Transaminase Inhibition by 2-Hydroxyglutarate Impairs Glutamate Biosynthesis and Redox Homeostasis in Glioma.

IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas.

Gold Catalyzed 7-endo-dig Hydroaminations Yielding 1,4-Diazepineones.

Strategies to access the 1,4-diazepindiones heterocyclic core of the TAN-1057 family of natural products revealed a successful gold-catalyzed hydroamination of yneamide tethered amines. The precursor amino-yneamides are derived from easily accessible 1,2-diamines and alkynoic acids and are efficiently cyclized to the corresponding diazepineones.

Dissecting the Nucleoside Antibiotics as Universal Translation Inhibitors.

Without question, natural products have provided the lion share of leads, if not drugs themselves, for the treatment of bacterial infections. The bacterial arms race, fueled by selection and survival pressures has delivered a natural arsenal of small molecules targeting the most essential of life processes. Antibiotics that target these critical intracellular processes face the formidable defense of both penetrating a bacterial cell membrane and avoiding efflux to exert their effect. These challenges are especially effective in Gram-negative (Gram-(-)) bacteria, which have a double membrane structure and efficient efflux systems from the combination of outer-membrane porins and inner membrane proton pumps. In this landscape of offense and defense, our clinically used antibiotics have only successfully targeted three intracellular processes for therapeutic intervention in Gram-(-) bacteria: dihydrofolate biosynthesis, transcription, and translation. Not surprisingly, such critical survival machinery is a popular target for bacterial warfare, and eight of our 14 classes of commonly used antibiotics target translation with the bacterial ribosome remaining one the most vetted targets for antimicrobial therapy. On the plus side, its anionic character attracts cationic inhibitors, which are generally more capable of penetrating the bacterial cell wall, and clinical resistance rates are usually manageable as mutation of such a highly evolved machine is difficult. On the down side, this highly evolved machine renders it difficult to inhibit selectively, and the inhibition of prokaryotic translation versus both eukaryotic cellular and mitochondrial translation is critical for clinical development and minimization of undesired toxicities.A class of natural products known as the "nucleoside antibiotics" have historically been recognized as universal inhibitors of the ribosome and can inhibit translation in prokaryotes, eukaryotes, and archaea. While they have served an essential role in dissecting the biochemical underpinnings of the enzymatic functions of the ribosome, they have not proven therapeutically useful as they target the highly conserved rRNA in the P-site and are toxic to mammalian cells. In this Account, we describe our studies on the natural product amicetin, a nucleoside antibiotic that we have demonstrated to break the rule of being a universal translation inhibitor. While the cytosine of amicetin mimics C75 of the 3'-CCA tail of the P-site tRNA akin to other nucleoside antibiotics, we advance a hypothesis that amicetin's unique interaction with the ribosomal protein uL16 exploits an untapped mechanism for selectively targeting the bacterial ribosome. A complex molecule comprised of a nucleoside, carbohydrates and amino acids, amicetin is also chemically unstable. Our initial attempts to stabilize and simplify this scaffold are presented with the ultimate goal of rebuilding the compound with improved penetrance to bacterial cells. If successful, this scaffold would demonstrate a path forward for a new class of antibiotics capable of selectively targeting the ribosomal P-site.

Unifying the Aminohexopyranose- and Peptidyl-Nucleoside Antibiotics: Implications for Antibiotic Design.

In search of new anti-tuberculars compatible with anti-retroviral therapy we re-identified amicetin as a lead compound. Amicetin's binding to the 70S ribosomal subunit of Thermus thermophilus (Tth) has been unambiguously determined by crystallography and reveals it to occupy the peptidyl transferase center P-site of the ribosome. The amicetin binding site overlaps significantly with that of the well-known protein synthesis inhibitor balsticidin S. Amicetin, however, is the first compound structurally characterized to bind to the P-site with demonstrated selectivity for the inhibition of prokaryotic translation. The natural product-ribosome structure enabled the synthesis of simplified analogues that retained both potency and selectivity for the inhibition of prokaryotic translation.

Synthetic Ingenols Maximize Protein Kinase C-Induced HIV-1 Latency Reversal.

