The Interaction and Effect of a Small MitoBlock Library as Inhibitor of ALR Protein-Protein Interaction Pathway.

MIA40 and ALR of the MIA pathway mediate the import of protein precursors that form disulfides into the mitochondrial intermembrane space. This import pathway is suggested to be a linear pathway in which MIA40 first binds to the precursor via a disulfide linkage and oxidizes it. Subsequently, ALR re-oxidizes MIA40 and then ALR transfers electrons to terminal electron acceptors. However, the precise mechanism by which ALR and MIA40 coordinate translocation is unknown. With a collection of small molecule modulators (MB-5 to MB-9 and MB-13) that inhibit ALR activity, we characterized the import mechanism in mitochondria. NMR studies show that most of the compounds bind to a similar region in ALR. Mechanistic studies with small molecules demonstrate that treatment with compound MB-6 locks the precursor in a state bound to MIA40, blocking re-oxidation of MIA40 by ALR. Thus, small molecules that target a similar region in ALR alter the dynamics of the MIA import pathway differently, resulting in a set of probes that are useful for studying the catalysis of the redox-regulated import pathway in model systems.

N-terminal domains mediate [2Fe-2S] cluster transfer from glutaredoxin-3 to anamorsin.

In eukaryotes, cytosolic monothiol glutaredoxins are proteins implicated in intracellular iron trafficking and sensing via their bound [2Fe-2S] clusters. We define a new role of human cytosolic monothiol glutaredoxin-3 (GRX3) in transferring its [2Fe-2S] clusters to human anamorsin, a physical and functional protein partner of GRX3 in the cytosol, whose [2Fe-2S] cluster-bound form is involved in the biogenesis of cytosolic and nuclear Fe-S proteins. Specific protein recognition between the N-terminal domains of the two proteins is the mandatory requisite to promote the [2Fe-2S] cluster transfer from GRX3 to anamorsin.

Elucidating the Molecular Function of Human BOLA2 in GRX3-Dependent Anamorsin Maturation Pathway.

In eukaryotes, the interaction between members of the monothiol glutaredoxin family and members of the BolA-like protein family has been involved in iron metabolism. To investigate the still unknown functional role of the interaction between human glutaredoxin-3 (GRX3) and its protein partner BOLA2, we characterized at the atomic level the interaction of apo BOLA2 with the apo and holo states of GRX3 and studied the role of BOLA2 in the GRX3-dependent anamorsin maturation pathway. From these studies, it emerged that apo GRX3 and apo BOLA2 form a heterotrimeric complex, composed by two BOLA2 molecules and one GRX3 molecule. This complex is able to bind two [2Fe-2S](2+) clusters, each being bridged between a BOLA2 molecule and a monothiol glutaredoxin domain of GRX3, and to transfer both [2Fe-2S](2+) clusters to apo anamorsin producing its mature holo state. Collectively, the data suggest that the heterotrimeric complex can work as a [2Fe-2S](2+) cluster transfer component in cytosolic Fe/S protein maturation pathways.

The IR-¹5N-HSQC-AP experiment: a new tool for NMR spectroscopy of paramagnetic molecules.

A crucial factor for the understanding of structure-function relationships in metalloproteins is the identification of NMR signals from residues surrounding the metal cofactor. When the latter is paramagnetic, the NMR information in the proximity of the metal center may be scarce, because fast nuclear relaxation quenches signal intensity and coherence transfer efficiency. To identify residues at a short distance from a paramagnetic center, we developed a modified version of the ¹5N-HSQC experiment where (1) an inversion recovery filter is added prior to HSQC, (2) the INEPT period has been optimized according to fast relaxation of interested spins, (3) the inverse INEPT has been eliminated and signals acquired as antiphase doublets. The experiment has been successfully tested on a human [Fe2S2] protein which is involved in the biogenesis of iron-sulfur proteins. Thirteen HN resonances, unobserved with conventional HSQC experiments, could be identified. The structural arrangement of the protein scaffold in the proximity of the Fe/S cluster is fundamental to comprehend the molecular processes responsible for the transfer of Fe/S groups in the iron-sulfur protein assembly machineries.

