Metals in Medicine: Targets, Diagnostics, and Therapeutics icon

Metals in Medicine: Targets, Diagnostics, and Therapeutics

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Metals in Medicine: Targets, Diagnostics, and Therapeutics

June 28 & 29, 2000

Natcher Conference Center

National Institutes of Health

Bethesda, Maryland, U.S.A.


Introduction 3

Final Agenda 5

List of Speakers 9

List of Posters 11

Speaker Abstracts 17

Speaker Biosketches 45

Poster Abstracts 69

Additional Biosketches 103

List of Registrants 151


National Institute of General Medical Sciences

Center for Scientific Review, NIH

National Cancer Institute

National Institute of Diabetes and Digestive and Kidney Diseases

National Institute of Allergy and Infections Diseases

National Institute of Environmental Health Sciences

Office of Dietary Supplements, Office of the Director, NIH

^ Meeting Website:

Meeting Contact: Dr. Peter C. Preusch, NIGMS. E-mail:

Cover Art: Paul Ehrlich (1854-1915), a founding figure in medicinal chemistry and developer of Salvarsan (arsphenamine), one of the first effective chemotherapeutics -- an inorganic agent used in the treatment of syphilis. Structure of HMG1 domain A bound to cisplatin modified DNA, solved in the laboratory of Stephen J. Lippard.

^ Metals in Medicine: Targets, Diagnostics, and Therapeutics

INTRODUCTION: Why Metals, Why Here, Why Now?

The National Institute of General Medical Sciences and other components of the National Institutes of Health have provided significant support for research in the areas of bioinorganic chemistry and metallobiochemistry. Much of the proposed research in this area has been reviewed by the Metallobiochemistry study section of the Center for Scientific Review. It is important to insure that this basic research investment is translated into health benefits. A focused meeting on the NIH campus was conceived as a way to emphasize NIH interest in the area. Discussions with leaders in the field suggested that opportunities exist to increase the impact of the NIH investment in this area through additional research and enhanced participation of bioinorganic chemists in the pharmaceutical development process.

Recent advances have occurred in understanding the roles of metals in cell regulation, the complexities of metal metabolism, and the mechanisms of metalloenzymes. Metalloenzymes are well recognized as targets for conventional drug development. Advances have occurred in the ability to characterize these proteins and to model metal/ligand interactions in drug design. Advances in understanding metal trafficking and the roles of metals in disease have suggested targeting metals, themselves, as a useful therapeutic strategy. The success of cisplatin and its congeners suggests that metals can also be developed as therapeutic agents. The ability to synthesize sophisticated metal complexes has become well established. Advances have occurred in understanding and controlling the reactivity of metals in vitro and in vivo. Experience in the development of imaging and radiation therapy agents demonstrates that metal complexes can be tailored to provide clinically useful pharmacokinetic properties. Methods have been developed that allow metal complexes to target selected cellular and molecular processes.

The objectives of the meeting are to:

  • Present state-of-the-art research on metal-containing systems of medical importance;

  • Identify emerging areas of research opportunity;

  • Increase communication between scientists in academia, government, and industry;

  • Foster the formation of new collaborative research efforts in the field.

The scientific program is intended to examine the current role of bioinorganic chemistry in the pharmaceutical industry; examine opportunities for the development of inorganic complexes as therapeutic agents and diagnostics; and examine opportunities for the development of drugs that manipulate endogenous metal metabolism or otherwise exploit metal-containing targets. Issues in drug development, such as specificity, stability, and toxicity will be explored. The principles of medicinal chemistry applicable to the development and regulation of organic agents have been well developed. It is hoped that a statement of the principles of medicinal chemistry relevant to metals will emerge from this meeting, or at least, a statement of the research needed to elucidate those principles.

The meeting program is currently available interactively through the Metals in Medicine meeting Web site: This site will be maintained for at least several weeks after the meeting. A printable archive version of this meeting booklet in both Word and Adobe Acrobat formats will be available through the NIGMS Web site for the indefinite future. See: A report of this meeting will become available on the NIGMS site shortly after the meeting. Visit the NIGMS site for postings of additional information of interest as the year progresses. The meeting is being videocast live to the internet and the NIH local area network (MBONE) via: The videocast will be available as an archive rebroadcast within a few weeks of the meeting.

