkers. Methylglyoxal (MG) is a reactive product produced from protein, lipid, and sugar metabolism, providing a more comprehensive measure of metabolic changes compared to hyperglycemia alone. MG forms covalent adducts on nucleic and amino acids, termed MG-advanced glycation end products (AGEs) that associate with type 1 diabetes. Methods: We tested their ability to predict risk of disease and discriminate which individuals with autoimmunity will progress to type 1 diabetes. We measured serum MG-AGEs from 141 individuals without type 1 diabetes and 271 individuals with type 1 diabetes enrolled in the Fr1da cohort. Individuals with type 1 diabetes were at stages 1, 2, and 3. Results: We examined the association of MG-AGEs with type 1 diabetes. MG-AGEs did not correlate with HbA1c or differ between stages 1, 2, and 3 type 1 diabetes. Yet, RNA MG-AGEs were significantly associated with the rate of progression to stage 3 type 1 diabetes, with lower serum levels increasing risk of progression. Discussion: MG-AGEs were able to discriminate which individuals with autoantibodies would progress at a faster rate to stage 3 type 1 diabetes providing a potential new clinical biomarker for determining rate of disease progression and pointing to contributing metabolic pathways.

DJ-1 is not a deglycase and makes a modest contribution to cellular defense against methylglyoxal damage in neurons.

Human DJ-1 is a cytoprotective protein whose absence causes Parkinson's disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α-keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity.

Differential Kat3 Usage Orchestrates the Integration of Cellular Metabolism with Differentiation.

The integration of cellular status with metabolism is critically important and the coupling of energy production and cellular function is highly evolutionarily conserved. This has been demonstrated in stem cell biology, organismal, cellular and tissue differentiation and in immune cell biology. However, a molecular mechanism delineating how cells coordinate and couple metabolism with transcription as they navigate quiescence, growth, proliferation, differentiation and migration remains in its infancy. The extreme N-termini of the Kat3 coactivator family members, CBP and p300, by far the least homologous regions with only 66% identity, interact with members of the nuclear receptor family, interferon activated Stat1 and transcriptionally competent ß-catenin, a critical component of the Wnt signaling pathway. We now wish to report based on multiomic and functional investigations, utilizing p300 knockdown, N-terminal p300 edited and p300 S89A edited cell lines and p300 S89A knockin mice, that the N-termini of the Kat3 coactivators provide a highly evolutionarily conserved hub to integrate multiple signaling cascades to coordinate cellular metabolism with the regulation of cellular status and function.

Elevated glucose increases genomic instability by inhibiting nucleotide excision repair.

We investigated potential mechanisms by which elevated glucose may promote genomic instability. Gene expression studies, protein measurements, mass spectroscopic analyses, and functional assays revealed that elevated glucose inhibited the nucleotide excision repair (NER) pathway, promoted DNA strand breaks, and increased levels of the DNA glycation adduct N 2 -(1-carboxyethyl)-2'-deoxyguanosine (CEdG). Glycation stress in NER-competent cells yielded single-strand breaks accompanied by ATR activation, γH2AX induction, and enhanced non-homologous end-joining and homology-directed repair. In NER-deficient cells, glycation stress activated ATM/ATR/H2AX, consistent with double-strand break formation. Elevated glucose inhibited DNA repair by attenuating hypoxia-inducible factor-1α-mediated transcription of NER genes via enhanced 2-ketoglutarate-dependent prolyl hydroxylase (PHD) activity. PHD inhibition enhanced transcription of NER genes and facilitated CEdG repair. These results are consistent with a role for hyperglycemia in promoting genomic instability as a potential mechanism for increasing cancer risk in metabolic disease. Because of the pleiotropic functions of many NER genes beyond DNA repair, these results may have broader implications for cellular pathophysiology.

Protein-coated corrole nanoparticles for the treatment of prostate cancer cells.

