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.

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.

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.

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.

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.

Examination of a first-in-class bis-dialkylnorspermidine-terphenyl antibiotic in topical formulation against mono and polymicrobial biofilms.

Biofilm-impaired tissue is a significant factor in chronic wounds such as diabetic foot ulcers. Most, if not all, anti-biotics in clinical use have been optimized against planktonic phenotypes. In this study, an in vitro assessment was performed to determine the potential efficacy of a first-in-class series of antibiofilm antibiotics and compare outcomes to current clinical standards of care. The agent, CZ-01179, was formulated into a hydrogel and tested against mature biofilms of a clinical isolate of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa ATCC 27853 using two separate methods. In the first method, biofilms were grown on cellulose discs on an agar surface. Topical agents were spread on gauze and placed over the biofilms for 24 h. Biofilms were quantified and imaged with confocal and scanning electron microscopy. In the second method, biofilms were grown on bioabsorbable collagen coupons in a modified CDC biofilm reactor. Coupons were immersed in treatment for 24 h. The first method was limited in its ability to assess efficacy. Efficacy profiles against biofilms grown on collagen were more definitive, with CZ-01179 gel eradicating well-established biofilms to a greater degree compared to clinical standards. In conclusion, CZ-01179 may be a promising topical agent that targets the biofilm phenotype. Pre-clinical work is currently being performed to determine the translatable potential of CZ-01179 gel.

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.

In vivo efficacy of a unique first-in-class antibiofilm antibiotic for biofilm-related wound infections caused by Acinetobacter baumannii.

Wounds complicated by biofilms challenge even the best clinical care and can delay a return to duty for service members. A major component of treatment in wounded warriors includes infected wound management. Yet, all antibiotic therapy options have been optimized against planktonic bacteria, leaving an important gap in biofilm-related wound care. We tested the efficacy of a unique compound (CZ-01179) specifically synthesized to eradicate biofilms. CZ-01179 was formulated as the active agent in a hydrogel, and tested in vitro and in vivo in a pig excision wound model for its ability to treat and prevent biofilm-related wound infection caused by Acinetobacter baumannii. Data indicated that compared to a clinical standard-silver sulfadiazine-CZ-01179 was much more effective at eradicating biofilms of A. baumannii in vitro and up to 6 days faster at eradicating biofilms in vivo. CZ-01179 belongs to a broader class of newly-synthesized antibiofilm agents (referred to as CZ compounds) with reduced risk of resistance development, specific efficacy against biofilms, and promising formulation potential for clinical applications. Given its broad spectrum and biofilm-specific nature, CZ-01179 gel may be a promising agent to increase the pipeline of products to treat and prevent biofilm-related wound infections.

A Direct C11 Alkylation Strategy on the Saxitoxin Core: A Synthesis of (+)-11-Saxitoxinethanoic Acid.

The bis-guanidinium ion family of natural products are revered for their utility in the study of ion channel physiology. While many congeners have been isolated with various oxidation and sulfation patterns, only two members of this family have been isolated bearing a carbon-carbon bond at C11, namely 11-saxitoxinethanoic acid and zetekitoxin AB. Herein we described a synthetic platform capable of efficiently targeting (+)-saxitoxin and 11-saxitoxinethanoic acid with an embedded C11 carbon-carbon bond. We demonstrate that this strategy enables direct enolate coupling in both an inter- and intramolecular fashion to create the C11-C15 carbon-carbon bond.

Growth substrate may influence biofilm susceptibility to antibiotics.

The CDC biofilm reactor is a robust culture system with high reproducibility in which biofilms can be grown for a wide variety of analyses. Multiple material types are available as growth substrates, yet data from biofilms grown on biologically relevant materials is scarce, particularly for antibiotic efficacy against differentially supported biofilms. In this study, CDC reactor holders were modified to allow growth of biofilms on collagen, a biologically relevant substrate. Susceptibility to multiple antibiotics was compared between biofilms of varying species grown on collagen versus standard polycarbonate coupons. Data indicated that in 13/18 instances, biofilms on polycarbonate were more susceptible to antibiotics than those on collagen, suggesting that when grown on a complex substrate, biofilms may be more tolerant to antibiotics. These outcomes may influence the translatability of antibiotic susceptibility profiles that have been collected for biofilms on hard plastic materials. Data may also help to advance information on antibiotic susceptibility testing of biofilms grown on biologically relevant materials for future in vitro and in vivo applications.

In vivo analysis of a first-in-class tri-alkyl norspermidine-biaryl antibiotic in an active release coating to reduce the risk of implant-related infection.

