# Metals in Medicine: Targets, Diagnostics, and Therapeutics

**June 28-29, 2000**

-   Introduction

-   Factors Motivating Organization of the Meeting

-   Meeting Objectives

-   Meeting Organization, Advertising, and Participation

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-   Executive Summary

-   Metalloenzymes as Targets

-   Metallopharmaceutical Diagnostics and Radiotherapeutics

-   Metal Metabolism as a Research Target

-   Medicinal Inorganic Chemistry

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-   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

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-   Session 4: Metal Metabolism

-   Day 2 Morning Discussion

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-   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. An unknown number viewed the meeting on the
Internet or NIH local area network via the
[videocast.nih.gov]{.underline} Web site. The broadcast will remain
available on that site for the indefinite future. The meeting Program
Booklet, List of Registrants, and this report have been posted on the
NIGMS Metals in Medicine Web page:
<http://www.nigms.nih.gov/news/meetings/metals.html>.

**[Factors Motivating Organization of the Meeting]{.underline}**

-   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.

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-   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).

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-   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.

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-   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]{.underline}**

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?

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-   *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?

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-   *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]{.underline}**

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]{.underline}**

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]{.underline}**

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]{.underline}**

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]{.underline}**

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]{.underline}**

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.

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-   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.).

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-   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.

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-   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.

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-   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.

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-   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.

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-   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.

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-   Metal complexes can be potent and highly selective ligands of cell
    surface receptors.

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-   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.

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-   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.

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-   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.

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-   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.

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-   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.

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-   Chelator co-therapy can be useful to minimize toxic side effects in
    radiation therapy and chemotherapy using metal complexes.

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-   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.

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-   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

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-   Recruitment of cellular reagents as co-factors for metal-catalyzed
    reactions

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-   Design of reactions that recruit cellular amplification events such
    as apoptotic or other cell signaling pathways

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-   Development of both irreversible and reversible cellular
    modification strategies

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-   Design of agents that take advantage of triggering mechanisms to
    exploit cellular compartment transmembrane potential, redox
    potential, pH, and metal ion gradients

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-   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

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-   Understanding the role of ion pairing for polyoxymetalate anions and
    other complexes and how this may affect localization and activity

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-   Development of better *in vitro* screens and animal models to
    provide better prediction of human efficacy and toxicity

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-   Understanding metal activation of drugs *in vivo*, how metal is
    acquired, and how many drugs are affected by metal binding *in vivo*

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-   Development of complexes that are stable, but catalytically active,
    as radiosensitizers

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-   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

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-   Access to appropriate animal models for pharmacokinetic evaluations

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-   Access to GMP production and support for translational human
    research

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-   Management of the transition from academic basic research to
    industrial research and clinical trials

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-   Limited inorganic chemical expertise in the pharmaceutical industry

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-   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

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-   Refer proposals in this area to study sections that include
    expertise to review both the biology and the relevant chemistry

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-   Support and encourage the continued evaluation of metal complexes
    within the NCI Developmental Therapeutics Program (DTP)

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-   Support and encourage growth of the NCI Development of Clinical
    Imaging Drugs and Enhancers Program (DCIDE)

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-   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.

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-   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.

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-   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:*

1)  *The potentially high specificity of metal effects on cells and the
    cellular recruitment processes that recognize specific types of
    metal-DNA adducts*

2)  *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*

3)  *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:*

1)  *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*

2)  *Recruitment of cellular reagents (e.g., water, sulfhydryls, oxygen,
    superoxide) that can replace the unnatural oxidants and reductants
    used in many* in vitro *methods*

3)  *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)*

4)  *Design of reactions that recruit cellular amplification events,
    such as noted above for cisplatin, or set off chains of oxygen
    radical generation*

5)  *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:*

1)  *Potentially irreversible enzyme inhibitions*

2)  *Easily synthesized framework for structure/activity modifications*

3)  *Electrochemical control for potential cell redox poise regulated
    activation*

4)  *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. ^99m^Tc-SESTAMIBI, developed in the lab of Alan
Davidson, is a moderately hydrophobic cation. Other ^99m^Tc-hexakis
isonitrile complexes and ^68^Ga(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:

1)  *Major stumbling blocks occurring at the developmental and
    translational research level:*

-   *Evaluation of the toxicity of metal complexes and meeting FDA
    criteria*

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-   *Access to appropriate animal models*

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-   *Pharmacokinetic evaluations*

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-   *Translational human research and required GMP production*

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-   *Managing the transition between academic and industry interests and
    infrastructures*

2)  *Suggestions for NIH support:*

-   *Support and encourage evaluation of metal complexes within the NCI
    Developmental Therapeutics Program (DTP)*

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-   *Support and encourage growth of the NCI Development of Clinical
    Imaging Drugs and Enhancers Program (DCIDE)*

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-   *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 (α-, β-, and γ-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:*

1)  *Many drugs act at cell surface receptors.*

2)  *Potent, selective molecules can be generated.*

3)  *Structural diversity can be explored with D,L forms of \>100
    natural and unnatural amino acids.*

4)  *Syntheses are amenable to automated, combinatorial solid phase
    methods.*

5)  *Radiolabeled (Tc) and non-radiolabeled isosteric complexes simplify
    PK/PD studies.*

6)  *Metallopeptides are stable and are excreted into urine unchanged.*

7)  *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.*

**Day 1\--Afternoon Session**

##### Session 2: Metal-Containing Targets of Drug Action

John Kozarich discussed drug development programs involving two
metallopeptidase targets, metallo beta-lactamases (a.k.a.,
carbapenamases) and peptide deformylase. Metallo beta-lactamases (as
opposed to the conventional serine protease type enzymes) have appeared
more recently and pose a threat to last line of defense antibiotics,
such as imipenem. They are dinuclear Zn enzymes with a bridging water
molecule. The Benkovic lab and Lippard lab have contributed studies of
the enzyme and active site models, respectively. Merck has used
high-throughput screening to identify lead compounds. A group of
biphenyl tetrazoles were identified that had originally been synthesized
as part of an angiotensin receptor antagonist development program (a
non-metal-containing target; however, see Abrams\' talk). Another class
was developed from a benzyl succinate lead. The most potent candidate
presented, a p-phenyl substituted dibenzyl succinate, has an IC~50~ in
the 100 pM range and strongly potentiates imipenem action in resistant
cells. High-resolution crystal structures show that in both cases the
inhibitor is bound to the Zn sites. In the succinate derivatives, both
carboxylate groups are liganded to Zn with one bonded to both Zn atoms,
replacing the bridging water molecule.

Peptide deformylase and methionine amino-peptidase are required for
sequential removal of the formyl group, then the methionine group, of
the translation initiator f-Met in prokaryotic protein biosynthesis.
Peptide deformylase is a member of the thermolysin family, containing a
single Zn atom coordinated by the HEXXH motif and a cysteine residue.
Actinonin is an acyl-hydroxamate antibiotic that was discovered in the
1960s. A crystal structure demonstrates that it binds to the Zn site.
Experiments have demonstrated that its mechanism of antibiotic action is
indeed inhibition of this enzyme. Unfortunately, resistance can be
acquired at very high frequency by mutations which eliminate formylation
of the initiator met-tRNA~f-met~ . Therefore, it does not appear that
peptide deformylase is in fact a useful antibiotic development target.
The methionine amino-peptidase, which is also a metalloenzyme, may be
more promising.