Antiretroviral therapy (ART) does not cure HIV-1 infection due to the persistence of proviruses in long-lived resting T cells. Strategies targeting these latently infected cells will be necessary to eradicate HIV-1 in infected individuals. Protein kinase C (PKC) activation is an effective mechanism to reactivate latent proviruses and allows for recognition and clearance of infected cells by the immune system. Several ingenol compounds, naturally occurring PKC agonists, have been described to have potent latency reversal activity. We sought to optimize this activity by synthesizing a library of novel ingenols via esterification of the C-3 hydroxyl group of the ingenol core, which itself is inactive for latency reversal. Newly synthesized ingenol derivatives were evaluated for latency reversal activity, cellular activation, and cytotoxicity alongside commercially available ingenols (ingenol-3,20-dibenzoate, ingenol 3-hexanoate, and ingenol-3-angelate) in HIV latency cell lines and resting CD4+ T cells from aviremic participants. Among the synthetic ingenols that we produced, we identified several compounds that demonstrate high efficacy and represent promising leads as latency reversal agents for HIV-1 eradication.

Regioselective Base-Mediated Cyclizations of Mono-N-acylpropargylguanidines.

A regioselective base-mediated cyclization of mono-N-acylpropargylguanidines is reported. A related Ag(I)-catalyzed hydroamination strategy was recently employed to yield N3-Cbz-protected ene-guanidines, which found utility in the synthesis of naamidine A. Herein, we report the base-catalyzed hydroamination of mono-N-acylpropargylguanidines, which proceeds with the opposite regiochemistry to deliver isomerized N2-acyl-2-aminoimidazoles with broad substrate scope, circumventing the problematic regiospecific acylation of free 2-aminoimidazoles.

Exploring hydroamination-cycloaddition-fragmentation sequences to access polycyclicguanidines and vinyl-2-aminoimidazoles.

The intramolecular hydroamination of a guanidine on an eneyne unit affords a guanidine-substituted diene capable of reacting with dienophiles. These substrates undergo [4+2]-cycloaddition reactions to generate a series of complex cyclic- and spirocyclic-guanidines. Select substrates can further undergo a ring opening-elimination cascade that ultimately reveals a vinyl-2-aminoimidazole. As such this cascade reaction may find application in the synthesis of oroidin-type natural products and their analogues.

Synthesis of Naamidine A and Selective Access to N(2)-Acyl-2-aminoimidazole Analogues.

A short and scalable synthesis of naamidine A, a marine alkaloid with a selective ability to inhibit epidermal growth factor receptor (EGFR)-dependent cellular proliferation, has been achieved. A key achievement in this synthesis was the development of a regioselective hydroamination of a monoprotected propargylguanidine to deliver N(3)-protected cyclic ene-guanidines. This permits the extension of this methodology to prepare N(2)-acyl analogues in a fashion that obviates the troublesome acylation of the free 2-aminoimidazoles, which typically yields mixtures of N(2)- and N(2),N(2)-diacylated products.

Preparation of a 1,2-isoxazolidine synthon for the synthesis of zetekitoxin AB.

A synthesis of the 1,2-isoxazolidine fragment of the potent voltage gated sodium channel blocker, zetekitoxin AB is described. The synthesis utilizes an intramolecular nitrone -olefin 1,3-dipolar cycloaddition to establish the stereochemistry of the cis-1,2-isoxazolidine. The oxidative cleavage of an all anti-triol with the excision of the central carbon is central to using α-D-glucopyranoside as a traceless stereochemical template. This route furnishes a suitably protected synthon for the synthesis of zetekitoxin AB.

Chemical genetic screen reveals a role for desmosomal adhesion in mammary branching morphogenesis.

During the process of branching morphogenesis, the mammary gland undergoes distinct phases of remodeling to form an elaborate ductal network that ultimately produces and delivers milk to newborn animals. These developmental events rely on tight regulation of critical cellular pathways, many of which are probably disrupted during initiation and progression of breast cancer. Transgenic mouse and in vitro organoid models previously identified growth factor signaling as a key regulator of mammary branching, but the functional downstream targets of these pathways remain unclear. Here, we used purified primary mammary epithelial cells stimulated with fibroblast growth factor-2 (FGF2) to model mammary branching morphogenesis in vitro. We employed a forward chemical genetic approach to identify modulators of this process and describe a potent compound, 1023, that blocks FGF2-induced branching. In primary mammary epithelial cells, we used lentivirus-mediated knockdown of the aryl hydrocarbon receptor (AHR) to demonstrate that 1023 acts through AHR to block branching. Using 1023 as a tool, we identified desmosomal adhesion as a novel target of AHR signaling and show that desmosomes are critical for AHR agonists to block branching. Our findings support a functional role for desmosomes during mammary morphogenesis and also in blocking FGF-induced invasion.

