Chemical Reactions of Indole Alkaloids That Enable Rapid Access to New Scaffolds for Discovery.

This graphical review provides a concise overview of indole alkaloids and chemical reactions that have been reported to transform both these natural products and derivatives to rapidly access new molecular scaffolds. Select biologically active compounds from these synthetic efforts are reported herein.

Design, synthesis and evaluation of halogenated phenazine antibacterial prodrugs targeting nitroreductase enzymes for activation.

It is of great importance to develop new strategies to combat antibiotic resistance. Our lab has discovered halogenated phenazine (HP) analogues that are highly active against multidrug-resistant bacterial pathogens. Here, we report the design, synthesis, and study of a new series of nitroarene-based HP prodrugs that leverage intracellular nitroreductase (NTR) enzymes for activation and subsequent release of active HP agents. Our goals of developing HP prodrugs are to (1) mitigate off-target metal chelation (potential toxicity), (2) possess motifs to facilitate intracellular, bacterial-specific HP release, (3) improve water solubility, and (4) prevent undesirable metabolism (e.g., glucuronidation of HP's phenol). Following the synthesis of HP-nitroarene prodrugs bearing a sulfonate ester linker, NTR-promoted release experiments demonstrated prodrug HP-1-N released 70.1% of parent HP-1 after 16 hours (with only 6.8% HP-1 release without NTR). In analogous in vitro experiments, no HP release was observed for control sulfonate ester compounds lacking the critical nitro group. When compared to parent HP compounds, nitroarene prodrugs evaluated during these studies demonstrate similar antibacterial activities in MIC and zone of inhibition assays (against lab strains and clinical isolates). In conclusion, HP-nitroarene prodrugs could provide a future avenue to develop potent agents that target antibiotic resistant bacteria.

First-in-class multifunctional TYMS nonclassical antifolate inhibitor with potent in vivo activity that prolongs survival.

Although thymidylate synthase (TYMS) inhibitors have served as components of chemotherapy regimens, the currently available inhibitors induce TYMS overexpression or alter folate transport/metabolism feedback pathways that tumor cells exploit for drug resistance, limiting overall benefit. Here we report a small molecule TYMS inhibitor that i) exhibited enhanced antitumor activity as compared with current fluoropyrimidines and antifolates without inducing TYMS overexpression, ii) is structurally distinct from classical antifolates, iii) extended survival in both pancreatic xenograft tumor models and an hTS/Ink4a/Arf null genetically engineered mouse tumor model, and iv) is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we verify the compound is a multifunctional nonclassical antifolate, and using a series of analogs, we identify structural features allowing direct TYMS inhibition while maintaining the ability to inhibit dihydrofolate reductase. Collectively, this work identifies nonclassical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile, highlighting the potential for enhanced cancer therapy.

Design, Synthesis, and Evaluation of Carbonate-Linked Halogenated Phenazine-Quinone Prodrugs with Improved Water-Solubility and Potent Antibacterial Profiles.

Pathogenic bacteria have devastating impacts on human health as a result of acquired antibiotic resistance and innate tolerance. Every class of our current antibiotic arsenal was initially discovered as growth-inhibiting agents that target actively replicating (individual, free-floating) planktonic bacteria. Bacteria are notorious for utilizing a diversity of resistance mechanisms to overcome the action of conventional antibiotic therapies and forming surface-attached biofilm communities enriched in (non-replicating) persister cells. To address problems associated with pathogenic bacteria, our group is developing halogenated phenazine (HP) molecules that demonstrate potent antibacterial and biofilm-eradicating activities through a unique iron starvation mode of action. In this study, we designed, synthesized, and investigated a focused collection of carbonate-linked HP prodrugs bearing a quinone trigger to target the reductive cytoplasm of bacteria for bioactivation and subsequent HP release. The quinone moiety also contains a polyethylene glycol group, which dramatically enhances the water-solubility properties of the HP-quinone prodrugs reported herein. We found carbonate-linked HP-quinone prodrugs 11, 21-23 to demonstrate good linker stability, rapid release of the active HP warhead following dithiothreitol (reductive) treatment, and potent antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis, and Enterococcus faecalis. In addition, HP-quinone prodrug 21 induced rapid iron starvation in MRSA and S. epidermidis biofilms, illustrating prodrug action within these surface-attached communities. Overall, we are highly encouraged by these findings and believe that HP prodrugs have the potential to address antibiotic resistant and tolerant bacterial infections.