*This work illustrated:*

1)  *The contributions of basic research on metalloenzyme
    structure/function from several NIH-supported labs to a
    pharmaceutical company research program*

2)  *Compounds developed for other purposes and not targeted toward
    metal-containing enzyme sites may in fact also interact with
    metalloenzyme active sites.*

3)  *The importance of validating drug targets before investing too much
    effort in discovering new inhibitors*

4)  *The utility of both screening approaches and structure-based
    rational drug design in the optimization metalloenzyme inhibitors*

Tom Poulos presented recent work from his lab and others on the
structure and function of nitric oxide synthase. This research seeks to
understand the electron transfer process between the two flavins,
tetrahydrobiopterin, and heme cofactors per monomer; regulation of the
three different isoforms; the reaction mechanism; and to develop isoform
selective inhibitors. Crystal structures have been obtained for the heme
domains of all three isoforms (eNOS, nNOS, and iNOS). The enzyme forms a
dimer stabilized by a tetra-cysteine coordinated zinc atom at the
interface. Although spectroscopically and mechanistically related to
P450, the NOS fold is different. All three structures are similar (rmsd
= 1.06 Å). The active sites are nearly identical (except for an asp/asn
substitution at one site). Sequence variations in the pterin site are
somewhat greater. Structures have been obtained for a variety of bound
arginine and pterin analogs, and other inhibitors. H-bonding and
ion-pairing with glutamate and also one of the heme propionate groups
hold the arginine substrate in close proximity to the heme iron. The
pterin is located near the dimer interface, but also binds to the same
heme propionate group. Arginine analogs such as nitro-arginine bind with
the nitro-group extending over and blocking oxygen access to the heme.
Isothioureas bind in place of the arginine guanidino group, and utilize
similar H-bonding and ion-pairing contacts. Some mechanism-based
(suicide substrate) inhibitors have been discovered. Several of these
cause the heme to oxidize itself, yielding biliverdin, others cause
protein modifications. 2-bromo-7-nitro-indazole appears to bind as an
electrostatic analog of the guanadino group, but causes changes in
position of the propionate group that also result in loss of pterin
binding and binding of a second molecule of inhibitor at the pterin site
instead (in iNOS, but not in nNOS). Most other inhibitors bind without
causing major structural changes.

*Future directions:*

*The active sites are relatively open, and the sequences diverge more,
farther away from the active site. It may be possible to exploit
secondary contacts outside the site (e.g., neighboring histidine
residues) to generate selective inhibitors. Site-directed mutagenesis
and rational drug design will be useful to test the utility of targeting
the pterin site. Few inhibitors have been found to function by binding
directly to the heme iron atom. This may be a way to generate binding
affinity, but since the sites are so similar, may not be a useful
strategy to generate isoform selective inhibitors.*

Ken Merz provided a review of molecular modeling methods applicable to
metal-containing systems. Issues include: i) appropriate electrostatic
representation of metals, ii) representation of long-range electrostatic
effects, iii) inclusion of dynamic behavior, iv) ligand exchange, v)
calculation of complex electronic structures, and vi) representing
environmental \"solvation\" effects. Modeling of metal sites in proteins
is a different problem than modeling the behavior of the overall
metalloprotein; however, both depend on how accurately one needs to
represent the metal and its interaction with the protein. Purely
molecular mechanical bonded model approaches may be adequate for fixed
coordination, structural questions, but are not adequate for questions
about energetics, reactivity, and coordination changes. Local
environmental effects, solvation, dynamics, etc., significantly alter
charge densities so that fixed formal charges are inadequate. For
example, calculations for the zinc atom (formal charge = +2.0) in
carbonic anhydrase show variations between a charge of +0.6 and 0.0 on a
psec time scale. Combined quantum mechanical/molecular mechanical
methods that use QM to model the metal site and MM methods to model the
surrounding protein provide improved results. The appropriate way to
interface these calculational regimes remains an area of research.
Problems include how to handle boundary effects, finite size of the QM
region, neglect of long-range electrostatic interactions, choice of
semi-empirical QM method, limitations of the gas-phase QM calculation
(e.g., removal of active site geometric constraints) Fully QM and QM/QM
approaches have been developed using density matrix minimization,
localized orbitals, divide-and-conquer or onion-skin approaches, and
parallel computation. These methods have provided accurate calculations
for systems as large as 5,000 atoms, but will not allow MD simulations.

*In discussion, the question of accessibility of methods and the utility
of commercial software programs for the average chemist was raised. Most
off-the-shelf programs are not parameterized for metals. Docking
programs for looking at substrate-protein interactions mostly do not
include good ways to handle small-molecule ligand binding to metal
sites. This would seem to be an opportunity for small businesses to make
a real contribution to the field. Additional ways to validate
computational results beyond basic structural questions are also
needed.*

Benjamin Burke provide a discussion of structure-based drug design in
the development of matrix metalloprotease inhibitors. These
zinc-containing enzymes have recently been among the hottest
pharmaceutical targets (e.g., relevant to basement membrane invasion and
metastasis, tumor angiogenesis, and connective tissue breakdown in
arthritis). Approximately 23 MMPs are known, including gelatinases,
collagenases, thermolysin, stromalysins, and membrane-bound proteases.
They are regulated by a self-inhibitory pro-domain, activated in
cascades by other MMPs and other proteases, transcriptionally regulated
by cytokines and growth hormones, and subject to inhibition by tissue
inhibitors (TIMPs). Although differing in substrate specificity, the
MMPs are structurally homologous, containing: an N-terminal
self-inhibitory domain, a C-terminal specificity domain, and a catalytic
Zn (and Ca) domain. The alpha/beta sandwich catalytic domain is fully
active. Two calcium atoms and a structural zinc site contribute to the
stability of the structure. The active site zinc is coordinated by three
histidines with a nearby glutamate residue essential for activity.
Backbone carbonyl groups are also involved in both the catalytic
mechanism and inhibitor binding. Inhibitors have targeted the
selectivity specifying P1\' binding sites, while also providing zinc
ligation by oxygen, nitrogen, and/or sulfur atoms. Hydroxamate, reverse
hydroxymate, carboxylate, phosphinate, amino carboxylate, and
sulfodiimine compounds have been developed by several groups. Both
tetrahedral and bipyramidal (TS mimic) coordination have been observed.
Electrostatic and H-bonding interactions at other sites, shifts in pKa,
and strain, influence the crystallographically observed distortion from
ideal geometries and binding affinities. Calorimetry, NMR, and
calculational methods were used to examine the energetics and suggest
the next compounds to make. MMP inhibitors were shown to inhibit tumor
growth and neovascularization. Side effects in cancer therapy (e.g.,
joint pain) were reduced by minimizing activity against MMP1 through
P1\' site moiety design.

*Future directions:*

*The number of MMPs is not fully known. Regulation and activation
cascades are not fully understood. Knockout mouse models may not be
possible in all cases; therefore, specific inhibitors will be useful
tools to sort this out. Ligand coordination modeling in metalloproteins
is still difficult. Energetic calculations need to be improved to
account for changes in H-bonding, protonation, pKa, torsional strain,
and charge on zinc. It is difficult to make comparisons across compound
classes. Earlier fundamental studies on the mechanism of thermolysin
were useful. Additional studies on the basic chemistry of small
chelators focused on pH effects would be useful.*

##### Session 3: Radiology, Imaging, and Photodynamic Therapy

Shuang Liu provided a discussion of radioimaging agents. ^99m^Technetium
(t~1/2~ = 6.02 hr, 140 keV γ-emission, readily available, and low cost),
is the radioisotope of choice. Two strategies were discussed: i) the
integrated approach, where the metal complex, itself, contains targeting
information to provide localization of the radiolabel, and ii) the
bifunctional chelator (BFC) approach, where the chelator provides a
binding site for the metal and a means of attaching the radiolabel to
some targeting moiety (e.g., antibodies, peptides, peptidomimetics,
non-peptide small molecules). Illustrative targeting moieties used by
Dupont, Diatide, various other companies, and academic researchers
include: somatostatin analogs, RGD peptides, LTB~4~ antagonists, folate
antagonists, fMLFK chemotactic peptide, and vitronectin receptor
antagonists. Generally, antagonists are preferred because free ligand
causes fewer side effects than with agonists. Tc-cores have been based
of the chemistry of Tc(III), Tc(IV), \[TcO\]^+3^, \[TcO~2~\]^+^,
\[Tc≡N\], \[Tc(CO)~3~\]^+^, and \[TcN=N-R\]. A wide range of linker and
chelation chemistry has been employed.

