Marvin J. Miller

Studies and syntheses of siderophores, microbial iron chelators, and analogs as potential drug delivery agents.

Siderophores (microbial iron chelators) play an extremely important role in microbial pathogenicity. Microbial uptake of siderophore-iron complexes through active transport systems allow microbes to survive and proliferate even under iron deficient environments during invasion of a host. Due to their structural complexity, unique iron (III) chelation, acquisition properties, and their therapeutic potential, siderophores have attracted much attention in a broad range of disciplines. Tremendous progress has been made in siderophore syntheses, in determination of the structures and functions of outer membrane receptors (e.g. FhuA and FepA), and in the mechanistic insight into siderophore-iron-mediated active transport processes. One of the important practical applications of this active transport system is development of species-selective active drug transport (the Trojan Horse approach) to potentially treat infections due to drug resistant strains of microbes. Siderophore-drug conjugates have shown great potential in active drug delivery to target pathogenic microbes.

The Nitrosocarbonyl Hetero-Diels–Alder Reaction as a Useful Tool for Organic Syntheses

Organic transformations that result in the formation of multiple covalent bonds within the same reaction are some of the most powerful tools in synthetic organic chemistry. Nitrosocarbonyl hetero-Diels-Alder (HDA) reactions allow for the simultaneous stereospecific introduction of carbon-nitrogen and carbon-oxygen bonds in one synthetic step, and provide direct access to 3,6-dihydro-1,2-oxazines. This Review describes the development of the nitrosocarbonyl HDA reaction and the utility of the resulting oxazine ring in the synthesis of a variety of important, biologically active molecules.

Orally active vasopressin V1a receptor antagonist, SRX251, selectively blocks aggressive behavior

Arginine vasopressin functions as a neurochemical signal in the brain to affect social behavior. There is an expanding literature from animal and human studies showing that vasopressin, through the vasopressin 1A receptor (V1A), can stimulate aggressive behavior. Using a novel monocylic beta lactam platform, a series of orally active vasopressin V1a antagonists was developed with high affinity for the human receptor. SRX251 was chosen from this series of V1a antagonists to screen for effects on serenic activity in a resident-intruder model of offensive aggression. Resident, male Syrian golden hamsters were given oral doses of SRX251 or intraperitoneal Manning compound, a selective V1a receptor antagonist with reduced brain penetrance, at doses of 0.2 microg, 20 microg, 2 mg/kg or vehicle. When tested 90-120 min later, SRX251, but not Manning compound, caused a significant dose-dependent reduction in offensive aggression toward intruders as measured by latency to bite and number of bites. The reduction in aggression persisted for over 6 h and was no longer present 12 h post treatment. SRX251 did not alter the amount of time the resident investigated the intruder, olfactory communication, general motor activity, or sexual motivation. These data corroborate previous studies showing a role for vasopressin neurotransmission in aggression and suggest that V1a receptor antagonists may be used to treat interpersonal violence co-occurring with such illness as ADHD, autism, bipolar disorder, and substance abuse.

A dual read-out assay to evaluate the potency of compounds active against Mycobacterium tuberculosis.

Tuberculosis is a serious global health problem caused by the bacterium Mycobacterium tuberculosis. There is an urgent need for discovery and development of new treatments, but this can only be accomplished through rapid and reproducible M. tuberculosis assays designed to identify potent inhibitors. We developed an automated 96-well assay utilizing a recombinant strain of M. tuberculosis expressing a far-red fluorescent reporter to determine the activity of novel compounds; this allowed us to measure growth by monitoring both optical density and fluorescence. We determined that optical density and fluorescence were correlated with cell number during logarithmic phase growth. Fluorescence was stably maintained without antibiotic selection over 5 days, during which time cells remained actively growing. We optimized parameters for the assay, with the final format being 5 days’ growth in 96-well plates in the presence of 2% w/v DMSO. We confirmed reproducibility using rifampicin and other antibiotics. The dual detection method allows for a reproducible calculation of the minimum inhibitory concentration (MIC), at the same time detecting artefacts such as fluorescence quenching or compound precipitation. We used our assay to confirm anti-tubercular activity and establish the structure activity relationship (SAR) around the imidazo[1,2-a]pyridine-3-carboxamides, a promising series of M. tuberculosis inhibitors.

Exploiting bacterial iron acquisition: siderophore conjugates

Siderophores are chelators synthesized by bacteria and fungi to sequester iron, which is essential for virulence and pathogenicity. Since the process involves active transport, which is highly regulated, remarkably efficient and often microbially selective, it has been exploited as a Trojan Horse method for development of microbe-selective antibiotics. Siderophores also have significant potential for the development of imaging contrast agents and diagnostics for pathogen-selective detection. These promising results demonstrate the versatility of natural and synthetic microbial iron chelators and their potential therapeutic applications.