Bis-aryloxadiazoles as effective activators of the aryl hydrocarbon receptor.

Bis-aryloxadiazoles are common scaffolds in medicinal chemistry due to their wide range of biological activities. Previously, we identified a 1,2,4-bis-aryloxadiazole that blocks mammary branching morphogenesis through activation of the aryl hydrocarbon receptor (AHR). In addition to defects in mammary differentiation, AHR stimulation induces toxicity in many other tissues. We performed a structure activity relationship (SAR) study of 1,2,4-bis-aryloxadiazole to determine which moieties of the molecule are critical for AHR activation. We validated our results with a functional biological assay, using desmosome formation during mammary morphogenesis to indicate AHR activity. These findings will aid the design of oxadiazole derivative therapeutics with reduced off-target toxicity profiles.

Development of a screen to identify selective small molecules active against patient-derived metastatic and chemoresistant breast cancer cells.

INTRODUCTION: High failure rates of new investigational drugs have impaired the development of breast cancer therapies. One challenge is that excellent activity in preclinical models, such as established cancer cell lines, does not always translate into improved clinical outcomes for patients. New preclinical models, which better replicate clinically-relevant attributes of cancer, such as chemoresistance, metastasis and cellular heterogeneity, may identify novel anti-cancer mechanisms and increase the success of drug development. METHODS: Metastatic breast cancer cells were obtained from pleural effusions of consented patients whose disease had progressed. Normal primary human breast cells were collected from a reduction mammoplasty and immortalized with human telomerase. The patient-derived cells were characterized to determine their cellular heterogeneity and proliferation rate by flow cytometry, while dose response curves were performed for chemotherapies to assess resistance. A screen was developed to measure the differential activity of small molecules on the growth and survival of patient-derived normal breast and metastatic, chemoresistant tumor cells to identify selective anti-cancer compounds. Several hits were identified and validated in dose response assays. One compound, C-6, was further characterized for its effect on cell cycle and cell death in cancer cells. RESULTS: Patient-derived cells were found to be more heterogeneous, with reduced proliferation rates and enhanced resistance to chemotherapy compared to established cell lines. A screen was subsequently developed that utilized both tumor and normal patient-derived cells. Several compounds were identified, which selectively targeted tumor cells, but not normal cells. Compound C-6 was found to inhibit proliferation and induce cell death in tumor cells via a caspase-independent mechanism. CONCLUSIONS: Short-term culture of patient-derived cells retained more clinically relevant features of breast cancer compared to established cell lines. The low proliferation rate and chemoresistance make patient-derived cells an excellent tool in preclinical drug development.

Cationic dirhodium carboxylate-catalyzed synthesis of dihydropyrimidones from propargyl ureas.

Cationic Rh(II) complexes are able to catalyze the regioselective hydroamination of propargyl ureas in a 6-endo fashion. This transformation permits access to interesting substitution patterns of dihydropyrimidines which have found use as nucleotide exchange factor inhibitors.

Ex vivo comparison of V.A.C.® Granufoam Silver™ and V.A.C.® Granufoam™ loaded with a first-in-class bis-dialkylnorspermidine-terphenyl antibiofilm agent.

Implementation of negative pressure wound therapy (NPWT) as a standard of care has proven efficacious in reducing both the healing time and likelihood of nosocomial infection among pressure ulcers and traumatic, combat-related injuries. However, current formulations may not target or dramatically reduce bacterial biofilm burden following therapy. The purpose of this study was to determine the antibiofilm efficacy of an open-cell polyurethane (PU) foam (V.A.C.® Granufoam™) loaded with a first-in-class compound (CZ-01179) as the active release agent integrated via lyophilized hydrogel scaffolding. An ex vivo porcine excision wound model was designed to perform antibiofilm efficacy testing in the presence of NPWT. PU foam samples loaded with a 10.0% w/w formulation of CZ-01179 and 0.5% hyaluronic acid were prepared and tested against current standards of care: V.A.C.® Granufoam Silver™ and V.A.C.® Granufoam™. We observed statistically significant reduction of methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii biofilms with the CZ-01179 antibiofilm foam in comparison to current standard of care foams. These findings motivate further development of an antibiofilm PU foam loaded with CZ-01179.