*Important issues in radiopharmaceutical design include: selection of
Tc-core and BFC strategy, choice of targeting molecules, ease of
synthesis, stability to target linkage and radiolabeling steps,
immunogenicity, pharmacokinetics, and characterization methods,
including attention to possible isomerism. Pharmacokinetic preferences
include: fast receptor binding, slow dissociation, fast blood clearance,
short blood residence time, and renal clearance. Selection from a
variety of cationic, anionic, neutral, and enzymatically cleavable
linker units can tailor the pharmacokinetic properties. Metal chelates
must be thermodynamically stable and kinetically inert, should minimize
the formation of isomers, possess high labeling efficiency, and have
high hydrophilicity, which facilitates renal versus hepatobiliary
excretion. Development depends on a better understanding of both biology
and coordination chemistry (particularly at tracer levels of material),
and the application of appropriate analytical methods.*

Carolyn Anderson provided a discussion of radiation therapy using
targeted metal complexes. Advantages include: i) administration of
higher radiation doses to achieve greater efficacy; ii) matching the
type and energy of the ionizing radiation delivered (e.g., β, α, or
Auger electron) to the size of the tumor, and iii) use of the same or
analogous agents for imaging and therapy to allow prediction of tumor
response and toxic side effects. Some of the metals used in targeted
radiotherapy include: Re, Y, In, Lu, and Cu. Examples were presented
based on somatostatin analogs linked to DTPA, TETA, or DOTA chelators.
Several molecules from various academic and corporate programs are
currently in Phase I/II clinical trials. Promising results have been
achieved with minimal overall toxicity. Dose-limiting renal toxicity is
due to radiation exposure, not to metal exposure *per se*. Cu-64 decay
modes are useful for both PET imaging and therapy, and the isotope can
be economically produced using local facilities. Progress in the
Washington University program on Cu-64 somatostatin analogs includes:
synthesis, receptor binding assays, intracellular localization,
biodistribution and targeted therapy in tumor-bearing rats, *in vivo*
metabolism, toxicity studies in rats, dosimetry in rodents and primates,
and a clinical PET imaging trial. Useful diagnostic imaging results in
humans and promising therapeutic effects in animal models have been
demonstrated. The mechanism of action appears to involve
receptor-mediated endocytosis, dissociation of the metal from the
chelator, and delivery to the nucleus, where isotopes such as Cu-64 that
emit Auger electrons as well as other radiation were shown to be
particularly effective.

*Considerations in targeted radiotherapy include:*

1)  *Tumor type\--which receptors/antigens are present and which
    molecules will target the tumor?*

2)  *Tumor location and effect on normal tissue uptake and radiation
    exposure*

3)  *Tumor size and decay scheme of radionuclide*

4)  *Matching of radiopharmaceutical and radionuclide half-life*

5)  *Cost and availability of isotope*

*Challenges include the development of thermodynamically stable and
kinetically inert complexes for a wide variety of radiometals, retention
of biological targeting activity during synthesis, optimization of tumor
uptake and minimization of non-target tissue effects, understanding how
small changes in radiometal or BFC affect biodistribution, understanding
how intracellular metabolism and trafficking affect delivery of metals
to the nucleus, and understanding better the mechanism of cell killing.*

Randall Lauffer provided a discussion of MRI contrast agents. The
current market is about \$600 million and has almost doubled since 1994.
MRI contrast agents offer several advantages over other imaging methods.
They are non-radioactive, and administered at concentrations of 0.1 to 1
mM, whereas iodine-based x-ray contrast agents require 1-10 mM and
radiation exposure. Functional components of an imaging agent \[e.g.,
Schering AG\'s Magnevist (Gd-DTPA)\] are a Gd ion (large S value) held
tightly by a chelating agent to prevent Gd toxicity, and may also
include a targeting moiety. Signal is derived from the Gd ion and from
proton signals of spin-coupled water molecules. The biophysical
properties of the complexes (electron spin relaxation, rotational
correlation, and exchangeable water residence times) can be tuned to
match medical imaging needs. Development issues include achieving
sufficient relaxation rates changes in the target tissue, and safety
(non-toxic, readily excreted, and stable complexes). Other practical
concerns include cost, administration, viscosity, and timing. EPIX has
been developing non-covalently targeted MRI agents. Potential advantages
are: i) low retention of gadolinium in the body, ii) low immunogenicity,
iii) tunable binding affinity to allow a convenient imaging time window,
and iv) higher magnetic efficiency (protein binding was shown to
increase relaxation changes 10x). MS-325 is a
diphenylcyclohexyl-phosphate derivative of Gd-DTPA designed to target
serum albumin as a blood pool marker. It is being tested in MRI cardiac
angiography and as a monitor of tissue ablation in focused ultrasound
surgery (protein denaturation results in dissociation of the marker and
thus changed relaxation). Another agent has been developed as a pro-drug
with a phosphate group that blocks protein binding until acted upon by a
phosphatase. This agent may be useful for imaging in the lymphatic
system A third agent was presented that utilizes a fibrin binding
peptide to image clot formation.

*Future research opportunities in the area of MRI contrast agents
include:*

1)  *Improved understanding of lanthanide coordination chemistry*

2)  *Development of new chelates*

3)  *Analysis of structures and spin dynamics*

4)  *Understanding of structure activity relationships for water
    exchange and T1 relaxation rates.*

*Regarding practical problems in developing MRI agents, the following
points were made. Academic research is the cradle of creativity, but
translating ideas to the marketplace can only be done by industry, and
requires lots of money. However, finding translatable licensing
opportunities is difficult. Advantages of the industrial research
setting are highly motivated employees, focused multidisciplinary teams,
careful treatment of proprietary information, and regulatory expertise.
All this takes a lot of money. Suggestions included increased funding
for basic and applied research, and promotion of responsible treatment
of proprietary information. The present limited level of SBIR funding is
not sufficient to accomplish the job and the turnaround time is too
slow. Increasing the level of funding for companies to at least \$4
million over two years and decreasing review time to three months with
minimal disclosure of proprietary material was suggested.*

Richard Miller presented work from Pharmacyclics that derives from the
research of John Sessler on expanded porphyrins (Texaphryins). These
include gadolinium motexafin (Xcytrin, a radiation enhancer), and
lutetium motexafin (Lutrin, a photosensitizer). The actions of these
complexes are dependent on their redox and photochemical properties, in
the first case through depletion of cellular reductants, in the other
case through both depletion of cellular reductants and generation of
singlet oxygen. Both compounds are selectively concentrated in tumors
and localize in the mitochondria of cells, where they may have effects
on cellular bioenergetics. Radiation therapy is thought to act through
generation of hydroxyl radical and superoxide-mediated DNA damage.
Cellular reductants protect the cell by quenching initially formed DNA
radicals. Xcytrin has been shown to catalyze ascorbate and NAD(P)H
oxidation in vitro and to lower reductant levels in cells. Enhanced
apoptosis has been demonstrated after radiation treatment in a tumor
bearing mouse model. Clinical trials have demonstrated enhanced benefits
of radiation therapy with Xcytrin co-therapy, particularly in treatment
of brain metastases derived from lung and breast cancers. Localization
of the drug can be monitored by MRI due to relaxation effects of the Gd
atom, and the sensitivity of tumor imaging is enhanced. Normal tissue
uptake was limited to liver and kidney. Dose-limiting renal toxicity was
reversible. Blood clearance was rapid, but the drug accumulated in
target tissue such that efficacy and also imageability of the tumor
increased with multiple doses. Lutrin has shown efficacy in cutaneous
breast cancer. Limiting toxicity was due to skin necrosis and pain in
the treatment area. The ability to deliver light internally through
catheters, plus the ability of long-wavelength light to penetrate tissue
means that photodynamic therapy (PDT) is not limited to the skin.
NCI-sponsored clinical trials are in progress for PDT of prostate
cancer, cervical neoplasia, and esophageal cancer. Other applications of
Texaphrins are being developed for atherosclerosis and ophthalmologic
applications.

*Research objectives in this area included:*

1)  *Exploitation of the unique redox and photochemical properties of
    metals*

2)  *Modification of the porphyrin nucleus to bind metals with a larger
    radius than that of iron*

3)  *Optimization of drug delivery and tumor localization*

4)  *Red shift of absorption wavelength to increase tissue penetration
    in PDT*

5)  *Pharmacokinetic optimization of dose and radiation (or light
    irradiation) timing protocols.*

Bruce Averill gave a brief presentation of recent results, including
crystallographic studies, on proteolytic activation of purple acid
phosphatase, a diiron enzyme. In the unactivated state, a surface-loop
asparate residue is postulated to interact with either a histidine or
asparagine near the Fe^+2^ site, and reduces the nucleophilicity of
hydroxide coordinated at the Fe^+3^ site. Proteolytic cleavage
eliminates these interactions.