Development of novel therapeutic strategies to eradicate malignant tumors is of paramount importance in cancer research. In a recent study, we have introduced a facile protocol for the preparation of corrole-protein nanoparticles (NPs). These NPs consist of a corrole-core coated with protein. We now report that a novel lipophilic corrole, (2)Ga, delivered as human serum albumin (HSA)-coated NPs, displayed antineoplastic activity towards human prostate cancer DU-145 cells. Cryo-TEM analysis of these NPs revealed an average diameter of 50.2 ± 8.1 nm with a spherical architecture exhibiting low polydispersity. In vitro cellular uptake of (2)Ga/albumin NPs was attributable to rapid internalization of the corrole through ligand binding-dependent extracellular release and intercalation of the corrole cargo into the lipid bilayer of the plasma membrane. This finding is in contrast with a previously reported study on corrole-protein NPs that displayed cellular uptake via endocytosis. Investigation of the non-light-induced mechanism of action of (2)Ga suggested the induction of necrosis through plasma membrane destabilization, impairment of calcium homeostasis, lysosomal stress and rupture, as well as formation of reactive oxygen species (ROS). (2)Ga also exhibited potent light-induced cytotoxicity through ROS generation. These findings demonstrate a rapid cellular uptake of (2)Ga/protein NPs along with targeted induction of tumor cell necrosis.

Metal-Assisted Protein Quantitation (MAPq): Multiplex Analysis of Protein Expression Using Lanthanide-Modified Antibodies with Detection by Inductively Coupled Plasma Mass Spectrometry.

Understanding the complex relationships between genomics, transcriptomics, and proteomics requires the development of more sensitive and rapid methods of multiplexed protein analysis. This is necessary to understand the relationship between cellular responses to environmental stresses, disease progression, and/or drug treatment; however, most methods are limited by low sensitivity, nonspecificity, and minimal multiplexing capacity. To more fully explore the relationship between multiple cellular pathways, we have developed a novel antibody-based multiplex assay using inductively coupled plasma mass spectrometry (ICP-MS), which we term metal-assisted protein quantitation (MAPq). MAPq utilizes lanthanide-conjugated antibodies to simultaneously quantify up to 35 proteins with low pg/mL sensitivity. This method is especially advantageous for low-abundance proteins, a significant limitation of many multiplex MS methods. We observed a limit of detection of 0.5 pg/mL and a limit of quantitation of 5 pg/mL with virtually no background signal. We applied this method to both cultured cells and mouse tissues to investigate changes in low-abundance nuclear and cytoplasmic proteins following drug or environmental stresses. MAPq was found to be at least 10 times more sensitive than Western blots and could detect quantitative changes in protein expression not readily observed using conventional approaches.

DNA Adducts as Biomarkers To Predict, Prevent, and Diagnose Disease-Application of Analytical Chemistry to Clinical Investigations.

Characterization of the chemistry, structure, formation, and metabolism of DNA adducts has been one of the most significant contributions to the field of chemical toxicology. This work provides the foundation to develop analytical methods to measure DNA adducts, define their relationship to disease, and establish clinical tests. Monitoring exposure to environmental and endogenous toxicants can predict, diagnose, and track disease as well as guide therapeutic treatment. DNA adducts are one of the most promising biomarkers of toxicant exposure owing to their stability, appearance in numerous biological matrices, and characteristic analytical properties. In addition, DNA adducts can induce mutations to drive disease onset and progression and can serve as surrogate markers of chemical exposure. In this perspective, we highlight significant advances made within the past decade regarding DNA adduct quantitation using mass spectrometry. We hope to expose a broader audience to this field and encourage analytical chemistry laboratories to explore how specific adducts may be related to various pathologies. One of the limiting factors in developing clinical tests to measure DNA adducts is cohort size; ideally, the cohort would allow for model development and then testing of the model to the remaining cohort. The goals of this perspective article are to (1) provide a summary of analyte levels measured using state-of-the-art analytical methods, (2) foster collaboration, and (3) highlight areas in need of further investigation.

Structures and Spectroscopic Properties of Metallocorrole Nanoparticles.

In aqueous media, hydrophobic metallocorroles form nanoparticles that are potential theranostic anticancer agents. We have analyzed the electronic and Raman spectra of Al(III), Ga(III), and Au(III) corrole nanoparticles (and made comparisons with DFT-validated assignments of the IR spectra of corresponding monomers) in order to estimate the strengths of corrole-corrole electronic couplings in these assemblies. We find that these spectra are virtually unchanged upon aggregation, confirming that the intermolecular interactions in these nanoparticles are very weak.

MLS128 antibody-induced suppression of colon cancer cell growth is mediated by a desmocollin and a 110 kDa glycoprotein.

