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Metals in Medicine
by National Institute of General Medical Sciences
Monoatomics are firmly established in the health and wellness industry. Traditional medical science is starting to see metals for what they truely are.
This was originally a 38 page word .doc put out by the National Institute of General Medical Sciences from one of thier past conferences..
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Metals in Medicine: Targets, Diagnostics, and Therapeutics
- Introduction
- Factors Motivating Organization of the Meeting
- Meeting Objectives
- Meeting Organization, Advertising, and Participation
- Executive Summary
- Metalloenzymes as Targets
- Metallopharmaceutical Diagnostics and Radiotherapeutics
- Metal Metabolism as a Research Target
- Medicinal Inorganic Chemistry
- Opportunities
- Challenges
- General Comments and Recommendations
- Detailed Meeting Report
- Session 1: Molecular and Cellular Targets of Metal Action
- Day 1 Morning Session Discussion
- Session 2: Metal-Containing Targets of Drug Action
- Session 3: Radiology, Imaging, and Photodynamic Therapy
- Day 1 Afternoon Discussion
- Session 4: Metal Metabolism
- Day 2 Morning Discussion
- Session 5: Metallotherapeutics and Disease
- Day 2 Afternoon Discussion and Overall Meeting Discussion
Introduction
A meeting, entitled, "Metals in Medicine: Targets, Diagnostics, and Therapeutics", was held June 28-29, 2000, in the Natcher Conference Center on the NIH campus, in Bethesda, Maryland. This meeting, sponsored by the National Institute of General Medical Sciences (NIGMS), the National Cancer Institute (NCI), the National Institute of Allergy and Infectious Diseases (NIAID), the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the National Institute of Environmental Health Sciences (NIEHS), the Center for Scientific Review (CSR), and the NIH Office of Dietary Supplements generated substantial interest in the scientific community. In addition to the 25 invited speakers, another 53 people volunteered abstracts for poster presentations. A total of 352 people registered for the meeting. Actual attendance on-site was 235.
Factors Motivating Organization of the Meeting
- NIGMS and other NIH components provide significant support for research in bioinorganic chemistry and metallobiochemistry. Most currently supported research is focused on metalloprotein structure/function studies and metalloproteins are clearly important targets for drug development.
- Discussions with leaders in the scientific community suggested additional areas of emerging scientific opportunity (e.g., roles of metals in cellular regulation, mechanisms of metal trafficking, opportunities to modulate normal and aberrant metal metabolism).
- Discussions also raised the question of why so few inorganic drugs are on the market and whether this relates to insurmountable obstacles in development of such agents or limited participation of inorganic chemists in the pharmaceutical industry.
- Advances in synthesis and control of inorganic complex reactivity and in understanding the reactions of metals in vivo, plus the successful development of metallopharmaceuticals in several diagnostic and therapeutic areas, suggest that additional research opportunities might exist in this area.
Meeting Objectives
Three general topics were explored:
- Current utilization of bioinorganic chemistry and metallobiochemistry basic research discoveries in the pharmaceutical industry. What is the role of metalloenzyme structure/function insights in drug discovery and development? What is the role of inorganic compounds as leads in drug discovery? What is the industry experience? What is the industry attitude?
- Obstacles and opportunities for the development of additional metallopharmaceuticals. What progress has been made in selectivity? What do we know about metal toxicity? Is this an insurmountable obstacle or a matter of bad publicity? What has been learned from the successful development of radiotherapy and imaging agents? What promising results have been obtained in specific disease areas? What are the issues surrounding metals as dietary supplements? What types of research are needed to exploit the unique properties of metals for potential pharmaceutical benefit?
- Opportunities for the development of agents that target metal metabolism and metal-regulated cellular processes. What new cellular processes are being discovered? What potential do they have for drug development? What are the roles of essential elements in the body? What are the causes of metal toxicity? What successes have been achieved for correcting aberrant metal metabolism and toxic metal exposures?