Dimerization of the 4Ig isoform of B7-H3 in tumor cells mediates enhanced proliferation and tumorigenic signaling.

B7-H3 (CD276) has two isoforms (2Ig and 4Ig), no confirmed cognate receptor, and physiological functions that remain elusive. While differentially expressed on many solid tumors correlating with poor survival, mechanisms of how B7-H3 signals in cis (tumor cell) versus in trans (immune cell co-regulator) to elicit pro-tumorigenic phenotypes remain poorly defined. Herein, we characterized a tumorigenic and signaling role for tumor cell-expressed 4Ig-B7-H3, the dominant human isoform, in gynecological cancers that could be abrogated upon CRISPR/Cas9 knockout of B7-H3; tumorigenesis was rescued upon re-expression of 4Ig-B7-H3. Size exclusion chromatography revealed dimerization states for the extracellular domains of both human 4Ig- and murine 2Ig-B7-H3. mEGFP lifetimes of expressed 4Ig-B7-H3-mEGFP fusions determined by FRET-FLIM assays confirmed close-proximity interactions of 4Ig-B7-H3 and identified two distinct homo-FRET lifetime populations, consistent with monomeric and homo-dimer interactions. In live cells, bioluminescence imaging of 4Ig-B7-H3-mediated split luciferase complementation showed dimerization of 4Ig-B7-H3. To separate basal from dimer state activities in the absence of a known receptor, C-terminus (cytosolic) chemically-induced dimerization of 4Ig-B7-H3 increased tumor cell proliferation and cell activation signaling pathways (AKT, Jak/STAT, HIF1α, NF-κß) significantly above basal expression of 4Ig-B7-H3 alone. These results revealed a new, dimerization-dependent intrinsic tumorigenic signaling role for 4Ig-B7-H3, likely acting in cis, and provide a therapeutically-actionable target for intervention of B7-H3-dependent tumorigenesis.

Adipose Triglyceride Lipase Is a Therapeutic Target in Advanced Prostate Cancer That Promotes Metabolic Plasticity.

Lipid metabolism plays a central role in prostate cancer. To date, the major focus has centered on de novo lipogenesis and lipid uptake in prostate cancer, but inhibitors of these processes have not benefited patients. A better understanding of how cancer cells access lipids once they are created or taken up and stored could uncover more effective strategies to perturb lipid metabolism and treat patients. Here, we identified that expression of adipose triglyceride lipase (ATGL), an enzyme that controls lipid droplet homeostasis and a previously suspected tumor suppressor, correlates with worse overall survival in men with advanced, castration-resistant prostate cancer (CRPC). Molecular, genetic, or pharmacologic inhibition of ATGL impaired human and murine prostate cancer growth in vivo and in cell culture or organoids under conditions mimicking the tumor microenvironment. Mass spectrometry imaging demonstrated that ATGL profoundly regulates lipid metabolism in vivo, remodeling membrane composition. ATGL inhibition induced metabolic plasticity, causing a glycolytic shift that could be exploited therapeutically by cotargeting both metabolic pathways. Patient-derived phosphoproteomics identified ATGL serine 404 as a target of CAMKK2-AMPK signaling in CRPC cells. Mutation of serine 404 did not alter the lipolytic activity of ATGL but did decrease CRPC growth, migration, and invasion, indicating that noncanonical ATGL activity also contributes to disease progression. Unbiased immunoprecipitation/mass spectrometry suggested that mutation of serine 404 not only disrupts existing ATGL protein interactions but also leads to new protein-protein interactions. Together, these data nominate ATGL as a therapeutic target for CRPC and provide insights for future drug development and combination therapies. SIGNIFICANCE: ATGL promotes prostate cancer metabolic plasticity and progression through both lipase-dependent and lipase-independent activity, informing strategies to target ATGL and lipid metabolism for cancer treatment.

Production, isolation, and shipment of clinically relevant quantities of astatine-211: A simple and efficient approach to increasing supply.