Meeting organized by NIGMS staff. Contact: Peter C. Preusch, Ph.D., Pharmacology, Physiology, and Biological Chemistry Division, National Institute of General Medical Sciences, NIH, 45 Center Drive, MSC 6200, Bethesda, Maryland, USA, 20892-6200. Phone: 301-594-5938. Fax: 301-480-2802. Email:

Input from other sponsoring NIH components, and suggestions from a substantial number of the meeting participants and other active researchers in the field, are gratefully acknowledged. The contributions of the discussion chairpersons, Stephen J. Lippard, John Kozarich, Thomas Meade, Thomas V. O'Halloran, and Nicholas P. Farrell, are particularly acknowledged.



Targets, Diagnostics, and Therapeutics

DAY 1 - June 28, 2000

7:30 Registration

8:00 Introduction: Peter Preusch, Marvin Cassman

(National Institute of General Medical Sciences, NIH)

8:15 ^ Keynote Address: Case History and Recent Advances in Understanding Cisplatin

Stephen Lippard (Massachusetts Institute of Technology)

Session 1: Molecular and Cellular Targets of Metal Action

9:00 Metal-Catalyzed Cleavage of Nucleic Acids

Cynthia Burrows (University of Utah)

9:30 Selective Inhibition of Human -Thrombin by Co(III) Schiff Base Complexes

Thomas Meade (California Institute of Technology)

10:00 Coffee Break

10:30 Metal Complexes and Drug Transport

David Piwnica-Worms (Washington University)

11:00 Metal Complexes as Receptor Ligands

Shubh Sharma (Palatin Technologies, Inc.)

11:30 Session Discussion - Stephen Lippard (Discussion Leader)

12:00 Lunch Break

^ Session 2: Metal-Containing Targets of Drug Action

1:00 Metalloenzyme Drug Development Targets at Merck

John Kozarich (Merck & Company)

1:30 Nitric Oxide Synthase

Thomas Poulos (University of California-Irvine)

2:00 Molecular Modeling Of Metalloenzymes: Current Status And Future Prospects

Kenneth Merz (Pennsylvania State University)

2:30 Modeling Matrix Metalloproteases: A Case Study

Benjamin Burke (Agouron Pharmaceuticals, Inc.)

3:00 Coffee Break

^ Session 3: Radiology, Imaging, and Photodynamic Therapy

3:30 Fundamentals of Receptor-Based Metalloradiopharmaceuticals

Shuang Liu (DuPont Pharmaceuticals Co.)

4:00 Radiolabeled Somatostatin Analogs for Targeted Radiotherapy of Cancer

Carolyn Anderson (Washington University)

4:30 Biophysical Tuning and Targeting of Gadolinium(III) Chelates for MRI

Randall Lauffer (EPIX Medical, Inc.)

5:00 Metallotexaphyrins: New Drugs with Diverse Applications in Cancer Treatment

Richard Miller (Pharmacyclics, Inc.)

5:30 Discussion Session - John Kozarich, Thomas Meade (Discussion Leaders)

6:00 Poster Session and Evening Reception

7:30 Meeting Adjourned for the Day

DAY 2 - June 29, 2000

Session 4: Metal Metabolism

8:30 Intracellular Trafficking Pathways: Inorganic Cell Biology of Copper and Zinc

Thomas O'Halloran (Northwestern University)

9:00 Combinatorial RNA Regulation Of Bio-Iron And New Drug Targets

Elizabeth Theil (Children's Hospital of Oakland Research Institute)

9:30 Metal Chelation Therapy

Ken Raymond (University of California-Berkeley)

10:00 Coffee Break

10:30 Chromium as a Dietary Supplement

Richard Anderson (U.S. Department of Agriculture)

11:00 Mechanisms of Metal Toxicity

Max Costa (New York University)

11:30 Session Discussion - Thomas O'Halloran (Discussion Leader)

12:00 Lunch Break

^ Session 5: Metallotherapeutics and Disease

1:00 Vanadium and Diabetes

Christopher Orvig (University of British Columbia)

1:30 Computer-Aided Design Of Selective Synthetic Superoxide Dismutase

Enzyme Mimetics

Dennis Riley (Metaphore Pharmaceuticals, Inc.)

2:00 Polyoxometalate Nanocluster HIV-1 Protease Inhibitors: A New Mode of

Protease Inhibition

Craig Hill (Emory University)

2:30 NCI Developmental Therapeutics Program Experiences

Jill Johnson (National Cancer Institute, NIH)

3:00 Coffee Break

^ Session 6: Medicinal Chemistry of Metallopharmaceuticals

3:30 Principles of Medicinal Chemistry for Inorganics

Nicholas Farrell (Virginia Commonwealth University)

4:00 The US Drug Approval Process - Challenges for Metallopharmaceuticals

David Place (Food and Drug Administration)

4:30 The Bicyclam Story: From Metal Complexes to Ligands and Back to Metal


Michael Abrams (AnorMED, Inc.)