Meeting Organization, Advertising, and Participation
Suggestions for this meeting were solicited from several hundred individuals, including representatives from academia, small businesses, and major pharmaceutical companies. Mailing lists were compiled for applicants and grantees in the NIGMS bioinorganic chemistry portfolio, applications reviewed by the NIH metallobiochemistry study section, attendees at recent Metals in Biology Gordon Conferences, the 9th Annual ICBIC meeting, ACS Medicinal Chemistry Division meetings, and additional sources. The meeting flyer was mailed to approximately 2,000 people, e-mailed to several listserves (e.g., ACS Div Med Chem members and NIH SBIR/STTR applicants), posted on the Internet and linked by various sites. The meeting was advertised on the NIH campus by posters and e-mail. Pre-meeting and on-site registration totaled 352. Of these, about 100 were from the NIH intramural and extramural programs, 48 represented scientists from 38 different companies, and 10 were from the FDA. Reporters from C&E News and Science magazine registered and attended. See List of Registrants posted on the Web site.
Regrettably, few registrants were from the PHARMA companies. A limitation in organizing and advertising the program was the difficulty in identifying contacts within these organizations. Interviews with those who were contacted were useful in painting the current picture. In several companies, metal complexes and metallo-organic compounds had been removed from the libraries currently used in their high-throughput screening programs. The potential toxicity of metals was cited as a significant concern. Many of the projects involving metal-containing lead compounds, that had been in progress a few years back, had been dropped. Efficacy and pharmacokinetic issues, market factors, and shifting business interests, as well as toxicity concerns, contributed to those decisions. Many of those trained as inorganic chemists in academia were no longer involved with inorganic chemical problems. Nonetheless, senior managers were encouraging of an effort to determine what opportunities may exist in this area.
The meeting program, which includes the agenda, lists of speakers, posters, biosketches, and abstracts, can be viewed and/or downloaded on the NIGMS Metals in Medicine Web site (http://www.nigms.nih.gov/news/meetings/metals.html). Although the collection of presentations might be considered eclectic, they actually represent an emerging and cohesive field. The talks spanned a continuum of interests from the roles of essential elements in both normal and disease processes to the potential beneficial, as well as detrimental, effects of non-essential elements. They are joined by the necessity of bringing to bear on the problems both a deep understanding of inorganic chemistry and state-of-the-art research in biology.
Executive Summary
Opportunities exist to exploit inorganic chemistry in the discovery and development of pharmaceuticals. Inorganic chemists can usefully contribute to drug development programs involving metalloenzyme targets. Radiotherapeutics and imaging agents are an established and growing area of metallopharmaceutical development. Opportunities exist to take advantage of the unique properties of metals in the further development of metallotherapeutics. Fear of metal toxicity is a limiting factor, but may be, in part, a matter of perception. Not all metals are bad--not all metals are "heavy metals." Yet, even essential metals are toxic at certain levels and in some chemical forms. The key is to understand and control the interaction of the metal with the living system. Basic research is needed to achieve that understanding.
Metalloenzymes as Targets
Most currently supported NIH research is focused in this area and has had the most impact to date. A substantial fraction of all proteins are metalloproteins and a significant fraction may become drug targets. The general strategies for developing metalloenzyme inhibitors are similar to those applied to non-metalloenzymes. Progress has been made in target validation and applications of both high-throughput screening approaches and structure-based drug design. Compounds not originally synthesized with metalloenzyme targets in mind may be useful inhibitors. Inhibitors can be designed to specifically involve metal site ligation, but this is not necessarily a requirement for inhibitor potency and selectivity. Some further improvement in methods for molecular modeling of metal sites would be helpful. Many computational programs are not parameterized for metals. Docking programs do not handle small molecule binding to metal sites very well. Continuing to establish the physiological functions of metalloproteins is important. Specific inhibitors may be useful in this process, particularly in cases where knock-out mice are not likely to be viable. Fundamental studies on the mechanisms of metalloenzymes, including studies on small molecule chelators, have been useful. In summary, this is an area of on-going research activity that has enjoyed NIH-wide support that should be continued.