Prosthetic joint infection (PJI) is a well-known and persisting problem. Active release coatings have promise to provide early protection to an implant by eradicating small colony biofilm contaminants or planktonic bacteria that can form biofilm. Traditional antibiotics can be limited as active release agents in that they have limited effect against biofilms and develop resistance at sub-lethal concentrations. A unique first-in-class compound (CZ-01127) was assessed as the active release agent in a silicone (Si)-based coating to prevent PJI in a sheep model of joint space infection. Titanium (Ti) plugs contained a porous coated Ti (PCTi) region and polymer-coated region. Plugs were implanted into a femoral condyle of sheep to assess the effect of the Si polymer on cancellous bone ingrowth, the effect of CZ-01127 on bone ingrowth, and the ability of CZ-01127 to prevent PJI. Microbiological results showed that CZ-01127 was able to eradicate bacteria in the local region of the implanted plugs. Data further showed that Si did not adversely affect bone ingrowth. However, bacteria that reached the joint space (synovium) were not fully eradicated. Outcomes suggested that the CZ-01127 coating provided local protection to the implant system in a challenging model, the design of which could be beneficial for testing future antimicrobial therapies for PJI. STATEMENT OF SIGNIFICANCE: Periprosthetic joint infection (PJI) is now commonplace, and constitutes an underlying problem that patients and physicians face. Active release antibiotic coatings have potential to prevent these infections. Traditional antibiotics are limited in their ability to eradicate bacteria that reside in biofilms, and are more susceptible to resistance development. This study addressed these limitations by testing the efficacy of a unique antimicrobial compound in a coating that was tested in a challenging sheep model of PJI. The unique coating was able to eradicate bacteria and prevent infection in the environment adjacent to the implant. Bacteria that escaped into the joint space still caused infection, yet benchmark data can be used to optimize the coating and translate it toward clinical use.

In vitro testing of a first-in-class tri-alkylnorspermidine-biaryl antibiotic in an anti-biofilm silicone coating.

Biofilm-related infection is among the worst complication to prosthetic joint replacement procedures; once established on the implant surface, biofilms show strong recalcitrance to clinical antibiotic therapy, frequently requiring costly revision procedures and prolonged systemic antibiotics for their removal. A well-designed active release coating might assist host immunity in clearing bacterial contaminants within the narrow perioperative window and ultimately prevent microbial colonization of the joint prosthesis. A first-in-class compound (CZ-01127) was tested as the active release agent in a silicone (Si) coating using an in vitro dynamic flow model of surgical site contamination and compared with analogous coatings containing clinical gold-standard antibiotics vancomycin and gentamicin; the CZ-01127 coating outperformed both vancomycin and gentamicin coatings and was the only to decrease the methicillin-resistant Staphylococcus aureus (MRSA) inocula below detectable limits for the first 3 days. The antimicrobial activity of CZ-01127, and for comparison vancomycin and gentamicin, were characterized against both planktonic and biofilm MRSA using the minimum inhibitory concentration (MIC) assay, serial passages, and serial dilution tests against established biofilms grown with a CBR 90 CDC biofilm reactor. Despite a similar MIC (1 µg/ml) and behavior in a 25-day serial passage analysis, CZ-01127 displayed much greater bactericidal activity against established biofilms and was the only to decrease biofilm colony forming units (CFUs) below detectable limits at the highest concentration tested (500 µg/ml). Coating release profiles were characterized using ATR-FTIR and displayed burst release kinetics within the decisive period of the perioperative window suggesting the silicon carrier is broadly useful for screening antibiotic compound for local delivery applications. STATEMENT OF SIGNIFICANCE: With an aging population, an increasing number of people are undergoing total joint replacement procedures in which diseased joint tissues are replaced with permanent metallic implants. Some of these procedures are burdened by costly and debilitating infections. A promising approach to prevent infections is the use of an antimicrobial coating on the surface of the implant which releases antibiotics into the surgical site to prevent infection. In this study, we tested a new antibiotic compound formulated in a silicone coating. Data showed that this compound was more effective at killing pathogenic methicillin resistant Staphylococcus aureus (MRSA) bacteria than two clinical gold-standard antibiotics-vancomycin and gentamicin-and could be a promising agent for antimicrobial coating technologies.

A Cancer-Selective Zinc Ionophore Inspired by the Natural Product Naamidine A.

We present data demonstrating the natural product mimic, zinaamidole A (ZNA), is a modulator of metal ion homeostasis causing cancer-selective cell death by specifically inducing cellular Zn2+-uptake in transformed cells. ZNA's cancer selectivity was evaluated using metastatic, patient-derived breast cancer cells, established human breast cancer cell lines, and three-dimensional organoid models derived from normal and transformed mouse mammary glands. Structural analysis of ZNA demonstrated that the compound interacts with zinc through the N2-acyl-2-aminoimidazole core. Combination treatment with ZnSO4 strongly potentiated ZNA's cancer-specific cell death mechanism, an effect that was not observed with other transition metals. We show that Zn2+-dyshomeostasis induced by ZNA is unique and markedly more selective than other known Zn2+-interacting compounds such as clioquinol. The in vivo bioactivity of ZNA was also assessed and revealed that tumor-bearing mice treated with ZNA had improved survival outcomes. Collectively, these data demonstrate that the N2-acyl-2-aminoimidazole core of ZNA represents a powerful chemotype to induce cell death in cancer cells concurrently with a disruption in zinc homeostasis.