Tom Meade presented a short video on the use of MRI contrast agents to
visualize gene expression and cell signaling activity. These experiments
are based on agents that are capped to prevent water exchange (and hence
water relaxation effects) at the Gd site until modified by enzymatic
cleavage or ligand binding. They have been used to map the anatomical
expression of genes that are important in development.

**Day 1\--Afternoon Discussion**

*Discussion focused on the reasons that more inorganic agents have not
been developed as pharmaceuticals. The reality is that most chemists
working for companies are organic chemists by training and naturally
look in that direction for leads. There is a perception, right or wrong,
that metal-containing agents are more toxic. Metal-containing agents
have generally been excluded from the high-throughput library screening
programs that have become the major paradigm for drug lead discovery.
Metal-based drug discovery may be more amenable to a rational design
approach. Industry has been involved in this area in the past and
remains very active in some specialty areas. If sufficient potential
benefits can be identified, there would be a willingness to explore
metal-containing leads.*

*Discussion recapitulated the question about the quality of metal site
modeling and the need for better programs and more easily accessible
properly parameterized programs. Discussion also emphasized the
continued need for basic research into the coordination chemistry of
metals and SAR for ligand control of metal properties. Such work is
applicable in all areas of metallobiochemistry and medicinal inorganic
chemistry.*

**Day 2\--Morning Session**

##### Session 4: Metal Metabolism

Tom O\'Halloran discussed the emerging view that cells are not bags of
buffer in which metals freely diffuse between thermodynamically
controlled binding sites. Rather, a series of membrane metal
transporters, metal chaperones, and assembly complexes have recently
been identified that regulate the uptake and delivery of metal ions to
specific sites. Despite reasonably high total concentrations (0.02-0.75
mM for Cu, Zn, Fe), the concentration of free metal is vanishingly small
(less than one free atom per cell). In this context, thermodynamics of
metal binding (which can be very tight, e.g., K~d~ up to 10^-40^ for
mercury binding to vicinyl thiol groups), gives way to kinetics where
dissociation rates can, nonetheless, be rapid. Specific examples were
presented based on work in collaboration with the labs of Culotta,
Penner-Hahn, and Rosenzweig, on the exchange of copper between the Ctr1
uptake pump, Atx1 chaperone, and ccc2 vesicular pump of yeast.
Structural studies suggest an exchange mechanism in docked complexes
involving a sequence of di- and tri-thiol coordinated intermediates. A
similar pathway has been shown for delivery of copper to Cu,Zn-SOD via
another chaperone (CCS) that, itself, includes an Atx1-like domain.
Exchange in this case is not thermodynamically driven (K~eq~ ≈ 1.5), and
both are tight-binding. However, the exchange is rapid, k~f~ \>\> 30

M^-1^sec^-1^. The hypothesis is that protein/protein interaction lowers
the activation barrier for dissociation by distortion of the metal
binding site. Regulation of zinc in cells also appears to result in low
free concentrations. In this case, zinc-containing vesicles are
important. Fluorescent sensor compounds (Zinquin and derivatives) have
been used to image zincergic neurons in the hippocampal region of the
brain and the appearance of vesicles in other cell types as well.

*Opportunities and obstacles:*

1)  *Metal trafficking is emerging as a new area of cell biology and a
    potential site for therapeutic interventions.*

2)  *Improved metal ion selective sensors (particularly ratiometric
    fluorescent probes) are needed in varying affinity ranges to study
    metal concentrations and fluxes in cells.*

3)  *Improved understanding of metal metabolism may be relevant to
    treatment of diseases such as Menkes and Wilson\'s diseases, and
    ALS.*

Liz Theil provided an overview of iron metabolism, then focused on the
regulation of iron-responsive protein synthesis by iron response element
(IRE) recognition by IRE-binding proteins (IRPs). IREs have been
identified in iron storage, transport, and utilizing enzymes (e.g.,
ferritin, transferrin receptor, DMT1, ferroportin, δ-ALA synthase,
m-aconitase). Multiple IRPs have been identified. These are themselves
iron-binding proteins and may have other cellular functions (e.g.,
cytoplasmic aconitase). Many are regulated by cellular kinases as well
as iron levels. Various IRPs have differential effects on various
IRE-containing proteins such that a given iron level produces multiple
partial activations. Understanding iron regulation is relevant to
diseases of iron accumulation (e.g., sickle cell anemia, thalassemia,
hemochromatosis) and also to diseases of iron deficiency, that together
may affect 20-25% of the population. An objective is to increase
translation of ferritin from the large untranslated mRNA pool to enhance
iron storage capacity. IREs are hairpin loop structures that may occur
at either 3\'- or 5\'-ends of mRNA. They may affect ribosome binding,
message stability, or both. Details of IRE structure/function, dynamics,
and stability have been studied by NMR spectroscopy and metal-based
cleavage agents (e.g., Cu(phen)~2~, Ru(tipy)(bipy), Rh(phen)(phe)),
which result in selective modification of only certain IREs based on
shape complementarity. Studies on the structure and function of the iron
storage protein ferritin were described with the aim of facilitating
iron chelator removal of trapped iron. Ferritin is an oligomeric protein
encapsulating a nanoscale volume in which mineral iron-oxide is
reversibly deposited. Mutational studies of the proposed
iron-entrance/exit channel suggest that large scale opening/closing
motions may be important in function (e.g., perhaps for docking of iron
delivery molecules).

*Conclusions:*

1)  *Messenger RNA is a good drug target.*

2)  *Three-dimensional structures are stable enough for tight
    recognition and binding.*

3)  *Cell specificity of mRNA expression, in contrast to DNA, enhances
    biological selectivity.*

4)  *Backbone cleavage can provide high efficacy and information on the
    binding site.*

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4)  *Targets are present in many EST databases.*

5)  *mRNA translational regulation is emerging as relatively common
    mechanism of control.*

6)  *IREs are ideal for selectivity studies to model other RNAs.*

Ken Raymond provided a discussion of iron chelation therapy. The daily
iron requirement for humans is low, in part, because there is no
excretion mechanism. Iron levels are regulated by duodenal absorption.
However, several diseases result in iron overload. Hemochromatosis is
the most common hereditary disorder in the U.S. and results in excess
dietary iron absorption. Accumulation of the excess iron in the liver
leads to cirrhosis and hepatocarcinoma. Thalassemia(s) result from
defects in hemoglobin chain synthesis, requiring frequent transfusions
of normal hemoglobin carrying red blood cells. Excessive iron
accumulates in the reticuloendothelial cells of the spleen. Thalassemia
is widespread in the tropics. Dietary management and phlebotomy were the
principal ways to reduce iron loads until introduction of
desferrioximine B chelation therapy in the last decade. Unfortunately,
this drug is slow acting, rapidly excreted, and is not orally active.
Bacterial siderophores (iron transport molecules) have provided leads
for chelator development, including the discovery of desferrioximine.
Others include enterobactin, cepabactin, and deferipron, which utilize
catechol, hydroxypyrimidate, and hydroxypyridinone chelating groups,
respectively. Dr. Raymond has created a series of hydroxypyridinone
derivatives that improve efficacy and lower toxicity of this class by
combining multiple chelating units in a single molecule \[e.g.,
TREN-(Me-3,2-HOPO)\]. This creates a hexadentate ligand that binds iron
much more tightly by forming a 1:1 complex, as opposed to the 3:1
complexes formed by bidentate ligands, such as desferroximine and
deferipron. The new compounds have been successfully tested in mouse and
rat models of iron overload.

Conclusions from these studies include:

1)  Thermodynamically predicted greater effectiveness of higher dentate
    chelators was confirmed by animal testing results.

2)  The multidentate chelators showed low toxicity and no significant
    removal of iron from normal animals (i.e., they did not remove iron
    from essential sites).

3)  Iron removal was equal to or better that for desferrioximine, the
    currently used therapeutic, when both were injected.

4)  TREN-(Me-3,2-HOPO) was active when administered either by injection
    or orally.