Metallopharmaceutical Diagnostics and Radiotherapeutics
Radioimaging and radiation therapy agents, MRI contrast agents, and photodynamic therapy agents represent particularly successful examples of metallopharmaceutical development. Multiple agents are on the market already or in clinical trials. Targeted radiation therapy provides several advantages, including increased efficacy, reduced toxicity, and the ability to use the same or analogous agents for imaging, pharmacokinetic studies, and treatment. Issues include selection of metal center core chemistry, choice of targeting molecules, ease of synthesis, stability to target linkage and radiolabeling steps, characterization methods, immunogenicity, and pharmacokinetics. Challenges include the development of thermodynamically stable and kinetically inert complexes with optimized uptake by target tissues and minimal uptake by non-target tissues. Basic research is needed to understand the mechanisms of intracellular delivery of radiometals and the mechanisms of cell killing. In the case of MRI agents, complexes must be stable, yet also allow for exchange of bound water molecules. Basic research is needed to understand the structure/activity relationships for water exchange and spin relaxation. The challenges in radiosensitization and photodynamic therapy are more akin to those listed below in the section "Medicinal Inorganic Chemistry" in that complexes must not only be stable, but catalytically active. In summary, this is an area of rapid growth that can benefit from enhanced research. By long standing agreement, the areas discussed here are mainly in the purview of the NCI, although other Institutes' research areas may benefit from and contribute to the basic research base in this area.
Metal Metabolism as a Research Target
Metal metabolism is emerging as an exciting area of cell biology and a potential site for therapeutic interventions. Normal metal metabolism appears to maintain free metal ion concentrations at a very low level and to deliver metals very selectively to their sites of action, while maintaining tight control over their reactivity. Aberrant metal metabolism contributes to pathological conditions. Intercepting normal metallation reactions may also be a way to control metalloprotein activity. Improved metal ion sensors are needed to study cellular metal ion localization. The macromolecular players and vesicular compartments involved in metal ion homeostasis and metal trafficking are only just being learned. Roles of metals in other aspects of cell regulation, signal transduction, and cell-cell signalling are just coming to light. Metal responsive transcriptional and translational regulators, and mRNAs, may be important therapeutic targets and generalizable models. At least some of the mechanistic roles of many essential trace elements are known, but for several, they remain completely unclear. In some cases, definitive daily requirements are unknown and potential benefits of supplementation are hotly debated. Validated measures of metal status are needed. Analytical tools are needed that reflect biologically important pools and chemical speciation. Biomarkers of exposure and mechanisms of toxicity due to metals in the environment need to be understood. Biomarkers for variable susceptibility in the population are needed. Opportunities exist for the application of microarray technologies to many questions in metal metabolism. In summary, this is an expanding area of biomedical research.
Medicinal Inorganic Chemistry
This is a multidisciplinary field combining elements of chemistry, pharmacology and toxicology, biochemistry, biophyiscs, and medicinal chemistry. Medicinal inorganics have an appreciable current impact and significant growth potential. The hit rates in drug screening programs and the success rates of metallopharmaceutical advanced clinical leads are comparable to those of traditional organic agents. The regulatory process and expectations are the same for both metal and non-metal containing agents. A limiting factor may be the relatively limited expertise within the U.S. Food and Drug Association (FDA) and within most pharmaceutical companies in the area of inorganic chemistry. It would be incorrect to say the major pharmaceutical companies have not expressed any interest. However, it does appear that the bar is set somewhat higher for medicinal inorganics. Control of metal reactivity to improve specificity and reduce metal toxicity is the major obstacle to development of metallopharmaceuticals. Yet, this may in part be a matter of perception. The goals of improved specificity and reduced toxicity are not fundamentally different than in the case of organic drug development, however, the knowledge base and personnel infrastructure are much less fully developed. Government programs may need to reach further to off-set industrial caution in this area. Numerous opportunities and challenges exist.
Opportunities
- The unique properties of metal complexes may offer advantages in the discovery and development of new drugs. These unique properties include redox activity, Lewis acidity, electrophilicity, access to cationic/anionic/radical species, flexible bond orders, unique geometries, easily accessed structure/activity variations, and magnetic, spectroscopic, and radioactive signatures.