Protein glycosylation is a diverse form of post-translational modification. Two to three consecutive O-linked N-acetylgalactosamines (Tn-antigens) are recognized by antibodies such as MLS128. MLS128 mAb inhibited cell growth and bound to a 110 kDa glycoprotein (GP) in LS180 and HT29 colon cancer cells. However, purification and identification of the 110 kDa GP was unsuccessful due to its low abundance. The present study used a highly sophisticated and sensitive mass spectrometry method to identify proteins immunoprecipitated with MLS128 and separated by two-dimensional gel electrophoresis. Three desmosome components were identified. Of these, desmocollin and desmoglein shared many similar characteristics, including molecular mass, pI, and potential Tn-antigen sites. Western blotting analyses of LS180 cell lysates revealed a common 110 kDa band recognized by MLS128 and anti-desmocollin, but not by anti-desmoglein. Immunofluorescence microscopy of LS180 cells revealed that desmocollin is membrane-bound, while desmoglein is primarily localized in the cytosol. Confocal microscopy demonstrated colocalization of the desmocollin-specific antibody with the MLS128 antibody on the cell membrane, suggesting that desmocollin may contain Tn-antigens recognized by MLS128. Treatment of LS180 cells with siRNA to knock down desmocollin expression or a desmocollin-specific antibody decreased cell viability, suggesting a critical role for this protein in cell growth and survival. N-glycosidase F digestion of the 110 kDa GP and desmocollin suggested that although both proteins contain N-glycosylation sites, they are not identical. These findings suggest that desmocollin colocalizes with the 110 kDa GP and that growth inhibition induced by the MLS128 antibody may be mediated through a mechanism that involves desmocollin.

Cell-Penetrating Protein/Corrole Nanoparticles.

Recent work has highlighted the potential of metallocorroles as versatile platforms for the development of drugs and imaging agents, since the bioavailability, physicochemical properties and therapeutic activity can be dramatically altered by metal ion substitution and/or functional group replacement. Significant advances in cancer treatment and imaging have been reported based on work with a water-soluble bis-sulfonated gallium corrole in both cellular and rodent-based models. We now show that cytotoxicities increase in the order Ga < Fe < Al < Mn < Sb < Au for bis-sulfonated corroles; and, importantly, that they correlate with metallocorrole affinities for very low density lipoprotein (VLDL), the main carrier of lipophilic drugs. As chemotherapeutic potential is predicted to be enhanced by increased lipophilicity, we have developed a novel method for the preparation of cell-penetrating lipophilic metallocorrole/serum-protein nanoparticles (NPs). Cryo-TEM revealed an average core metallocorrole particle size of 32 nm, with protein tendrils extending from the core (conjugate size is ~100 nm). Optical imaging of DU-145 prostate cancer cells treated with corrole NPs (≤100 nM) revealed fast cellular uptake, very slow release, and distribution into the endoplasmic reticulum (ER) and lysosomes. The physical properties of corrole NPs prepared in combination with transferrin and albumin were alike, but the former were internalized to a greater extent by the transferrin-receptor-rich DU-145 cells. Our method of preparation of corrole/protein NPs may be generalizable to many bioactive hydrophobic molecules to enhance their bioavailability and target affinity.

Inhibition of GLO1 in Glioblastoma Multiforme Increases DNA-AGEs, Stimulates RAGE Expression, and Inhibits Brain Tumor Growth in Orthotopic Mouse Models.

Cancers that exhibit the Warburg effect may elevate expression of glyoxylase 1 (GLO1) to detoxify the toxic glycolytic byproduct methylglyoxal (MG) and inhibit the formation of pro-apoptotic advanced glycation endproducts (AGEs). Inhibition of GLO1 in cancers that up-regulate glycolysis has been proposed as a therapeutic targeting strategy, but this approach has not been evaluated for glioblastoma multiforme (GBM), the most aggressive and difficult to treat malignancy of the brain. Elevated GLO1 expression in GBM was established in patient tumors and cell lines using bioinformatics tools and biochemical approaches. GLO1 inhibition in GBM cell lines and in an orthotopic xenograft GBM mouse model was examined using both small molecule and short hairpin RNA (shRNA) approaches. Inhibition of GLO1 with S-(p-bromobenzyl) glutathione dicyclopentyl ester (p-BrBzGSH(Cp)2) increased levels of the DNA-AGE N²-1-(carboxyethyl)-2'-deoxyguanosine (CEdG), a surrogate biomarker for nuclear MG exposure; substantially elevated expression of the immunoglobulin-like receptor for AGEs (RAGE); and induced apoptosis in GBM cell lines. Targeting GLO1 with shRNA similarly increased CEdG levels and RAGE expression, and was cytotoxic to glioma cells. Mice bearing orthotopic GBM xenografts treated systemically with p-BrBzGSH(Cp)2 exhibited tumor regression without significant off-target effects suggesting that GLO1 inhibition may have value in the therapeutic management of these drug-resistant tumors.