*Design criteria for new iron chelators include:*

1)  *selectivity for Fe+3, not Ca, Mg, Cu, or Zn*

2)  *High affinity and thus ability to compete with* in vivo
    *complexation sites*

3)  *Stability* in vivo

4)  *Low toxicity*

5)  *Rapid excretion as the iron complex*

6)  *Oral activity*

7)  *Low synthetic cost*

Richard Anderson provided a discussion of chromium intake levels and
potential beneficial effects of chromium supplementation. Many effects
related to glucose and lipid metabolism, and insulin action, have been
noted. It appears from a few cases involving complete parenteral
nutrition that some level of chromium is essential. However, the
required daily amount is not known. The current Estimated Safe and
Adequate Daily Dietary Intake (ESADDI) amount is 50-200 micrograms. The
EPA reference dose for Cr^+3^ toxicity is 70,000 micrograms. However,
Cr^+6^ is toxic (see discussion by Costa below) and the difference may
be due to differences in bioavailability and cell uptake, rather than
the innocuousness of Cr^+3^, *per se*. Actual average daily intakes in
the U.S. population are 33 microgram in males and 25 micrograms in
females. In a controlled diet study providing intakes less than 20
micrograms per day for 5 weeks, little effect was found in most
subjects. However, glucose tolerance was decreased in patients with
initial borderline hypoglycemia. Chromium requirements may be increased
in diabetes. Several clinical trials have suggested the efficacy of
chromium supplementation in patients with Type I, Type II, gestationally
induced, and steroid-induced, diabetes. Various markers (e.g., glucose
tolerance, fasting blood glucose, urinary glucose levels, hemoglobin
AIC, and circulating insulin) have been monitored. Potential benefits
have also been observed for lipid level risk factors for cardiovascular
disease. However, as noted in the discussion, an approximately equal
number of trials have demonstrated no beneficial effect. An important
consideration is the poor bioavailability of chromium supplements
(0.5-2% of dose for the most available chemical forms and completely
unavailable in other forms). Measurement of urinary Cr loss is a
valuable measure of bioavailability. Another complication is the lack of
a simple relationship between hypoglycemia and blood or urinary chromium
levels. In fact, blood Cr levels and urinary losses may be elevated in
diabetes. Urinary losses are also influenced by physical stress or
corticosteroid treatments. *In vitro* and animal studies have
demonstrated: i) increases in insulin binding, ii) increases in insulin
receptor number, iii) increased insulin internalization, iv) increases
in receptor phosphorylation, and v) inhibition of receptor
dephosphorylation. All of these effects increase insulin sensitivity. A
low molecular weight chromium (LMWCr) factor has been isolated by John
Vincent, which stimulates insulin receptor tyrosine kinase and may also
inhibit phosphatase activity. Whether this observation adequately
explains the clinical data and whether this is the only role of chromium
in the body remains to be seen.

*A lively discussion following Dr. Anderson\'s talk suggests the
following conclusions:*

1)  *The potential benefits of chromium supplementation remain
    controversial.*

2)  *A problem is the lack of a validated measure of chromium status.*

3)  *Analytical methods are needed that reflect the biologically
    important chromium pools.*

4)  *Molecular mechanisms of chromium action in the body are poorly
    understood.*

5)  *Availability and widespread use of chromium dietary supplements by
    the population underscores the need for additional research.*

6)  *The low bioavailability of chromium contributes to confusion in the
    results of clinical trials, but may also be a major factor in
    safety.*

7)  *The extremely slow ligand exchange kinetics of Cr^+3^ dominates its
    chemistry, but perhaps should not be allowed to completely dominate
    thinking about its biology. There are things a metal (or metal
    complex absorbed from the diet) could conceivably do in the body
    that are not limited by slow ligand exchange.*

Max Costa provided balance to the program by covering two examples of
known metal toxicity, due to particulate nickel sulfides and high
valence chromium. Both are carcinogenic, but by different mechanisms.
Crystalline NiS and Ni~2~S~2~ particles with negative surface charges
are phagocytosed by cells and degraded in low pH vacuoles to release
soluble Ni ions into the cell at very high (\>0.25 M) concentrations.
Soluble nickel compounds, which cannot achieve such high concentrations,
are not very carcinogenic. The carcinogenic effect of nickel appears to
be due to the silencing of tumor suppressor genes by inducing DNA
methylation and inhibiting histone acetylation. Methylation of DNA, once
altered, is propogated upon cell replication, resulting in effectively
permanent mutagenesis. Acetylation of histones is necessary for
unwinding the contacts between adjacent nucleosome particles to allow
transcription. Nickel binds to a peptide sequence in histones adjacent
to the site of acetylation, which may normally bind copper or zinc ions.
Failure to unwind may affect a large number of genes. Other effects may
also be involved, and their interrelationships are not yet clear.

*Biomarkers of nickel exposure include:*

1)  *Decreased degradation of key transcription factors, that are
    regulated by ubiquitin-mediated pathways (e.g., HIF-alpha, p53,
    ATF), resulting in downstream gene activation*

2)  *Activation of very specific signaling cascades Ca//NFAT, NFkB, but
    not RARE, GRE CRE, P53, AP1*

3)  *Cap43 induction by nickel through both Ca and HIF-alpha*

4)  *DNA hypermethylation and inactivation of genes*

High valent chromium compounds are man-made oxidants (e.g., CrO~3~^-2^),
and are toxic environmental pollutants. CrO~3~^-2^ is an oxyanion, and
is efficiently transported into cells and subcellular compartments by
carriers that normally transport HPO~4~^-2^ and SO~4~^-2^. Once
internalized, it can react with cellular reductants to generate radical
species and then undergoes reduction to Cr^+3.^. Cr^+3^ forms tight
adducts with proteins, amino acids, glutathione, and DNA. A UvrABC DNA
repair assay for DNA adducts and PCR mapping methods revealed hot spots
for DNA damage, e.g., in the p53 tumor suppressor gene. Sensitivity of
the adducts to EDTA suggests that they are due to metal binding.

*Controversy over the toxicity of chromates may relate to variable
susceptibility in the population. Improved biomarkers of exposure,
effect, and of individual susceptibility are needed.*

*Biomarkers of chromate (VI) exposure and effect include:*

1)  *Cr in red blood cells (exposure); Chromate is actively taken up by
    RBC dianion transporters.*

2)  *DNA-protein cross-links and EDTA removal tests (exposure and
    effect)*

3)  *UvrABC/mapping of Cr adducts with ligation-mediated PCR (exposure
    and effect)*

*4) Amino acid/GSH, Cr(III) DNA adducts (exposure and effect)*

*Biomarkers of Genetic Susceptibility to Cr(VI):*

1)  5-methyl cytosine content in DNA increases adduct formation in
    opposite G residues.

*2) Cellular uptake of Cr(VI) is variable (WBC uptake* in vitro *test
may be useful).*

2)  *Differences in reduction kinetics of Cr(VI) to Cr(III), which may
    be mediated in part by variations in cytochrome P450s*

Christopher Frederickson provided a brief presentation on zincergic
neuron signaling. This process is analogous to other neurotransmitter
mechanisms. Zinc is stored in pre-synaptic vesicles, released in brief
pulses into the synaptic cleft, and acts on recognition sites in the
post-synaptic membrane. Zinc-containing nerves are anatomically widely
distributed in the brain. In normal brain tissue, most zinc is localized
to vesicles in the nerve terminals as visualized using EM histology or
the TSQ fluorescent indicator. In certain pathologies, e.g., a rat
convulsive model, zinc is depleted from the pre-synaptic terminals,
enters the post-synaptic cells via Zn-channels, and is associated with
death of the post-synaptic cells. The hypothesis is that zinc
excitotoxic reactions, analogous to glutamate excitotoxic pathways may
be important, and might be amenable to prevention by appropriate zinc
chelators.

*This field stands where the field of calcium signal transduction stood
a decade ago. There is a need for additional tools to study localized
zinc concentrations and fluxes and to intervene in zinc signaling
pathways. The macromolecular players involved in zinc signal
transduction pathways remain to be determined. The role of such pathways
in tissues other than the brain remains to be determined. The potential
involvement of abnormal zinc signaling and accumulations in various
pathologies remains to be fully clarified.*

Jie Liu provided an example of the application of microarray technology
to studying the problem of arsenite toxicity. In this work, RNA
extracted from control and arsenite transformed/tolerant cells was
converted to cDNAs which were used to probe a gene array. Changes in
expression levels were confirmed by RT-PCR and Western blots. Expression
levels of more than 70 genes were observed to have changed. The
presentation highlighted only the few changes most relevant to arsinite
tolerance. Upregulated genes included GSTs and GSH-arsenite adduct
transporters (MRP1,2 and MDR).