- Understanding the fundamental properties of metals and of metal-ligand chelation chemistry remains an important area for research. Examples include regulation of spin relaxation processes, complex stability, ligand exchange kinetics, and other physical chemical properties (electrochemical potentials, fluorescence quantum yields, ligand pKa values, etc.).
- Metal complexes are amenable to combinatorial synthetic methods, and an immense diversity of structural scaffolds can be achieved. Metal centers are capable of organizing surrounding atoms to achieve pharmacophore geometries that are not readily achieved by other means.
- The effects of metals can be highly specific and can be modulated by recruiting cellular processes that recognize specific types of metal-macromolecule interactions. Metals can be useful probes of cellular function. Understanding these interactions is paving the way toward rational design of metallopharmaceticals and implementation of new co-therapies.
- Metal-based agents can modify both DNA and RNA with a high degree of regiochemical, sequential, and conformational specificity. The next step is to demonstrate utility in vivo. Simply targeting DNA is no longer a sufficient rational for testing a compound (whether organic or inorganic). Cell selectivity in mRNA expression makes it an attractive target.
- Metal complex-based selective enzyme inhibition is an underexplored area. Metals may be useful in active site recognition and in bifunctional agents as secondary contacts to increase inhibitor affinity.
- Metal complexes can be substrates and inhibitors of membrane transport processes (e.g., MDR and pfMDR1), and can thus serve as useful probes, therapeutics, or co-therapeutics.
- Metal complexes can be potent and highly selective ligands of cell surface receptors.
- The influences of metals on the host of other known cellular targets remain largely unexplored, except in the context of metal toxicity. Studies of toxicity mechanisms may provide insights into potential therapeutic approaches.
- Essential metals are being developed as both drugs and dietary supplements. Several metals (e.g., vanadium and chromium) appear to have significant effects on complex metabolic diseases (e.g., diabetes). The mechanisms of these effects are still unclear.
- Metal complexes have been developed that are stable, yet have superoxide dismutase, catalase, and peroxidase catalytic activities. Such complexes may be useful in a host of oxygen radical-mediated disease processes.
- Metal complexes have shown potent anti-viral and anti-cancer activities in a variety of screens. The hit rates of metal complexes in these screens and the success rates of their further development are not different than for non-metal drugs.
- Metal complexation is the basis for chelation therapy to rectify abnormal metal accumulations or toxic metal exposures (e.g., iron overload; lead, cadmium, and mercury poisoning). Improved chelator designs are needed to enhance selectivity, affinity, stability, renal clearance, and oral activity, while maintaining low toxicity and low cost.
- Chelator co-therapy can be useful to minimize toxic side effects in radiation therapy and chemotherapy using metal complexes.
- Metal chelation chemistry may be important even in drugs that are not intentionally designed as metal chelators. A large fraction of drugs on the market are known or expected to bind metals with appreciable affinity. How this affects their actions is worthy of exploration.
- Metals currently have a significant market (and health benefit) impact, and significant growth potential. New agents are likely to find unique market niches due to unique mechanisms of action or pharmcokinetic properties that complement other therapeutics.
Challenges
- Delivery of metallopharmaceuticals into target cells and to specific intracellular sites
- Recruitment of cellular reagents as co-factors for metal-catalyzed reactions
- Design of reactions that recruit cellular amplification events such as apoptotic or other cell signaling pathways
- Development of both irreversible and reversible cellular modification strategies
- Design of agents that take advantage of triggering mechanisms to exploit cellular compartment transmembrane potential, redox potential, pH, and metal ion gradients
- Ligand design to optimize desired metal complex properties (thermodynamic and kinetic stability, hydrolytic stability, catalytic activity, molecular weight, charge, lipophilicity, water solubility, targeting functionalization, and ligand metabolism
- Understanding the role of ion pairing for polyoxymetalate anions and other complexes and how this may affect localization and activity
- Development of better in vitro screens and animal models to provide better prediction of human efficacy and toxicity
- Understanding metal activation of drugs in vivo, how metal is acquired, and how many drugs are affected by metal binding in vivo
- Development of complexes that are stable, but catalytically active, as radiosensitizers
- Development of complexes with improved quantum yield and favorable excitation spectra for photodynamic therapy
General Comments and Recommendations
Medicinal chemistry, and indeed chemistry in general, has been dominated by organic chemistry for much of this century. Many chemists receive only limited inorganic chemistry training. Not surprisingly, the medicinal chemistry departments within most pharmaceutical companies are oriented toward organic chemistry. Additional training of students in inorganic chemistry and particularly at the interface between inorganic chemistry and biology would be helpful.