Molecular characterization of myotonic dystrophy fibroblast cell lines for use in small molecule screening.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are common forms of adult onset muscular dystrophy. Pathogenesis in both diseases is largely driven by production of toxic-expanded repeat RNAs that sequester MBNL RNA-binding proteins, causing mis-splicing. Given this shared pathogenesis, we hypothesized that diamidines, small molecules that rescue mis-splicing in DM1 models, could also rescue mis-splicing in DM2 models. While several DM1 cell models exist, few are available for DM2 limiting research and therapeutic development. Here, we characterize DM1 and DM2 patient-derived fibroblasts for use in small molecule screens and therapeutic studies. We identify mis-splicing events unique to DM2 fibroblasts and common events shared with DM1 fibroblasts. We show that diamidines can partially rescue molecular phenotypes in both DM1 and DM2 fibroblasts. This study demonstrates the potential of fibroblasts as models for DM1 and DM2, which will help meet an important need for well-characterized DM2 cell models.

Pyrazine and Phenazine Heterocycles: Platforms for Total Synthesis and Drug Discovery.

There are numerous pyrazine and phenazine compounds that demonstrate biological activities relevant to the treatment of disease. In this review, we discuss pyrazine and phenazine agents that have shown potential therapeutic value, including several clinically used agents. In addition, we cover some basic science related to pyrazine and phenazine heterocycles, which possess interesting reactivity profiles that have been on display in numerous cases of innovative total synthesis approaches, synthetic methodologies, drug discovery efforts, and medicinal chemistry programs. The majority of this review is focused on presenting instructive total synthesis and medicinal chemistry efforts of select pyrazine and phenazine compounds, and we believe these incredible heterocycles offer promise in medicine.

Modular Synthetic Routes to Fluorine-Containing Halogenated Phenazine and Acridine Agents That Induce Rapid Iron Starvation in Methicillin-Resistant Staphylococcus aureus Biofilms.

During infection, bacteria use an arsenal of resistance mechanisms to negate antibiotic therapies. In addition, pathogenic bacteria form surface-attached biofilms bearing enriched populations of metabolically dormant persister cells. Bacteria develop resistance in response to antibiotic insults; however, nonreplicating biofilms are innately tolerant to all classes of antibiotics. As such, molecules that can eradicate antibiotic-resistant and antibiotic-tolerant bacteria are of importance. Here, we report modular synthetic routes to fluorine-containing halogenated phenazine (HP) and halogenated acridine (HA) agents with potent antibacterial and biofilm-killing activities. Nine fluorinated phenazines were rapidly accessed through a synthetic strategy involving (1) oxidation of fluorinated anilines to azobenzene intermediates, (2) SNAr with 2-methoxyaniline, and (3) cyclization to phenazines upon treatment with trifluoroacetic acid. Five structurally related acridine heterocycles were synthesized using SNAr and Buchwald-Hartwig approaches. From this focused collection, phenazines 5g, 5h, 5i, and acridine 9c demonstrated potent antibacterial activities against Gram-positive pathogens (MIC = 0.04-0.78 µM). Additionally, 5g and 9c eradicated Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis biofilms with excellent potency (5g, MBEC = 4.69-6.25 µM; 9c, MBEC = 4.69-50 µM). Using real-time quantitative polymerase chain reaction (RT-qPCR), 5g, 5h, 5i, and 9c rapidly induce the transcription of iron uptake biomarkers isdB and sbnC in methicillin-resistant S. aureus (MRSA) biofilms, and we conclude that these agents operate through iron starvation. Overall, fluorinated phenazine and acridine agents could lead to ground-breaking advances in the treatment of challenging bacterial infections.

A Modular Synthetic Route Involving N-Aryl-2-nitrosoaniline Intermediates Leads to a New Series of 3-Substituted Halogenated Phenazine Antibacterial Agents.