*Microarray technologies have yet to be widely applied to problems in
metal metabolism and metal toxicity. They have much potential to help
sort out the multi-factorial mechanisms of metals in biological
systems.*

**Day 2\--Morning Discussion**

*Questions were raised about differences in the results obtained by
O\'Halloran showing the occurrence of zinc vesicles in non-neural cells
and the results of Frederickson, who has only observed them in neural
tissue. Differences in reagents and methods (Zinquin versus TSQ
fluorescent agents and the silverstain method used in EM studies), hence
differences in sensitivity, were noted. A concern in using chelators is
whether they reflect the localization of Zn* in situ *without perturbing
it (e.g., by artificially localizing it to vesicles that sequester the
chelator). Another issue is the integrity of the system studied. Do
cells in culture reflect the normal state of cells in tissues? Do sample
preparation methods affect the results (e.g., Frederickson sees Zn in
endosomes in cells that have been damaged)? Permeabilization of cells
that may be required for some* in vitro *studies may alter cellular zinc
compartmentation. In situ electron beam x-ray microanalysis might be a
useful tool for some vesicle sizes.*

*The idea that proteins may catalyze copper transfer rates brought up a
question about small molecule effectors of the process. Thus far, there
is no evidence of this. It was noted that catalysis of metal transfer
rates is not unique to protein bound metals, rather it is the norm in
ligand exchange chemistry.*

A discussion of chelation therapy developed around the tradeoff between
the thermodynamic advantage of larger multi-dentate molecules and the
problem of oral drug bioavailability. Preliminary results in rodent
models may not be sufficiently predictive of oral bioavailability in
humans. The average molecular weight of drugs currently on the market is
about 260 daltons. The sizes of leads in the pipeline is increasing to
an average of about 400 daltons. The potential of chelator co-therapy to
minimize toxic side effects was discussed. This is already done in the
case of some radiation therapies and in some cases with cisplatin to
protect non-target tissues and scavenge residual metals after the
planned treatment.

*The problem of identifying the true metal associated with the* in vivo
*functional form of enzymes versus metal exchanged into enzymes upon
purification was discussed. This may be a particular problem in
overexpression systems. How proteins insert the correct metal (when and
where in the cell) is incompletly understood. In some cases, this can
clearly be post-translational, in others it may be co-translational. In
the case of heme proteins, metal insertion into protoporphyrins occurs
in the mitochondria, and insertion of the heme unit into protein occurs
post-translationally.*

*A continued discussion on the potential benefits of chromium
supplementation suggests that the basic science is not yet available to
make clear determinations. There have not yet been definitive studies
establishing dose/response and mechanisms of action. A large single
study was suggested for consideration.*

**Day 2\--Afternoon Session**

##### Session 5: Metallotherapeutics and Disease

Chris Orvig discussed the therapeutic potential of vanadium compounds
for diabetes. Such compounds may be useful adjuvants in Type I and
useful treatments in Type II diabetes. They are orally active, but not
effective in the complete absence of insulin. A vast literature of *in
vitro* insulin-like effects has been reported for vanadate ion
(VO~4~^-3^). Initial observations on the activity of sodium vanadate *in
vivo* were published in 1985. Other oxidation states (e.g.,
peroxyvanadates and vanadyl (VO^+2^) compounds), have also shown
activity. The Orvig lab has been pursuing bis(maltolato)-oxo-vanadium
(BMOV), which is a chelated form of the vanadyl ion. It is neutral,
water soluble, and small (mw = 317), all of which favor bioavailability.
It is easy to prepare and is based on maltol, which is an FDA-approved
food additive. It was shown to provide rapid and sustained correction of
hyperglycemia in the STZ rat model of diabetes. This effect was due to
enhanced insulin responsiveness, rather than altered insulin levels. It
was 2-3 times more potent that vanadyl sulfate, a difference that
correlates with the demonstrated higher bioavailability of BMOV using
^48^V tracers. Although oxidative metabolites are formed, activity
resides with the parent compound. BMOV appears to act mainly as a
vanadyl delivery vehicle since dissociation occurs in the plasma. EPR
studies demonstrate transfer of VO^+2^ to serum transferrin can occur.
The ethyl-maltol derived analog of BMOV is currently in Phase I clinical
trials. BMOV has been marketed as a dietary supplement under the trade
names \"Supervanadyl Fuel\" and \"Gluconase\".

*The mechanism of vanadium compound action in diabetes is not yet clear.
Vanadate is known to be a phosphate mimic. Vanadyl is able to bind to
various divalent metal binding sites. Vanadium compounds may alter the
redox poise responsiveness of the insulin receptor kinase and may
inhibit the receptor phosphatase. Vanadium is a trace element in the
diet and as in the case of many trace elements, it is difficult to
establish the range of optimum physiological response. Important
questions remain about speciation, oxidation state, ligation,
bioavailability, and how these are altered by ligand designs. The
existence of the dietary supplement industry has resulted in marketing
of vanadyl compounds (including BMOV) in the absence of the studies
needed to establish them as drugs.*

*Orvig pointed out that the actions of metals in most cases means the
action metal complexes and the real research has to do with ligand
design. This requires good synthetic organic chemistry as well as an
understanding of coordination chemistry. Design issues include:
stability (thermodynamic and kinetic, and particularly, hydrolytic),
molecular weight, charge, lipophilicity, water solubility,
functionalization for targeting, and metabolism. Naturally occurring
ligands and previously approved materials such as maltol can simplify
the regulatory process somewhat.*

Dennis Riley discussed the design chemistry involved in development of
the Metaphore superoxide dismutase mimics. Superoxide anion and hydrogen
superoxide (pKa ≈ 5) are implicated in a very large number of
pathological processes. Three types of superoxide dismutases are known,
containing Cu, Fe, or Mn as the redox-active center. These same metals
are active in model complexes. Mn compounds were selected for
development because the toxicity of Mn aquo complexes is lower than that
of Fe or Cu aquo complexes. Mn is an essential trace element, and the
body has mechanisms for its regulation and elimination. A
penta-aza-crown compound, identified as an early lead, showed activity
20% of the enzymatic rates. Design objectives were to increase stability
of the complexes, yet also increase catalytic activity. This was
accomplished through understanding the mechanism of reaction and
optimizing the geometry of the complexes. The rate limiting step in the
Mn catalyzed superoxide dismutase reaction is oxidation of Mn^+2^ to
Mn^+3^. The reductive half-reaction is facile and need not be specific
to superoxide to be useful. Complex stability was increased by
increasing the rigidity and pre-organization of the free ligand toward
its metal-bound geometry. Catalytic activity was optimized by minimizing
the structural reorganization upon changes in Mn oxidation state and
enhancing the rate of proton-coupled outer sphere electron transfer from
Mn bound H~2~O to HO~2~• to yield H~2~O~2~. The present generation of
mimics are stable and active in vivo. Efficacy has been demonstrated in
a rat ischemia/reperfusion model. Data on additional indications for the
Metaphore SOD mimics was presented in a poster by Daniela Salvemini.

*This work illustrated the application of molecular mechanics to ligand
design and the application of basic principles in inorganic chemistry to
generating stable, yet catalytically active metallotherapeutics. Other
factors considered in the discussion included reactivity with species
other than superoxide, and effects of design on overall mid-point
potentials.*

Susan Doctrow made a short presentation (actually later in the program)
on Mn Salen complexes that are being developed by Eukarion, Inc, as SOD
and catalase mimics. These have shown activity in cellular and rodent
models for stroke, myocardial infarction, inflammatory diseases, and
neurodegenerative diseases. Experimental results were reported for
studies in Mn-SOD knock-out and the human FALS mutant Cu,Zn-SOD mouse
models. In both cases, improvements in lifespan, markers of oxidative
stress, and measures of neurodegeneration were observed.