Significant stumbling blocks occur at all stages, but particularly at the developmental and translational research level. Issues include:
- Appropriate handling of intellectual property issues so that licenseable discoveries in academia can be developed
- Access to appropriate animal models for pharmacokinetic evaluations
- Access to GMP production and support for translational human research
- Management of the transition from academic basic research to industrial research and clinical trials
- Limited inorganic chemical expertise in the pharmaceutical industry
- Limited experience of FDA staff and reviewers with relevant methods and issues in metallopharmaceutical characterization
Ways that NIH can help:
- Increase support for basic research in medicinal inorganic chemistry
- Refer proposals in this area to study sections that include expertise to review both the biology and the relevant chemistry
- Support and encourage the continued evaluation of metal complexes within the NCI Developmental Therapeutics Program (DTP)
- Support and encourage growth of the NCI Development of Clinical Imaging Drugs and Enhancers Program (DCIDE)
- Link programs in discovery and development of metallopharmaceuticals with the Mouse Models of Human Cancer Consortium
Mechanisms of support that the community might more fully utilize:
- Research training of inorganic chemists can be supported through the NIGMS Chemistry/Biology Interface (see http://grants.nih.gov/grants/guide/pa-files/PA-92-063.html) and other training programs.
- Collaborative research efforts can be supported through the NIGMS "Glue Grant" (see http://www.nigms.nih.gov/funding/gluegrants.html) and Program Project grant mechanisms.
- Corporate research can be supported through regular R01 grant mechanisms and through the SBIR/STTR program. Flexibility in the level and duration of support is available within both grant mechanisms.
Detailed Meeting Report
This report blends information from the talks, abstracts, discussions, and editorial comments without attributing specific comments to any one individual. In some cases, material has been transplanted from the section of the meeting in which it actually was presented to the general discussion sessions to make broadly relevant comments more easily accessible. Statements, particularly about efficacy, should not be interpreted as government policies or endorsements. The material presented here represents individual statements and opinions, not the results of any consensus conference activity. All material should be considered in the context of the focus of the meeting on basic research needs and opportunities. *Material that suggests general principles, future directions, obstacles and opportunities, or recommends NIH actions, is presented in italics.
Day 1--Morning Session
Session 1. Molecular and Cellular Targets of Metal Action
Steve Lippard provided the keynote address with a review of the history of cisplatin and recent advances from his laboratory. He noted that the discovery of cisplatin by Rosenberg was serendipitous. Some 3-4,000 platinum compounds have since been screened and yielded a modest number of additional agents, notably carboplatin which is now the dominant drug used in the clinic. Both are pro-drugs that undergo ligand exchange to yield active aquated cationic complexes, which react with DNA to yield particularly 1,2-intrastrand G-G cross-links.
Work in the Lippard lab and by other groups has established a potential mechanism of cisplatin action and provided detailed structural information on a key cisplatin-DNA-HMG1 A-domain complex. Cisplatin cross-linking of adjacent guanines in the major grove distorts DNA to widen the minor grove and create a hydrophobic notch between the cross-linked G residues. This motif is recognized by high mobility group (HMG) proteins, which bind in the minor grove with a phenylalanine side-chain intercalated into the notch. Binding of the HMG protein reduces distortion at the platinum site away from its preferred geometry in cross-linked DNA alone, and contributes to the strength of interaction. HMG binding protects the site of cross-linking from the normal excision repair mechanisms. The mechanism of cell death appears to involve cessation of transcription and consequent activation of additional signaling pathways. Inhibition of transcription may involve over-stabilization of TATA box binding protein or FACT (SSRP1/SPt16) transcription complexes, which are known to bind tightly to cisplatin-modified sites in DNA. The extraordinary success of cisplatin in treating testicular cancer appears to reflect the high level of HMG2 in that tissue.