Pathogenic bacteria demonstrate incredible abilities to evade conventional antibiotics through the development of resistance and formation of dormant, surface-attached biofilms. Therefore, agents that target and eradicate planktonic and biofilm bacteria are of significant interest. We explored a new series of halogenated phenazines (HP) through the use of N-aryl-2-nitrosoaniline synthetic intermediates that enabled functionalization of the 3-position of this scaffold. Several HPs demonstrated potent antibacterial and biofilm-killing activities (e.g., HP 29, against methicillin-resistant Staphylococcus aureus: MIC = 0.075 µM; MBEC = 2.35 µM), and transcriptional analysis revealed that HPs 3, 28, and 29 induce rapid iron starvation in MRSA biofilms. Several HPs demonstrated excellent activities against Mycobacterium tuberculosis (HP 34, MIC = 0.80 µM against CDC1551). This work established new SAR insights, and HP 29 demonstrated efficacy in dorsal wound infection models in mice. Encouraged by these findings, we believe that HPs could lead to significant advances in the treatment of challenging infections.

Evolution of Resistance to Phenazine Antibiotics in Staphylococcus aureus and Its Role During Coinfection with Pseudomonas aeruginosa.

In the niches that Staphylococcus aureus and Pseudomonas aeruginosa coinhabit, the later pathogen produces phenazine antibiotics to inhibit the growth of S. aureus. Recently, a group of halogenated phenazines (HPs) has been shown to have potent antimicrobial activities against Staphylococci; however, no HP-resistant mutant has been reported. Here, we demonstrate that S. aureus develops HP-resistance via single amino acid change (Arg116Cys) in a transcriptional repressor TetR21. RNA-seq analysis showed that the TetR21R116C variation caused drastic up-regulation of an adjacent gene hprS (halogenated phenazine resistance protein of S. aureus). Deletion of the hprS in the TetR21R116C background restored bacterial susceptibility to HP, while hprS overexpression in S. aureus conferred HP-resistance. The expression of HprS is under tight transcriptional control of the TetR21 via direct binding to the promoter region of hprS. The R116C mutation in TetR21 significantly reduced its DNA binding affinity. Moreover, natural phenazine antibiotics (phenazine-1-carboxylic acid and pyocyanin) and a HP analog (HP-22) are ligands for the TetR21, regulating its repressor activity. Combining homology analysis and LC-MS/MS assay we demonstrated that HprS is a phenazine efflux pump. To the best of our knowledge, we provide the first report of phenazine efflux pump in S. aureus. Interestingly, the TetR21R116C variation has been found in some clinical S. aureus isolates, and a laboratory strain of S. aureus with TetR21R116C variation showed enhanced growth competitiveness toward P. aeruginosa and promoted coinfection with P. aeruginosa in the host environment, demonstrating significance of the mutation in host infections.

Progress towards a stable cephalosporin-halogenated phenazine conjugate for antibacterial prodrug applications.

Resistant bacteria successfully evade the action of conventional antibiotic therapies during infection, often leading to significant illness and death. Our lab has discovered halogenated phenazine (HP) analogues which demonstrate potent antibacterial activities through a unique iron-starving mechanism. Herein, we describe synthetic efforts towards a stable cephalosporin-HP conjugate prodrug with the aim of translating HPs into useful clinical agents. Cephalosporin-antibiotic conjugates offer multiple advantages for antibacterial design, including the release of active agents through the targeting of intracellular cephalosporinase following selective ring-opening of the beta-lactam warhead. During these studies, carbonate-linked cephalosporin-HP conjugate 16 was synthesized; however, we were unable to successfully remove the ester group required for cephalosporinase processing. Cephalosporin-HP 16 was then utilized as a probe to investigate the stability of the carbonate linker in antibacterial assays and, as predicted, this compound proved to be inactive against Staphylococcus aureus (MIC > 100 µM). The lack of 16's antibacterial activity can be attributed to the carbonate linker remaining intact throughout the MIC assay, thus not liberating the active HP moiety. These efforts have led to a more stable cephalosporin-HP conjugate joined through a carbonate linker compared to a highly unstable ether linked analogue we previously reported.