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.

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.

Cancer-associated 2-oxoglutarate analogues modify histone methylation by inhibiting histone lysine demethylases.

Histone lysine demethylases (KDMs) are 2-oxoglutarate-dependent dioxygenases (2-OGDDs) that regulate gene expression by altering chromatin structure. Their dysregulation has been associated with many cancers. We set out to study the catalytic and inhibitory properties of human KDM4A, KDM4B, KDM5B, KDM6A and KDM6B, aiming in particular to reveal which of these enzymes are targeted by cancer-associated 2-oxoglutarate (2-OG) analogues. We used affinity-purified insect cell-produced enzymes and synthetic peptides with trimethylated lysines as substrates for the in vitro enzyme activity assays. In addition, we treated breast cancer cell lines with cell-permeable forms of 2-OG analogues and studied their effects on the global histone methylation state. Our data show that KDMs have substrate specificity. Among the enzymes studied, KDM5B had the highest affinity for the peptide substrate but the lowest affinity for the 2-OG and the Fe2+ cosubstrate/cofactors. R-2-hydroxyglutarate (R-2HG) was the most efficient inhibitor of KDM6A, KDM4A and KDM4B, followed by S-2HG. This finding was supported by accumulations of the histone H3K9me3 and H3K27me3 marks in cells treated with the cell-permeable forms of these compounds. KDM5B was especially resistant to inhibition by R-2HG, while citrate was the most efficient inhibitor of KDM6B. We conclude that KDM catalytic activity is susceptible to inhibition by tumorigenic 2-OG analogues and suggest that the inhibition of KDMs is involved in the disease mechanism of cancers in which these compounds accumulate, such as the isocitrate dehydrogenase mutations.

Mutant IDH1 Promotes Glioma Formation In Vivo.

Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated gene in grade II-III glioma and secondary glioblastoma (GBM). A causal role for IDH1R132H in gliomagenesis has been proposed, but functional validation in vivo has not been demonstrated. In this study, we assessed the role of IDH1R132H in glioma development in the context of clinically relevant cooperating genetic alterations in vitro and in vivo. Immortal astrocytes expressing IDH1R132H exhibited elevated (R)-2-hydroxyglutarate levels, reduced NADPH, increased proliferation, and anchorage-independent growth. Although not sufficient on its own, IDH1R132H cooperated with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote glioma development in vivo. These tumors resembled proneural human mutant IDH1 GBM genetically, histologically, and functionally. Our findings support the hypothesis that IDH1R132H promotes glioma development. This model enhances our understanding of the biology of IDH1R132H-driven gliomas and facilitates testing of therapeutic strategies designed to combat this deadly disease.

Blueberry Metabolites Attenuate Lipotoxicity-Induced Endothelial Dysfunction.

SCOPE: Lipotoxicity-induced endothelial dysfunction is an important vascular complication associated with diabetes. Clinical studies support the vascular benefits of blueberry anthocyanins, but the underlying mechanism is unclear. The hypothesis that metabolites of blueberry anthocyanins attenuate lipotoxicity-induced endothelial dysfunction was tested. METHODS AND RESULTS: Human aortic endothelial cells (HAECs) were treated for 6 h with either: (i) the parent anthocyanins (malvidin-3-glucoside and cyanidin-3-glucoside); or (ii) the blueberry metabolites (hydroxyhippuric acid, hippuric acid, benzoic acid-4-sulfate, isovanillic acid-3-sulfate, and vanillic acid-4-sulfate), at concentrations known to circulate in humans following blueberry consumption. For the last 5 h HAECs were treated with palmitate or vehicle. HAECs treated with palmitate displayed elevated reactive oxygen species generation, increased mRNA expression of NOX4, chemokines, adhesion molecules, and IκBα, exaggerated monocyte binding, and suppressed nitric oxide production. Of note, the damaging effects of palmitate were ameliorated in HAECs treated with blueberry metabolites but not parent anthocyanins. Further, important translational relevance of these results was provided by our observation that palmitate-induced endothelial dysfunction was lessened in arterial segments that incubated concurrently with blueberry metabolites. CONCLUSION: The presented findings indicate that the vascular benefits of blueberry anthocyanins are mediated by their metabolites. Blueberries might complement existing therapies to lessen vascular complications.

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.