Glutamine-dependent anapleurosis dictates glucose uptake and cell growth by regulating MondoA transcriptional activity.

Glucose and glutamine are abundant nutrients required for cell growth, yet how cells sense and adapt to changes in their levels is not well understood. The MondoA transcription factor forms a heterocomplex with its obligate partner Mlx to regulate approximately 75% of glucose-dependent transcription. By mediating glucose-induced activation of thioredoxin-interacting protein (TXNIP), MondoA:Mlx complexes directly repress glucose uptake. We show here that glutamine inhibits transcriptional activation of TXNIP by triggering the recruitment of a histone deacetylase-dependent corepressor to the amino terminus of MondoA. Therefore, in the presence of both glucose and glutamine, TXNIP expression is low, which favors glucose uptake and aerobic glycolysis; the Warburg effect. Consistent with MondoA functioning upstream of TXNIP, MondoA knockdown reduces TXNIP expression, elevates glucose uptake and stimulates cell proliferation. Although glutamine has many intracellular fates, a cell permeable analog of a tricarboxylic acid cycle (TCA) intermediate, alpha-ketoglutarate, also blocks the transcriptional activity of MondoA at the TXNIP promoter and stimulates glucose uptake. Together our data suggest that glutamine-dependent mitochondrial anapleurosis dictates glucose uptake and aerobic glycolysis by blocking MondoA:Mlx-dependent transcriptional activation of TXNIP. We propose that this previously unappreciated coordination between glutamine and glucose utilization defines a metabolic checkpoint that restricts cell growth when subthreshold levels of these essential nutrients are available.

Antibiofilm potential of a negative pressure wound therapy foam loaded with a first-in-class tri-alkyl norspermidine-biaryl antibiotic.

Negative-pressure wound therapy (NPWT) is commonly utilized to treat traumatic injuries sustained on the modern battlefield. However, NPWT has failed to decrease the incidence of deep tissue infections experienced by Wounded Warriors, despite attempts to integrate common antimicrobials, like Ag+ nanoparticles, into the wound dressing. The purpose of this study was to incorporate a unique antibiofilm compound (CZ-01179) into the polyurethane matrix of NPWT foam via lyophilized hydrogel scaffolding. Foam samples with 2.5%, 5.0%, and 10.0% w/w CZ-01179 were produced and antibiofilm efficacy was compared to the current standards of care: V.A.C.® GRANUFOAM SILVER™ and V.A.C.® GRANUFOAM™. Gravimetric analysis and elution kinetics testing confirmed that this loading technique was both repeatable and controllable. Furthermore, zone of inhibition and antibiofilm efficacy testing showed that foam loaded with CZ-01179 had significantly increased activity against planktonic and biofilm phenotypes of methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii compared to the clinical standards. These findings motivate additional ex vivo and in vivo work with NPWT foam loaded with CZ-01179 with the overall objective of reducing NPWT-associated infections that complicate battlefield-related and other wounds.

De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma.

Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they seem to be less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1-mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH-mutant gliomas. Mechanistically, this reflects an obligate dependence of glioma cells on the de novo pyrimidine synthesis pathway and mutant IDH's ability to sensitize to DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.

A stereocontrolled synthesis of (+)-saxitoxin.

A concise stereoselective total synthesis of (+)-saxitoxin is described. A silver(I)-initiated hydroamination cascade constructs the bicyclic guanidinium ion core from a alkynyl bisguanidine. This sequence creates two C-N bonds, one C-O bond, and three rings and forms a single stereoisomer in a single synthetic transformation. This process enabled us to complete the synthesis of (+)-saxitoxin in 14 steps from N-Boc-l-serine methyl ester.

Chlorotrimethylsilane activation of acylcyanamides for the synthesis of mono-N-acylguanidines.

A simple and efficient one-pot method for the synthesis of monoprotected guanidines is presented. Treatment of an acylcyanamide with chlorotrimethylsilane generates a reactive N-silylcarbodiimide capable of guanylating a variety of amines. Typically the reaction is complete in 15 min for primary and secondary aliphatic amines at rt. Hindered amines and anilines are also competent nucleophiles but require extended reaction times.