Yohimbine as a Starting Point to Access Diverse Natural Product-Like Agents with Re-programmed Activities against Cancer-Relevant GPCR Targets.

G protein-coupled receptors (GPCRs) constitute the largest protein superfamily in the human genome. GPCRs play key roles in mediating a wide variety of physiological events including proliferation and cancer metastasis. Given the major roles that GPCRs play in mediating cancer growth, they present promising targets for small molecule therapeutics. One of the principal goals of our lab is to identify complex natural products (NPs) suitable for ring distortion, or the dramatic altering of the inherently complex architectures of NPs, to rapidly generate an array of compounds with diverse molecular skeletal systems. The overarching goal of our ring distortion approach is to re-program the biological activity of select natural products and identify new compounds of importance to the treatment of disease, such as cancer. Described herein are the results from biological screens of diverse small molecules derived from the indole alkaloid yohimbine against a panel of GPCRs involved in various diseases. Several analogues displayed highly differential antagonistic activities across the GPCRs tested. We highlight the re-programmed profile of one analogue, Y7g, which exhibited selective antagonistic activities against AVPR2 (IC50 = 459 nM) and OXTR (IC50 = 1.16 µM). The activity profile of Y7g could correlate its HIF-dependent anti-cancer activity to its GPCR antagonism since these receptors are known to be upregulated in hypoxic cellular environments. Our findings demonstrate that the ring distortion of yohimbine can lead to the identification of new compounds capable of interacting with distinct cancer-relevant targets.

Efficacy data of halogenated phenazine and quinoline agents and an NH125 analogue to veterinary mycoplasmas.

BACKGROUND: Mycoplasmas primarily cause respiratory or urogenital tract infections impacting avian, bovine, canine, caprine, murine, and reptilian hosts. In animal husbandry, mycoplasmas cause reduced feed-conversion, decreased egg production, arthritis, hypogalactia or agalactia, increased condemnations, culling, and mortality in some cases. Antibiotics reduce transmission and mitigate clinical signs; however, concerning levels of antibiotic resistance in Mycoplasma gallisepticum and M. capricolum isolates exist. To address these issues, we evaluated the minimum inhibitory concentrations (MICs) of halogenated phenazine and quinoline compounds, an N-arylated NH125 analogue, and triclosan against six representative veterinary mycoplasmas via microbroth or agar dilution methods. Thereafter, we evaluated the minimum bactericidal concentration (MBC) of efficacious drugs. RESULTS: We identified several compounds with MICs ≤25 µM against M. pulmonis (n = 5), M. capricolum (n = 4), M. gallisepticum (n = 3), M. alligatoris (n = 3), M. agassizii (n = 2), and M. canis (n = 1). An N-arylated NH125 analogue, compound 21, served as the most efficacious, having a MIC ≤25 µM against all mycoplasmas tested, followed by two quinolines, nitroxoline (compound 12) and compound 20, which were effective against four and three mycoplasma type strains, respectively. Nitroxoline exhibited bactericidal activity among all susceptible mycoplasmas, and compound 21 exhibited bactericidal activity when the MBC was able to be determined. CONCLUSIONS: These findings highlight a number of promising agents from novel drug classes with potential applications to treat veterinary mycoplasma infections and present the opportunity to evaluate preliminary pharmacokinetic indices using M. pulmonis in rodents as an animal model of human infection.

Preventing Morphine-Seeking Behavior through the Re-Engineering of Vincamine's Biological Activity.

Innovative discovery strategies are essential to address the ongoing opioid epidemic in the United States. Misuse of prescription and illegal opioids (e.g., morphine, heroin) has led to major problems with addiction and overdose. We used vincamine, an indole alkaloid, as a synthetic starting point for dramatic structural alterations of its complex, fused ring system to synthesize 80 diverse compounds with intricate molecular architectures. A select series of vincamine-derived compounds were screened for both agonistic and antagonistic activities against a panel of 168 G protein-coupled receptor (GPCR) drug targets. Although vincamine was without an effect, the novel compound 4 (V2a) demonstrated antagonistic activities against hypocretin (orexin) receptor 2. When advanced to animal studies, 4 (V2a) significantly prevented acute morphine-conditioned place preference (CPP) and stress-induced reinstatement of extinguished morphine-CPP in mouse models of opioid reward and relapse. These results demonstrate that the ring distortion of vincamine offers a promising way to explore new chemical space of relevance to opioid addiction.