Craig Hill presented data on the potential of polyoxometallate complexes
as broad spectrum antiviral agents. These are nanoscale assemblies of
early transition metals (e.g., vanadium, tungsten, molybdenum) with
oxygen to form a variety of cage-like structures. Single or multiple
sites of heterometal atom substitution may be entrapped or exposed on
selected surfaces and vertices. Potential advantages are low cost of
synthesis, self-assembly, and highly versatile combinatorial approaches,
including variations in elemental composition, redox potentials,
acidities, solubilities, shape, size, charge, and catalytic activity.
Approximately 300 have been screened and about 80% are active in cell
culture models with some fairly potent examples against HIV and CMV.
Unfortunately, the only clinical trial to date was conducted with a
material that, in hind-sight, is one of the most toxic members of the
class. This may have thrown an early damper on the entire area. Studies
of HIV protease inhibition have revealed compounds with K~i~ ≈ 1 nM and
EC~50~ in cells of about 1 μM. Furthermore, they appear to be active by
different mechanisms than the current HIV protease inhibitors and thus
might be active against resistant strains. Molecular modeling for a
K^+^~7~(P~2~NbW~17~O~62~)^-7^ salt suggests binding of the oxyanion to a
cationic pocket on the outer surface of the protein in the hinge region
of the active site flaps, rather than binding within the active site.

*Opportunities and obstacles for the future:*

1)  Synthesis, purification, and characterization of these highly varied
    species present a chemical challenge.

2)  *Clinical tests of an early lead may have been premature.*

3)  *The role of ion-pairing with these polyanions* in vivo *is not
    understood.*

4)  *Additional pharmacokinetic data is needed. How stable are they? How
    are they handled in the body? Does the presence of metals pose an
    insurmountable public relations obstacle?*

5)  *So many of the compounds are active in cell cultures that such
    results provide limited guidance for further development. Additional
    studies in animal models are needed.*

Jill Johnson provided an overview of the NCI Developmental Therapeutics
Program and their experience screening metal-containing agents for both
cancer and AIDS therapy. The program, which began in 1955, has gone
through several evolutions. Until recently an \"empirical drug
discovery\" paradigm has been used based on screening for
anti-proliferative effects in animal models (1975-1989) or a 60
cell-line screen (1990-present) with implanted hollow-fiber
(1995-present) and human xenograft mouse models (1990-present) as the
secondary screens. During this time 14,900 metal-containing compounds
have been tested (including a wide range of elements). Of those, 1,242
were selected as active in the mouse *in vivo* screen, 191 were selected
in the 60 cell-line screen. Nine were investigated as clinical
candidates. Five resulted in INDs. Two resulted in NDAs and are
currently used mainstream therapies (cisplatin and carboplatin). Reasons
for dropping clinical candidates included: i) stability, formulation, or
other pharmaceutical development issues; ii) renal toxicity or
neurotoxicity; and iii) insufficient advantage over current drugs. For
comparison, of the 550,000 total compounds tested by the DTP, 14,475
were active in mouse screen models and 7,741 (out of 77,000) were active
in the 60 cell-line assay; 59 INDs and 11 NDAs resulted. Since 1997, the
NCI has also operated a screen for compounds active against the
cytotoxic effects of HIV in CEM cells. Of 80,000 compounds tested, 4,050
(or about 5% were active). Of the compounds tested, 2,291 have included
metals. Of those, 136 (about 6%) were active, two became clinical
candidates. Both were dropped due to toxicity problems. The one was a
polyoxometallate, and about 80 others were tested. These were found to
be strongly active *in vitro*, but too toxic in animal models *in vivo*.
If a way around the toxicity problem can be found, interest in these
compounds would be very high. However, these obstacles in drug
development are not unique to metal-containing agents. The frequencies
of success in screens and also of failure in clinical development are
not different than the experience with development of non-metal
containing agents.

The model for drug development within the NCI program has changed to
focus on mechanism-oriented rational drug discovery based on known
molecular targets. Comparisons of compound activity against the 60
cell-line screen have developed correlations between cellular
sensitivity and molecular target expression (e.g., using the COMPARE
algorithm). Microarray expression and specific target activity assay
programs to characterize the levels of 300-400 targets in the 60
cell-line screen are underway. A yeast assay screen against a large
number of mutants has been developed to further help identify targets of
compound action. Interestingly, metals as a whole, and even the group of
platinum compounds tested, do not cluster well in these studies. This
implies that different metals act through different mechanisms of
action. In the new drug development model, it is not sufficient to find
anti-proliferative activity and test additional chemically related
compounds. It is necessary to demonstrate and optimize activity against
a specific target. Furthermore, DNA targeting, *per se*, is no longer
considered a sufficiently good rationale. A more specific cellular
effect of DNA modifying agents must be demonstrated. These same
constraints, however, operate equally in the selection of non-metal
containing candidates. The rapid access to intervention development
(RAID) program has been set up to assist investigators in moving their
leads to an IND. Information on this program, data on the library of
tested compounds, and the COMPARE program are available at:
[http://dtp.nci.nih.gov](http://dtp.nci.nih.gov/).

*Take home messages:*

1)  *A metal is not a metal is not a metal.*

2)  *Metals fared about as well as non-metals.*

3)  *Metals remain viable candidates for testing and development;
    however, the rationale for considering all compounds has become more
    stringent.*

4)  *Future developments must be based on rational targeted design.*

5)  *The greatest need is for improved ability to predict human activity
    from cell culture and animal studies.*

Nicholas Farrell provided a summary of some general principles (see
below) and specific examples from his own work on platinum anticancer
drugs. Most platinum compounds that have been tested are derivatives of
cisplatin. Although they may show altered pharmacokinetics, they are
likely to act in the same way. With the intent to develop compounds with
new activities, the Farrell lab has developed compounds designed to form
different types of cross-links (e.g., long range inter- and intra-strand
cross links) by combining two mono-chloro Pt-based DNA binding groups
with a diamine linker. Variations have examined leaving group, linker
chemistry, chain length, steric effects, H-bonding, and charge. The most
successful compound (BBR3464) contains an non-reactive
tetra-amino-platinum unit in the linker, but this can be replaced with
other positively charged units. This compound is active at nM
concentrations *in vitro*, positive in animal and human tumor xenograft
screens (against 15 types of tumor), and showed an acceptable
pre-clinical toxicology profile. Phase I human trials were completed and
Phase II trials are underway. Conclusions from Phase I included an
acceptable maximum tolerated dose limit, an altered dose-limiting
toxicity in comparison with cisplatin (diarrhea and myelosuppression as
opposed to renal toxicity), linear pharmacokinetics with a long t~1/2~,
and preliminary indications of efficacy in GI malignancy (colon cancer,
pancreatic cancer, and associated hepatic metastases). Studies on the
mechanism of action showed formation of the predicted long-range
cross-links, which are not recognized by HMG1 (which recognizes
cisplatin cross-links). Tissue selectivity and downstream signaling
effects are also different than for cisplatin.

Another effort has been directed toward examining trans-platinum
derivatives. Although earlier studies had shown these are less active
than cis-platinium derivatives, this is partly because they are too
reactive. By using planar amine ligands to slow down the reactions,
potency was increased. The types of cross-links formed and the mechanism
of action differ. In these compounds, protein-associated strand breaks
were found that are similar to those produced by DNA topoisomerase
inhibitors such as camptothecin.

Application of the COMPARE program to the NCI data on platinum complexes
reveals several well separated clusters with different spectra of
activity against the 60 cell lines. These results clearly show that a
monolithic model of platinum compound action is inadequate, and that
multiple mechanisms of action are more likely.