Recognizing the importance of HMG proteins and that the steroids estrogen and progesterone elevate HMG expression in steroid responsive tissues has led to the demonstration of steroid potentiation of cisplatin/carboplatin action in BG-1 ovarian cancer cells. Phase I clinical trials for estrogen/carboplatin co-therapy in ovarian cancer are just getting started.
This work illustrated:
- The potentially high specificity of metal effects on cells and the cellular recruitment processes that recognize specific types of metal-DNA adducts
- The utility of metals as probes of cellular mechanisms and the ability to rationally design therapeutic interventions by understanding the mechanism of action of the drug
- The understanding at a detailed structural level required to design, rather than screen for, new agents of potentially lower toxicity that might mimic the action of cisplatin
Cynthia Burrows provided a useful overview of nucleic acid cleavage and base modification chemistry. Many such reactions have been developed that target DNA or RNA by sugar oxidation, phosphodiester hydrolysis, or base modification and deglycosylation. The first group includes agents such as iron-bleomycin, manganese porphyrins, iron-EDTA, copper phenanthrolines, nickel-peptides, and ruthenium complexes. The second group includes divalent metals (Co, Cu, Zn) and various lanthanides. Mechanisms of action parallel those of DNA hydrolyzing enzymes; i.e., water activation, phosphoryl group polarization, and leaving group stabilization. The third group includes the type of chemistry pioneered by the Maxim and Gilbert method for DNA sequencing, wherein base modification and deglycosylation leads to strand cleavage (e.g., upon treatment with piperidine.) Such reactions and also a battery of photochemical nucleic acid cleavage reactions have been used to sequence nucleic acids and to study their conformations in vitro.
Work in the Burrows lab has focused on square planar nickel complexes. These are of interest both because of the toxicity and carcinogenicity of nickel, and because its redox chemistry can be highly regulated by bound ligands. Sequence-selective cleavages have been demonstrated using Ni-peptides. In addition to metal-centered redox reactions, metal-activated ligand-centered modification reactions have been observed for nickel salen complexes. Burrows has combined these concepts to design a molecule containing a salicylaldehyde redox active motif and a peptide molecular targeting motif. The reaction with DNA in several steps can site-selectively introduce a salicylaldehyde group into DNA, which may be used to conjugate additional groups. The redox chemistry involved has used the nickel-catalyzed oxidation of sulfite by oxygen to generate monoperoxysulfate, which provides the oxidative reagent in situ.
Challenges for the future:
- Delivery of nucleic acid modifying complexes into target cells; Need for further development of both improved transmembrane delivery in general and selective targeting to specific cell types
- Recruitment of cellular reagents (e.g., water, sulfhydryls, oxygen, superoxide) that can replace the unnatural oxidants and reductants used in many in vitro methods
- Development of non-reparable DNA-modifying chemistry (e.g., presently many reagents are available that achieve single-strand cleavage, but fewer achieve less readily repaired double-strand cleavage)
- Design of reactions that recruit cellular amplification events, such as noted above for cisplatin, or set off chains of oxygen radical generation
- Development of methods of further enhancing site specificity
Tom Meade discussed results from his lab in collaboration with Harry Gray's group and Redox Pharmaceuticals on cobalt acacaciden complexes as enzyme inhibitors. Such complexes are potent antiviral agents in vitro and are progressing in clinical trials. Because of the potential toxicity of the first generation compounds, initial applications have been for topical delivery. The mechanism appears to involve dissociation of the axial ligands to yield a coordinatively unsaturated site, which binds tightly to histidine residues. By appropriate modification of the acacaciden unit and axial ligands, the reactivity can be regulated, including the rate of activation of Co+3 complexes by reduction to the Co+2 state. Selectivity has been achieved by attaching the complexes to peptides, nucleic acids, or cell delivery vehicles. Demonstration examples included inhibitions of carbonic anhydrase, thermolysin, thrombin, chymotrypsin, and alpha-lytic protease. Complexes linked to appropriate double-stranded DNA fragments were able to bind Zn-finger domains and displace the zinc by competing for the coordinated histidine residues. Selective inhibition of a single specific Zn-finger protein in a mixture could be shown. These reagents have been applied as research tools in studies of development to knock-out selected transcripts. Cell-selective killing has been achieved by using transferrin receptor-mediated delivery of complexes attached to polylysine particles. Attachment of Gd complexes to the same particles allowed demonstration that the killed cells were those that had selectively taken up reagent.