The alpha emitter astatine-211 (211At) is a promising candidate for cancer treatment based on Targeted Alpha (α) Therapy (TAT). A small number of facilities, distributed across the United States, are capable of accelerating α-particle beams to produce 211At. However, challenges remain regarding strategic methods for shipping 211At in a form adaptable to advanced radiochemistry reactions and other uses of the radioisotope. PURPOSE: Our method allows shipment of 211At in various quantities in a form convenient for further radiochemistry. PROCEDURES: For this study, a 3-octanone impregnated Amberchrom CG300M resin bed in a column cartridge was used to separate 211At from the bismuth matrix on site at the production accelerator (Texas A&M) in preparation for shipping. Aliquots of 6 M HNO3 containing up to ≈2.22 GBq of 211At from the dissolved target were successfully loaded and retained on columns. Exempt packages (<370 MBq) were shipped to a destination radiochemistry facility, University of Texas MD Anderson Cancer Center, in the form of a convenient air-dried column. Type A packages have been shipped overnight to University of Alabama at Birmingham. MAIN FINDINGS: Air-dried column hold times of various lengths did not inhibit simple and efficient recovery of 211At. Solution eluted from the column was sufficiently high in specific activity to successfully radiolabel a model compound, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1), with 211At. The method to prepare and ship 211At described in this manuscript has also been used to ship larger quantities of 211At a greater distance to University of Alabama at Birmingham. PRINCIPAL CONCLUSIONS: The successful proof of this method paves the way for the distribution of 211At from Texas A&M University to research institutions and clinical oncology centers in Texas and elsewhere. Use of this simple method at other facilities has the potential increase the overall availability of 211At for preclinical and clinical studies.

RAS-Driven Macropinocytosis of Albumin or Dextran Reveals Mutation-Specific Target Engagement of RAS p.G12C Inhibitor ARS-1620 by NIR-Fluorescence Imaging.

PURPOSE: Macropinocytosis serves as a highly conserved endocytotic process that has recently been shown as a critical mechanism by which RAS-transformed cells transport extracellular protein into intracellular amino acid pathways to support their unique metabolic needs. We developed NIR fluorescently labeled molecular imaging probes to monitor macropinocytosis-mediated uptake of albumin in a K-RAS-dependent manner. PROCEDURES: Using western blot analysis, immunofluorescence, and flow cytometry, albumin retention was characterized in vitro across several RAS-activated lung and pancreatic cancer cell lines. AF790-albumin was synthesized and administered to mice bearing K-RAS mutant xenograft tumors of H460 (K-RAS p.Q61H) and H358 (K-RAS p.G12C) non-small cell lung cancers on each flank. Mice were treated daily with 2 mg/kg of ARS-1620, a targeted RAS p.G12C inhibitor, for 2 days and imaged following each treatment. Subsequently, the mice were then treated daily with 10 mg/kg of amiloride, a general inhibitor of macropinocytosis, for 2 days and imaged. Intratumoral distribution of AF790-albumin was assessed in vivo using near-infrared (NIR) fluorescence imaging. RESULTS: Albumin retention was observed as a function of K-RAS activity and macropinocytosis across several lung and pancreatic cancer cell lines. We documented that ARS-1620-induced inhibition of K-RAS activity or amiloride-mediated inhibition of macropinocytosis significantly reduced albumin uptake. Tumor retention in vivo of AF790-albumin was both RAS inhibition-dependent as well as abrogated by inhibition of macropinocytosis. CONCLUSIONS: These data provide a novel approach using NIR-labeled human serum albumin to identify and monitor RAS-driven tumors as well as evaluate the on-target efficacy in vivo of inhibitors, such as ARS-1620.

Paramagnetic 1H NMR Spectroscopy to Investigate the Catalytic Mechanism of Radical S-Adenosylmethionine Enzymes.

Iron-sulfur clusters in radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chemically challenging reactions across all domains of life. Here we showed that 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals of metal-ligands is a powerful tool to monitor the binding of SAM and of the octanoyl-peptide substrate to the two [4Fe-4S] clusters of human lipoyl synthase. The paramagnetically shifted signals of the iron-ligands were specifically assigned to each of the two bound [4Fe-4S] clusters, and then used to examine the interaction of SAM and substrate molecules with each of the two [4Fe-4S] clusters of human lipoyl synthase. 1H NMR spectroscopy can therefore contribute to the description of the catalityc mechanism of radical SAM enzymes.