Product Studies and Mechanistic Analysis of the Reaction of Methylglyoxal with Deoxyguanosine.

Methylglyoxal (MG) is a highly reactive electrophile produced endogenously as a byproduct of glucose metabolism and protein catabolism and exogenously as a food contaminant. MG reacts spontaneously with proteins, lipids, and nucleic acids to form advanced glycation end products (AGEs), modifying or inhibiting their function. Protein AGEs are associated with pathological complications of diabetes, cancer, and neurodegenerative diseases, while the physiological impact of DNA, RNA, and lipid AGE formation is less well explored. Conflicting reports in the literature on the biologically significant DNA-AGE product distribution and mechanisms of formation prompted a re-examination of the reaction products of MG with dG, oligonucleotides, and plasmid DNA under varying conditions of MG:dG stoichiometry, pH, and reaction time. Major products identified using sequential mass fragmentation and authentic standards were N2-(1-carboxyethyl)-2'-dG (CEdG), N2-(1-carboxyethyl)-7-1-hydroxy-2-oxopropyl-dG (MG-CEdG), and 1,N2-(1,2-dihydroxy-2-methyl)ethano-2'-dG (cMG-dG). CEdG and MG-CEdG were observed in all DNA substrates, although cMG-dG was not detected to any significant extent in oligomeric or polymeric DNA. Product analyses of reactions under conditions of diminished water activity as well as results from H218O labeling indicated that MG hydration equilibria plays an important role in controlling product distribution. In contrast to previous reports, our data support independent mechanisms of formation of CEdG and cMG-dG, with the latter kinetic product undergoing reversible formation under physiological conditions.

Metabolic advantages and vulnerabilities in brain metastases.

Metabolic adaptations permit tumor cells to metastasize to and thrive in the brain. Brain metastases continue to present clinical challenges due to rising incidence and resistance to current treatments. Therefore, elucidating altered metabolic pathways in brain metastases may provide new therapeutic targets for the treatment of aggressive disease. Due to the high demand for glucose in the brain, increased glycolytic activity is favored for energy production. Primary tumors that undergo Warburg-like metabolic reprogramming become suited to growth in the brain microenvironment. Indeed, elevated metabolism is a predictor of metastasis in many cancer subtypes. Specifically, metabolic alterations are seen in primary tumors that are associated with the formation of brain metastases, namely breast cancer, lung cancer, and melanoma. Because of this selective pressure, inhibitors of key metabolic factors may reduce tumor cell viability, thus exploiting metabolic pathways for cancer therapeutics. This review summarizes the metabolic advantages and vulnerabilities of brain metastases.

DNA Advanced Glycation End Products (DNA-AGEs) Are Elevated in Urine and Tissue in an Animal Model of Type 2 Diabetes.

More precise identification and treatment monitoring of prediabetic/diabetic individuals will require additional biomarkers to complement existing diagnostic tests. Candidates include hyperglycemia-induced adducts such as advanced glycation end products (AGEs) of proteins, lipids, and DNA. The potential for DNA-AGEs as diabetic biomarkers was examined in a longitudinal study using the Leprdb/db animal model of metabolic syndrome. The DNA-AGE, N2-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) was quantified by mass spectrometry using isotope dilution from the urine and tissue of hyperglycemic and normoglycemic mice. Hyperglycemic mice (fasting plasma glucose, FPG, ≥ 200 mg/dL) displayed a higher median urinary CEdG value (238.4 ± 112.8 pmol/24 h) than normoglycemic mice (16.1 ± 11.8 pmol/24 h). Logistic regression analysis revealed urinary CEdG to be an independent predictor of hyperglycemia. Urinary CEdG was positively correlated with FPG in hyperglycemic animals and with HbA1c for all mice. Average tissue-derived CEdG was also higher in hyperglycemic mice (18.4 CEdG/106 dG) than normoglycemic mice (4.4 CEdG/106 dG). Urinary CEdG was significantly elevated in Leprdb/db mice relative to Leprwt/wt, and tissue CEdG values increased in the order Leprwt/wt < Leprwt/db < Leprdb/db. These data suggest that urinary CEdG measurement may provide a noninvasive quantitative index of glycemic status and augment existing biomarkers for the diagnosis and monitoring of diabetes.