Potential advantages of metal complexes as enzyme inhibitors include:
- Potentially irreversible enzyme inhibitions
- Easily synthesized framework for structure/activity modifications
- Electrochemical control for potential cell redox poise regulated activation
- Spectroscopic signatures that may be useful in studies on mechanism and distribution
David Piwnica-Worms discussed several areas of metal complex transport across cell membranes. 99mTc-SESTAMIBI, developed in the lab of Alan Davidson, is a moderately hydrophobic cation. Other 99mTc-hexakis isonitrile complexes and 68Ga(III) Schiff base phenolic complexes developed in the Piwnica-Worms group, TcQ58 (Mallinkcrodt), and Tetrafosmin (Amersham) behave similarly. They are freely membrane permeant by passive diffusion, and accumulate into cells and cellular compartments according to the Nernstian potential. However, they are also substrates for P-glycoprotein-mediated transport. They accumulate in tissues with low P-gp and high concentrations of mitochondria, such as the heart, and are excluded from P-gp expressing tissues such as the brain. These agents and others have been validated as PET and SPECT imaging probes of P-gp activity in patients and are useful prognostic indicators of cancer drug resistance. Passive diffusion of these agents back into cells makes them not only substrates for P-gp, but also effective inhibitors of the transport of other drugs--an objective of multidrug resistance reversal agent co-therapy. An example was given of the development of agents that are selective for pfMDR1, which is expressed by chloroquine-resistant strains of malaria. Another means of metal-complex transport across cells was developed using the HIV-tat membrane permeant peptide as a delivery vehicle for metal chelates. An agent with a caspase-3 cleavage site peptide as the linker between the tat peptide and the complex allowed imaging of tissues undergoing apoptosis in a mouse model in vivo.
Comments regarding the discovery and development of metallopharmaceuticals:
- Major stumbling blocks occurring at the developmental and translational research level:
- Evaluation of the toxicity of metal complexes and meeting FDA criteria
- Access to appropriate animal models
- Pharmacokinetic evaluations
- Translational human research and required GMP production
- Managing the transition between academic and industry interests and infrastructures
- Suggestions for NIH support:
- Support and encourage evaluation of metal complexes within the NCI Developmental Therapeutics Program (DTP)
- Support and encourage growth of the NCI Development of Clinical Imaging Drugs and Enhancers Program (DCIDE)
- Link programs in discovery and development of metallopharmaceuticals with the Mouse Models of Human Cancer Consortium (MMHCC)
Shubh Sharma presented work on the inhibition of melanocortin receptors by Re[V]O complexed peptides. This work illustrated how metals can coordinate ligands to produce receptor ligand geometries that cannot be readily accessed by other means. This allowed structure/activity exploration of pharmacophore space that could not otherwise be tested. Metal complexation also fixed peptide geometry, thus reducing the conformational ambiguities that occur for peptides and many peptidomimetics. Whether these complexes will be drugs themselves, or not, they may provide a useful stepping stone on the way to developing small molecule agonists and antagonists of peptide receptors. Many drugs act at cell surface receptors and many more receptor targets of unknown function are being discovered through genomics. It is easy to produce peptide leads that recognize these receptors, but difficult to convert them into small molecule drugs. Combinatorial peptide screening often generates antagonists, but not agonists. Metallopeptides can be useful in combinatorial approaches for random screening or in rational structure-based drug design (e.g., based on stabilizing reverse turns). Melanocortins (a-, b-, and g-MSH) are related to ACTH and secreted by the pituitary gland. They have multiple physiological effects. Presently five receptor sub-types are known that mediate effects on skin, the adrenal glands, and the brain, including effects regulating overall basal metabolic activity. Several metallopeptide templates have been explored resulting in receptor agonists, as well as antagonists, with nM binding affinities and receptor sub-type selectivities of several orders of magnitude.