5:00 Discussion Session & Closing Comments - Nicholas Farrel (Discussion Leader)

Peter Preusch & Michael Rogers (National Institute of General Medical Sciences)

5:30 Meeting Adjourned

^ Metals in Medicine: Targets, Diagnostics, and Therapeutics

LIST of SPEAKERS (Alphabetically)

Michael Abrams (AnorMED, Inc.)

Carolyn Anderson (Washington University)

Richard Anderson (United States Department of Agriculture)

Benjamin Burke (Agouron Pharmaceuticals, Inc.)

Cynthia Burrows (University of Utah)

Max Costa (New York University)

Nicholas Farrell (Virgina Commonwealth University)

Craig Hill (Emory University)

Jill Johnson (National Cancer Institute)

John Kozarich (Merck Research Labs)

Randall Lauffer (EPIX Medical, Inc.)

Stephen Lippard (Massachusetts Institute of Technology)

Shuang Liu (DuPont Pharmaceuticals Company)

Thomas Meade (California Institute of Technology)

Kenneth Merz (Pennsylvania State University)

Richard Miller (Pharmacyclics, Inc.)

Thomas O'Halloran (Northwestern University)

Chrisopher Orvig (University of British Columbia)

David Piwnica-Worms (Washington University)

David Place (Food and Drug Administration)

Thomas Poulos (University of California-Irvine)

Kenneth Raymond (University of California-Berkeley)

Dennis Riley (Metaphore Pharmaceuticals, Inc.)

Shubh Sharma (Palatin Technologies, Inc.)

Elizabeth Theil (Children's Hospital of Oakland Research Institute)

Additional Speakers Participating in the Discussion Sessions:

Bruce Averill (University of Amsterdam)

Christopher Frederickson (University of Texas Medical Branch-Galveston)

Jie Liu (National Institute of Environmental Health Sciences)

Susan Doctrow (Eukarion, Inc.)

Janet Morrow (State University of New York at Buffalo)

David Petering (University of Wisconsin-Milwaukee)


Submitting Author and Affiliation

* Indicates Authors Sharing a Posterboard.

Indicates Poster and Short Presentation

By Related Meeting Session:

Session 1. Molecular and Cellular Targets of Metal Action

  1. Binding of HMG-domain Proteins in Purine-Rich Platinated DNA

Seth M. Cohen, Massachusetts Institute of Technology*

  1. Laser-Induced Photocross-linking of Cisplatin-Modified DNA to Damage Recognition Proteins

Yuji Mikata, Massachusetts Institute of Technology*

  1. Computational Analysis of All the Possible Configurational/Binding Geometries of Co(III) Bleomycins

Marc Zimmer, Connecticut College

  1. The Bioinorganic-Biomedical Interface of the Redox Active Antitumor Agent Iron Bleomycin

David H. Petering, University of Wisconsin-Milwaukee

  1. 'Fe2+ + O2' is More Important than the Fenton Reaction in Initiating Biological Radical Oxidation

Steven Y. Qian, National Institutes of Health

  1. Oxidative Cleavage Of DNA Substrates Mediated By Copper Complexes With Pyridyl(Alkyl)Amine Ligands

Kristi J. Humphreys, Johns Hopkins University

  1. A Combinatorial Approach to the Discovery of New Metal Complexes for Peptide Cleavage

Kathryn B. Grant, Georgia State University

  1. Design Of A Squence-Specific Photonuclease From A Synthetic bZIP Metalloprotein.

Robin C. Lasey, Bowling Green State University

  1. Inactivation of mRNA through Cleavage of the 5'-Cap Structure by Metal Ion Complexes and Oligonucleotide Conjugates

Janet R. Morrow, State University of New York at Buffalo

Session 2. Metal Containing-Targets of Drug Action

  1. Divalent Metal Binding and Mechanism of Action of the Methionine Aminopeptidases from Escherichia Coli and Pyrococcus furiosus

Richard C. Holz, Utah State University*

  1. Redox Regulation of Signal Transduction via Calcineurin

Frank Rusnak, Mayo Clinic & Foundation*

  1. Mycobacterium tuberculosis Catalase/Peroxidase KatG in Isoniazid Activation and Resistance

Nancy L. Wengenack, Mayo Clinic & Foundation*

  1. Towards Understanding the Heme Environment Required for Activation of Isoniazid by M. Tuberculosis KatG

Kenton R. Rogers, North Dakota State University*

  1. Proteolytic Activation of the Catalytic Activity of Purple Acid Phosphatases: A Possible Regulatory Mechanism and Potential Therapeutic Target for Treatment of Osteoporosis and Other Disorders