Re-Engineering of Yohimbine's Biological Activity through Ring Distortion: Identification and Structure-Activity Relationships of a New Class of Antiplasmodial Agents.

Select natural products are ideal starting points for ring distortion, or the dramatic altering of inherently complex molecules through short synthetic pathways, to generate an array of novel compounds with diverse skeletal architectures. A major goal of our ring distortion approach is to re-engineer the biological activity of indole alkaloids to identify new compounds with diverse biological activities in areas of significance to human health and medicine. In this study, we re-engineered the biological activity of the indole alkaloid yohimbine through ring rearrangement and ring cleavage synthesis pathways to discover new series of antiplasmodial agents. One new compound, Y7j, was found to demonstrate good potency against chloroquine-resistant Plasmodium falciparum Dd2 cells (EC50 = 0.33 µM) without eliciting cytotoxicity against HepG2 cells (EC50 > 40 µM). Y7j demonstrated stage-specific action against parasites at the late ring/trophozoite stage. A series of analogues was synthesized to gain structure-activity relationship insights, and we learned that both benzyl groups of Y7j are required for activity and fine-tuning of antiplasmodial activities could be accomplished by changing substitution patterns on the benzyl moieties. This study demonstrates the potential for ring distortion to drive new discoveries and change paradigms in chemical biology and drug discovery.

Instructive Advances in Chemical Microbiology Inspired by Nature's Diverse Inventory of Molecules.

Natural product antibiotics have played an essential role in the treatment of bacterial infection in addition to serving as useful tools to explore the intricate biology of bacteria. Our current arsenal of antibiotics operate through the inhibition of well-defined bacterial targets critical for replication and growth. Pathogenic bacteria effectively utilize a diversity of mechanisms that lead to acquired resistance and/or innate tolerance toward antibiotic therapies, which can result in devastating consequences to human life. Several research groups have established innovative programs that work at the chemistry-biology interface to develop new molecules that aim to define and address concerns related to antibiotic resistance and tolerance. In this Review, we present recent progress by select research groups that highlight a diversity of integrated chemical biology and medicinal chemistry approaches aimed at the development and utilization of chemical tools that have led to promising new microbiological insights that may lead to significant clinical advances regarding the treatment of pathogenic bacteria.

Combination Treatment of Erythromycin and Furamidine Provides Additive and Synergistic Rescue of Mis-Splicing in Myotonic Dystrophy Type 1 Models.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disease that presents with clinical symptoms including myotonia, cardiac dysfunction and cognitive impairment. DM1 is caused by a CTG expansion in the 3' UTR of the DMPK gene. The transcribed expanded CUG repeat RNA sequester the muscleblind-like (MBNL) and up-regulate the CUG-BP Elav-like (CELF) families of RNA-binding proteins leading to global mis-regulation of RNA processing and altered gene expression. Currently, there are no disease-targeting treatments for DM1. Given the multi-step pathogenic mechanism, combination therapies targeting different aspects of the disease mechanism may be a viable therapeutic approach. Here, as proof-of-concept, we studied a combination of two previously characterized small molecules, erythromycin and furamidine, in two DM1 models. In DM1 patient-derived myotubes, rescue of mis-splicing was observed with little to no cell toxicity. In a DM1 mouse model, a combination of erythromycin and the prodrug of furamidine (pafuramidine), administered orally, displayed both additive and synergistic mis-splicing rescue. Gene expression was only modestly affected and over 40 % of the genes showing significant expression changes were rescued back toward WT expression levels. Further, the combination treatment partially rescued the myotonia phenotype in the DM1 mouse. This combination treatment showed a high degree of mis-splicing rescue coupled with low off-target gene expression changes. These results indicate that combination therapies are a promising therapeutic approach for DM1.

Recent Progress in Natural-Product-Inspired Programs Aimed To Address Antibiotic Resistance and Tolerance.