*Although the collection of presentations at the Metals in Medicine
meeting might be considered eclectic, they actually represent an
emerging and cohesive field. The talks span a continuum of interests
from essential elements, in both normal and disease processes, to
non-essential elements and the potential beneficial as well as
detrimental effects thereof. They are joined by the necessity of
bringing both a deep understanding of inorganic chemistry and
state-of-the-art research in biology to bear on the problems. They are
further characterized by a focus on basic research, but with a clear
vision of potential future medical application.*

*Medicinal Inorganic Chemistry is a multidisciplinary field combining
elements of*

*chemistry (synthesis, reactivity); pharmacology (pharmacokinetics,
toxicology); biochemistry (targets, structure, conformational changes);
and medicinal chemistry (therapeutics, pharmacodynamics, SAR).*

*Medicinal inorganics have an appreciable current market impact and
significant growth potential. Current sales, including imaging,
diagnostics, and therapeutics are on the order of \$2 billion per year.
Platinum-based chemotherapeutics are among the most successful of all
anti-cancer drugs with combined sales of \$700 million per year, second
only to Taxol. New compounds are likely to gain a unique market niche by
acting through different mechanisms and/or avoiding common clearance
mechanisms (e.g., MDR). Successful or at least promising results have
been achieved for various indications involving a significant number of
inorganic elements from the periodic table. Additional exploration
should continue to be fruitful. The success rate of
metallopharmaceutical advanced clinical leads is about the going rate
(e.g., 1:10 survive Phase I trials and 1:4 survive in Phase II).
However, a limiting factor may be the relatively limited expertise of
most pharmaceutical companies in the area of inorganic chemistry.*

*Drug development is a long and non-linear process. It would be
incorrect to say major pharmaceutical companies have no interest.
AstraZeneca, Warner-Lambert, Boehringer Manheim, Hoffman LaRoche,
NovusPharma, Johson Matthey, Bristol-Myers Squibb, Dupont, Merck, and
others were mentioned during the meeting as having collaborated or
contributed support at various points along the way. However, a
promising research lead, alone, does not a drug make. Issues of
pharmacokinetics, efficacy, toxicity, and economics, along with patents,
politics, and personalities, all factor into decisions about whether to
continue or to drop a project. There is at least a perception that the
bar is set higher for metal-containing drugs. The government-based
development programs may need to reach further to off-set industrial
caution in this area.*

David Place of the FDA provided a discussion of regulatory issues for
metallopharmaceuticals. The key point, however, is that there is really
no difference in the process or in the expectations for demonstration of
safety and efficacy that must be met. Phase 1 must establish safety.
Phase 2 must establish safety and efficacy in a defined population.
Phase 3 should provide definitive evidence of efficacy and safety. Phase
4 studies may be needed to resolve some post-approval questions. Several
Centers and Divisions within FDA have responsibility for regulation of
metallopharmaceuticals. The largest number have come to the Division of
Medical Imaging and Radiopharmaceutical Drug Products within the Center
for Drug Evaluation and Research.

*Important issues with respect to metallopharmaceuticals are chemical
characterization (e.g., complexes versus mixtures) and stability. New
Molecular Entities must be fully characterized. This designation affects
the number of years of exclusivity that may be received. Applicable
analytical and spectroscopic methods must be available and appropriate
animal models must be used for the pre-clinical pharm/tox studies. The
mode of physiological/pharmacological action is important. Which part is
the active unit (i.e., intact complex, part of ligand, the metal ion,
itself) should be established. Knowledge of contaminants and their
activity profiles is also important. Early planning is valuable, and
interactions with FDA staff is encouraged at multiple stages in the
development process. Inorganic chemistry training of FDA staff and
reviewers is variable and it may be useful to provide additional
material to help educate them about issues, such as \"exotic\"
analytical methods which may be unfamiliar in a system where the
majority of materials under consideration are organics.*

Mike Abrams presented results on the anti-HIV activity of bicyclams and
metallo-bicyclams. These materials were identified by a program of
screening metals and metal chelators. Bicyclam was identified as the
active 1% contaminant present in samples of cyclam and was shown to have
nanomolar activity against HIV with low cytotoxicity. The current
AnorMED lead compound AMD3100 (a para-substituted phenyl bridged
bicyclam) was shown to have a novel mechanism of action. It inhibits HIV
replication by binding to the chemokine receptor CXCR4, which is used as
a co-receptor by the virus for membrane fusion and cell entry in its
T-tropic stage. Binding of AMD3100 inhibits binding of an anti-CXCR4 mAB
and of the endogenous ligand SDF-1α. Zinc ions were shown to enhance the
binding of AMD3100 to the receptor and several other metal ion complexes
bind with affinity similar to AMD3100. Modeling and mutagenesis suggest
binding to the 7TM receptor at two sites involving asparate residue
stabilization of positive charges. Phase I trials were completed in
healthy volunteers in 1999. Phase II trials are in progress in HIV
patients.

*Discussion brought out the problem of knowing whether a metal chelator,
when tested in a metal-free form actually acts by picking up a metal* in
vitro *and* in vivo*. Is the activity of AMD3100, alone, due to that
species, or a metal chelate that may well form by picking up metals in
the buffer? It was pointed out that perhaps 40 percent of the drugs on
the market are potential metal chelators. Whether their pharmacokinetics
and/or pharmacodynamics may be affected by metal chelation in the body
is largely unknown. The availability of Zn, Fe, Cu in the cell may be
exploited in drug design by engineering specific chelating scaffolds. Zn
is known to inhibit some enzymes, such as serine proteases, which
contain suitable coordination sites. It may be possible to design
ligands that in conjunction with target protein residues create a zinc
binding inhibitor site.*

*Dr. Abrams offered the following general comments:*

*Reasons why major pharmaceutical companies have shown only modest
interest in the field include:*

1)  *A high degree of serendipity versus target-directed design in the
    past*

2)  *Concerns about metal accumulation versus long-term therapeutic
    requirements*

3)  *Perceptions of toxicity*

4)  *Concern about inadequate specificity*

5)  *Concern about reversibility (or lack thereof)*

6)  *Unfamiliarity\--few coordination chemists are employed in industry*

*Ways to make inorganic medicines more \"drug-like\" and of greater
interest include:*

1)  *Take advantage of the enormous growth in knowledge of drug targets
    to find unique applications.*

2)  *Make use of catalytic mechanisms that cannot be duplicated by other
    agents.*

3)  *Look for reversible competitive inhibitors of enzymes and ligands
    for receptors that take advantage of the unique properties of metal
    ions.*

4)  *Take advantage of opportunities to recruit endogenous metals to
    avoid having to administer metal-containing agents.*

**Day 2\--Afternoon Discussion and Overall Meeting Discussion**

*Various speakers identified ways that basic research in
metallobiochemistry and bioinorganic chemistry can contribute to the
development of future pharmaceuticals and the health benefits therein.
Studies of metalloenzyme structure and function, and of the roles of
metalloenzymes in biological systems, are a vigorous area of ongoing
activity. Emerging areas of interest include the roles of metals in cell
regulation and the pathways by which cells regulate metal ion
concentrations and delivery to selected metal sites. Opportunities exist
to target these processes to alter metalloenzyme synthesis and to alter
metal ion accumulations. Opportunities exist to exploit the unique
properties of metal containing compounds as pharmaceuticals. These
include unique physical properties, unique types of biomolecule
interactions, redox, and other reactivities.*

*A limiting factor in the development of metallopharmaceuticals is the
perception that metals are toxic. This is certainly true of some metals.
Even essential metals are toxic under some circumstances. However,
metals are not necessarily toxic under all circumstances. All metals are
clearly not heavy metals. To rule out a large fraction of the periodic
table,* a priori*, eliminates any potential benefit that may be
discovered among those elements. Understanding and learning to control
the actions of metals* in vivo *is the key to potential beneficial
applications of metals, and the key to avoiding metal toxicities.*

*An objective of the meeting was to build bridges between research
communities and between researchers in academica, industry, and
government. The meeting was at least partially successful in bringing
these groups together. NIGMS staff used this opportunity to point out
several research grant mechanisms that may be useful to foster the
collaborative research needed in this area.*

*See the NIGMS Web site under funding for information on:*

*1) Glue Grants: <http://www.nigms.nih.gov/funding/gluegrants.html>*

*2) Program Projects:
<http://www.nigms.nih.gov/funding/pa/progproj.html>*

*It was pointed out that work in this area requires review by both
chemists and biologists, and that both types of expertise were not often
present on the same panel. CSR staff pointed out that NIH review
activities are primarily proposal-driven. If increased numbers of
applications are received in this area, potential changes in the
distribution of applications to various study sections and the
memberships of the study sections will be adjusted to accommodate the
workload.*

*The report of this meeting will be considered by the National Advisory
General Medical Sciences Council at its meeting on September 13-15,
2000. Information is posted on the NIGMS Web site:*
<http://pub.nigms.nih.gov/council/>*.*

Report prepared by Peter C. Preusch, Ph.D., PPBC Division, NIGMS, NIH.

August 7, 2000.