Iron Transport-Mediated Drug Delivery: Practical Syntheses and In Vitro Antibacterial Studies of Tris-Catecholate Siderophore–Aminopenicillin Conjugates Reveals Selectively Potent Antipseudomonal Activity

An artificial tris-catecolate siderophore with a tripodal backbone and its conjugates with ampicillin and amoxicillin were synthesized. Both conjugates exhibited significantly enhanced in vitro antibacterial activities against Gram-negative species compared to the parent drugs, especially against Pseudomonas aeruginosa . The conjugates appeared to be assimilated by an induced bacterial iron transport process as their activities were inversely related to iron concentration. The easily synthesized tris-catecolate siderophore has great potential for future development of various drug conjugates to target antibiotic-resistant Gram-negative bacteria.

N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide as a new scaffold that provides rapid access to antimicrotubule agents: synthesis and evaluation of antiproliferative activity against select cancer cell lines.

A series of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamides was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC) and afforded inhibitors of cancer cell growth. For example, compound 13e had an IC(50) of 46 nM against MCF-7 human breast tumor cells. Structure-activity relationship (SAR) studies demonstrated that (i) meta-phenoxy substitution of the N-1-benzyl group is important for antiproliferative activity and (ii) a variety of heterocyclic substitutions for the aryl group of the arylamide are tolerated. In silico COMPARE analysis of antiproliferative activity against the NCI-60 human tumor cell line panel revealed a correlation to clinically useful antimicrotubule agents such as paclitaxel and vincristine. This in silico correlation was supported by (i) in vitro inhibition of tubulin polymerization, (ii) G(2)/M-phase arrest in HeLa cells as assessed by flow cytometry, and (iii) perturbation of normal microtubule activity in HeLa cells as observed by confocal microscopy. The results demonstrate that N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide is a readily accessible small molecule scaffold for compounds that inhibit tubulin polymerization and tumor cell growth.

Is drug release necessary for antimicrobial activity of siderophore-drug conjugates? Syntheses and biological studies of the naturally occurring salmycin “Trojan Horse” antibiotics and synthetic desferridanoxamine-antibiotic conjugates

The recent rise in drug resistance found amongst community acquired infections has sparked renewed interest in developing antimicrobial agents that target resistant organisms and limit the natural selection of immune variants. Recent discoveries have shown that iron uptake systems in bacteria and fungi are suitable targets for developing such therapeutic agents. The use of siderophore-drug conjugates as “Trojan Horse” drug delivery agents has attracted particular interest in this area. This review will discuss efforts in our research group to study the salmycin class of “Trojan Horse” antibiotics. Inspired by the natural design of the salmycins, a series of desferridanoxamine-antibiotic conjugates were synthesized and tested in microbial growth inhibition assays. The results of these studies will be related to understanding the role of drug release in siderophore-mediated drug delivery with implications for future siderophore-drug conjugate design.

Generation and exploration of new classes of antitubercular agents: The optimization of oxazolines, oxazoles, thiazolines, thiazoles to imidazo[1,2-a]pyridines and isomeric 5,6-fused scaffolds.

Tuberculosis (TB) is a devastating disease resulting in a death every 20s. Thus, new drugs are urgently needed. Herein we report ten classes of compounds-oxazoline, oxazole, thiazoline, thiazole, pyrazole, pyridine, isoxazole, imidazo[1,2-a]pyridine, imidazo[1,2-a]pyrimidine and imidazo[1,2-c]pyrimidine-which have good (micromolar) to excellent (sub-micromolar) antitubercular potency. The 5,6-fused heteroaromatic compounds were the most potent with MICs as low as <0.195 μM (9 and 11). Overall, the imidazo[1,2-a]pyridine class was determined to be most promising, with potency similar to isoniazid and PA-824 against replicating Mtb H(37)Rv, clinically relevant drug sensitive, multi- and extensively resistant Mtb strains as well as having good in vitro metabolic stability.

Utilization of microbial iron assimilation processes for the development of new antibiotics and inspiration for the design of new anticancer agents

Pathogenic microbes rapidly develop resistance to antibiotics. To keep ahead in the “microbial war”, extensive interdisciplinary research is needed. A primary cause of drug resistance is the overuse of antibiotics that can result in alteration of microbial permeability, alteration of drug target binding sites, induction of enzymes that destroy antibiotics (ie., beta-lactamase) and even induction of efflux mechanisms. A combination of chemical syntheses, microbiological and biochemical studies demonstrate that the known critical dependence of iron assimilation by microbes for growth and virulence can be exploited for the development of new approaches to antibiotic therapy. Iron recognition and active transport relies on the biosyntheses and use of microbe-selective iron-chelating compounds called siderophores. Our studies, and those of others, demonstrate that siderophores and analogs can be used for iron transport-mediated drug delivery (“Trojan Horse” antibiotics) and induction of iron limitation/starvation (Development of new agents to block iron assimilation). Recent extensions of the use of siderophores for the development of novel potent and selective anticancer agents are also described.