Bacteria utilize multiple mechanisms that enable them to gain or acquire resistance to antibiotic therapies during the treatment of infections. In addition, bacteria form biofilms which are surface-attached communities of enriched populations containing persister cells encased within a protective extracellular matrix of biomolecules, leading to chronic and recurring antibiotic-tolerant infections. Antibiotic resistance and tolerance are major global problems that require innovative therapeutic strategies to address the challenges associated with pathogenic bacteria. Historically, natural products have played a critical role in bringing new therapies to the clinic to treat life-threatening bacterial infections. This Perspective provides an overview of antibiotic resistance and tolerance and highlights recent advances (chemistry, biology, drug discovery, and development) from various research programs involved in the discovery of new antibacterial agents inspired by a diverse series of natural product antibiotics.

Phenazine Antibiotic-Inspired Discovery of Bacterial Biofilm-Eradicating Agents.

Bacterial biofilms are surface-attached communities of slow-growing and non-replicating persister cells that demonstrate high levels of antibiotic tolerance. Biofilms occur in nearly 80 % of infections and present unique challenges to our current arsenal of antibiotic therapies, all of which were initially discovered for their abilities to target rapidly dividing, free-floating planktonic bacteria. Bacterial biofilms are credited as the underlying cause of chronic and recurring bacterial infections. Innovative approaches are required to identify new small molecules that operate through bacterial growth-independent mechanisms to effectively eradicate biofilms. One source of inspiration comes from within the lungs of young cystic fibrosis (CF) patients, who often endure persistent Staphylococcus aureus infections. As these CF patients age, Pseudomonas aeruginosa co-infects the lungs and utilizes phenazine antibiotics to eradicate the established S. aureus infection. Our group has taken a special interest in this microbial competition strategy and we are investigating the potential of phenazine antibiotic-inspired compounds and synthetic analogues thereof to eradicate persistent bacterial biofilms. To discover new biofilm-eradicating agents, we have established an interdisciplinary research program involving synthetic medicinal chemistry, microbiology and molecular biology. From these efforts, we have identified a series of halogenated phenazines (HPs) that potently eradicate bacterial biofilms, and future work aims to translate these preliminary findings into ground-breaking clinical advances for the treatment of persistent biofilm infections.

Turning the Tide against Antibiotic Resistance by Evaluating Novel, Halogenated Phenazine, Quinoline, and NH125 Compounds against Ureaplasma Species Clinical Isolates and Mycoplasma Type Strains.

Escalating levels of antibiotic resistance in mycoplasmas, particularly macrolide resistance in Mycoplasma pneumoniae and M. genitalium, have narrowed our antibiotic arsenal. Further, mycoplasmas lack a cell wall and do not synthesize folic acid, rendering common antibiotics, such as beta-lactams, vancomycin, sulfonamides, and trimethoprim, of no value. To address this shortage, we screened nitroxoline, triclosan, and a library of 20 novel, halogenated phenazine, quinoline, and NH125 analogues against Ureaplasma species and M. hominis clinical isolates from urine. We tested a subset of these compounds (n = 9) against four mycoplasma type strains (M. pneumoniae, M. genitalium, M. hominis, and Ureaplasma urealyticum) using a validated broth microdilution or agar dilution method. Among 72 Ureaplasma species clinical isolates, nitroxoline proved most effective (MIC90, 6.25 µM), followed by an N-arylated NH125 analogue (MIC90, 12.5 µM). NH125 and its analogue had significantly higher MICs against U. urealyticum isolates than against U. parvum isolates, whereas nitroxoline did not. Nitroxoline exhibited bactericidal activity against U. parvum isolates but bacteriostatic activity against the majority of U. urealyticum isolates. Among the type strains, the compounds had the greatest activity against M. pneumoniae and M. genitalium, with 8 (80%) and 5 (71.4%) isolates demonstrating MICs of ≤12.5 µM, respectively. Triclosan also exhibited lower MICs against M. pneumoniae and M. genitalium Overall, we identified a promising range of quinoline, halogenated phenazine, and NH125 compounds that showed effectiveness against M. pneumoniae and M. genitalium and found that nitroxoline, approved for use outside the United States for the treatment of urinary tract infections, and an N-arylated NH125 analogue demonstrated low MICs against Ureaplasma species isolates.