Fighting Cancer with Corroles.

Corroles are exceptionally promising platforms for the development of agents for simultaneous cancer-targeting imaging and therapy. Depending on the element chelated by the corrole, these theranostic agents may be tuned primarily for diagnostic or therapeutic function. Versatile synthetic methodologies allow for the preparation of amphipolar derivatives, which form stable noncovalent conjugates with targeting biomolecules. These conjugates can be engineered for imaging and targeting as well as therapeutic function within one theranostic assembly. In this review, we begin with a brief outline of corrole chemistry that has been uniquely useful in designing corrole-based anticancer agents. Then we turn attention to the early literature regarding corrole anticancer activity, which commenced one year after the first scalable synthesis was reported (1999-2000). In 2001, a major advance was made with the introduction of negatively charged corroles, as these molecules, being amphipolar, form stable conjugates with many proteins. More recently, both cellular uptake and intracellular trafficking of metallocorroles have been documented in experimental investigations employing advanced optical spectroscopic as well as magnetic resonance imaging techniques. Key results from work on both cellular and animal models are reviewed, with emphasis on those that have shed new light on the mechanisms associated with anticancer activity. In closing, we predict a very bright future for corrole anticancer research, as it is experiencing exponential growth, taking full advantage of recently developed imaging and therapeutic modalities.

Cellular uptake and anticancer activity of carboxylated gallium corroles.

We report derivatives of gallium(III) tris(pentafluorophenyl)corrole, 1 [Ga(tpfc)], with either sulfonic (2) or carboxylic acids (3, 4) as macrocyclic ring substituents: the aminocaproate derivative, 3 [Ga(ACtpfc)], demonstrated high cytotoxic activity against all NCI60 cell lines derived from nine tumor types and confirmed very high toxicity against melanoma cells, specifically the LOX IMVI and SK-MEL-28 cell lines. The toxicities of 1, 2, 3, and 4 [Ga(3-ctpfc)] toward prostate (DU-145), melanoma (SK-MEL-28), breast (MDA-MB-231), and ovarian (OVCAR-3) cancer cells revealed a dependence on the ring substituent: IC50values ranged from 4.8 to >200 µM; and they correlated with the rates of uptake, extent of intracellular accumulation, and lipophilicity. Carboxylated corroles 3 and 4, which exhibited about 10-fold lower IC50values (<20 µM) relative to previous analogs against all four cancer cell lines, displayed high efficacy (Emax= 0). Confocal fluorescence imaging revealed facile uptake of functionalized gallium corroles by all human cancer cells that followed the order: 4 >> 3 > 2 >> 1 (intracellular accumulation of gallium corroles was fastest in melanoma cells). We conclude that carboxylated gallium corroles are promising chemotherapeutics with the advantage that they also can be used for tumor imaging.

Lanthanides: Applications in Cancer Diagnosis and Therapy.

Lanthanide complexes are of increasing importance in cancer diagnosis and therapy, owing to the versatile chemical and magnetic properties of the lanthanide-ion 4f electronic configuration. Following the first implementation of gadolinium(III)-based contrast agents in magnetic resonance imaging in the 1980s, lanthanide-based small molecules and nanomaterials have been investigated as cytotoxic agents and inhibitors, in photodynamic therapy, radiation therapy, drug/gene delivery, biosensing, and bioimaging. As the potential utility of lanthanides in these areas continues to increase, this timely review of current applications will be useful to medicinal chemists and other investigators interested in the latest developments and trends in this emerging field.

An in vitro enzymatic assay to measure transcription inhibition by gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles.

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy. Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action. Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control. The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide. This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.