Advantages of metallopeptides in drug discovery:
- Many drugs act at cell surface receptors.
- Potent, selective molecules can be generated.
- Structural diversity can be explored with D,L forms of >100 natural and unnatural amino acids.
- Syntheses are amenable to automated, combinatorial solid phase methods.
- Radiolabeled (Tc) and non-radiolabeled isosteric complexes simplify PK/PD studies.
- Metallopeptides are stable and are excreted into urine unchanged.
- Useful and unique geometric information can be obtained for rational drug design.
Janet Morrow provided a brief talk that illustrated the value of RNA as a target for drug development and the many steps in RNA metabolism that may be attacked. Non-redox active metal complexes have been attached to antisense oligonucleotides to generate selective RNA-cleavage agents. Work from the Morrow lab has focused on dinuclear metal centers and lanthanide (Eu) macrocyclic complexes to catalyze removal of the 5-CAP structure from mRNA. Approximately three-fold enhanced activity has been demonstrated in cells compared to the parent antisense nucleotide.
David Petering presented a brief talk on bleomycin-mediated cleavage of DNA. The presence of a DNA-binding domain, metal-binding domain, linker domain, and disaccharide unit, illustrate complexity in contrast to the simplicity of cisplatin. The structure of a Co+3-stabilized analog of the postulated Fe-peroxide intermediate in the reaction bound to DNA was presented (a similar structure has recently been presented by Kozarich and Stubbe). The peroxo group is buried, which raises questions about accessibility to reductants. Differences are observed in the activation of the complex at non-selective versus selective sites. This may be influenced by the presence of high concentrations of phosphate in the cell and formation of iron-phosphate complexes in the DNA-bound state. The hypothesis is that activation may occur at non-selective sites, then activated drug may migrate to selective cleavage sites.
Many questions remain about the mechanism of bleomycin action. Bleomycin is administered as the free ligand, and picks up metal in the cell, but from where? The iron-bound form is reduced at least twice during turnover, but by what--possibly ascorbate? The iron undergoes several coordination changes during the DNA binding, oxygen binding, and reduction steps. The mechanism of double-strand, as opposed to single-strand, cleavage is unknown.
Day 1--Morning Discussion
The morning session talks provided examples of known cellular processes and how metals may target them. It also provided examples of how metals have been useful probes of cellular function. Many additional cellular processes are known, and how metals may be used to target them remains to be explored. For example, the phosphorylation and sulfation of saccharides on cell surfaces determines many of their functions and these reactions seem ripe targets for metal-based probes. RNA, which assumes very specific three-dimensional folded structures, may be a very useful target. Quite a few tools have been developed as in vitro probes of RNA conformation, but problems such as intracellular delivery remain barriers to their development as drugs.
Molecular geometries may be accessible using metal complexes (e.g., metallopeptides) that are simply not accessible by other means and may have unique and useful properties (e.g., pharmaceutically favorable characteristics, ability to provide radiolabeled isosteres, in vivo protease stability). Metal complexes may provide unique reactivities as enzyme inhibitors and certain advantages regarding cellular/subcellular delivery and prodrug activation. For example, cisplatin is not affected by MDR, but rather by a complementary resistance mechanism. Hence, cisplatin therapy is a useful complementary therapy in MDR-dependent drug-resistant cancers. The differential transmembrane potentials across various cell types and subcellular compartments may provide a means of selectively delivering charged metal complexes to specific sites. The electrochemical potentials that exist in these different compartments (e.g., varying GSH levels), may afford selective redox-active triggering as well.
Although the focus has been on metal-containing agents, bleomycin is an example of a pro-drug given in the metal-free state, which is activated by picking up its endogenous metal in the body. It is not the only example, and it may be arguable whether this is a good generalizable strategy. It avoids concerns about administering a metal, but may be bad in that chelation of endogenous metals may alter metal metabolism in deleterious ways.
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