Bruce Averill, E.C. Slater Institute, University of Amsterdam

  1. Nature's Way to Make Methane

Stephen W. Ragsdale, University of Nebraska

  1. The Evolving Mechanism of Metallo-beta-lactamase

Walter Fast, Pennsylvania State University

  1. Structure and Chemistry of the Enzyme Active Site of Glyoxalase I

Uwe Richter, National Institute of Standards and Technology

  1. Engineering Type-1 Copper Centres In Redox Enzymes For Hot Wiring

Martin Ph. Verbeet, Leiden Institute of Chemistry

  1. Identifying And Designing Of Calcium-Binding Proteins

Jenny J. Yang, Georgia State University

  1. The Guanidine Moiety as a Ligand for Hemin

Dabney W. Dixon, Georgia State University*

  1. Nitric Oxide Dioxygenases: Efficient Catalysts for Nitric Oxide Metabolism in Microbes and Mammals

Paul R. Gardner, Children's Hospital Medical Center of Cincinnati*

Session 3. Imaging, Radiology, and Photodynamic Therapy

  1. Receptor Based Biosensor for Gallium Using Novel Therapeutic Immunoconjugate

Omowunmi A. Sadik, SUNY Binghamton

  1. Synthesis and Relaxometric Investigations of Starburst Dendrimer-DOTA-M (M=Gd(III), Mn(II), Fe(III))

L. Henry Bryant, Jr., National Institutes of Health

  1. Lung Cancer - From Radiodiagnosis to Radiotherapy with Novel Somatostatin Receptor-Binding Peptides

Rajeesh Manchanda, Diatide, Inc.

  1. MR Tracking of Cell Migration and Myelination Following Transplantation of Magtagged Glial Cells

Jeff W.M. Bulte, National Institutes of Health

  1. Seeing is Believing: Visualizing In Vivo Gene Expression and Secondary Messengers by Magnetic Resonance Imaging

Thomas J. Meade, California Institute of Technology

  1. The First Three-Dimensional Perfluorinated Phthalocyanine: Synthesis, Structure and Photodynamic Activity

Barbara A. Bench, Brown University

  1. Metalloenediynes for Thermal and Photochemical Control of Bergman Cyclization

Jeffrey M. Zaleski, Indiana University

Session 4. Metal Metabolism as a Therapeutic Target

  1. Synaptic Release of Free Zinc Ions in the Brain: Roles in Brain Function, Disease, and Injury

Christopher J. Frederickson, University of Texas Medical Branch-Galveston

  1. Fluorescent Peptidyl Platforms for Metal Ion Sensing in Aqueous Solution

Dierdre A. Pearce, Massachusetts Institute of Technology*

  1. The Shu S protein of Shigella dysenteriae is a Heme-Sequestering Protein that Binds and Protects DNA.

Angela Wilks, University of Maryland*

  1. Accelerating Iron Release from Transferrin

Wesley R. Harris, University of Missouri-St. Louis

  1. Fe(III) Coordination and Redox Properties of a Bacterial Transferrin

Alvin L. Crumbliss, Duke University

  1. Ferritin is a Nuclear Protein that Responds to Cell Stress and Protects DNA

Khristy Thompson, Pennsylvania State University at Hershey

  1. Investigation of Structure and Metal Binding Properties of Transferrins by Electropray Mass Spectrometry

Igor A. Kaltashov, University of Massachusetts

  1. Why is lead toxic? Unraveling the Molecular Mechanism(s) of Lead Poisoning.

Hilary A. Godwin, Northwestern Univeristy

  1. Lead-Filled Tetraplex: Solution and Solid State Studies of G-Quartets Bound to Pb+2

Jeffery T. Davis, University of Maryland

  1. Structural biology of the GMTCXXC consensus sequence in the first metal binding domain from the protein involved in Menkes disease, MerP, and synthetic peptides.

Tara M. DeSilva, University of Pennsylvania

  1. Wilson's Disease Protein: Ligand-Binding Properties and Copper-Dependent Domain-Domain Interactions

Svetlana Lutsenko, Oregon Health Sciences University

  1. Characterization of the Mouse Homologue of a Bacterial Arsenite Translocating ATPase

Hiranmoy Bhattacharjee, Wayne State University*

  1. Structure of the ArsA ATPase: the Catalytic Subunit of a Heavy Metal Resistance Pump

Tongqing Zhou, Wayne State University*

  1. Application of cDNA Microarray to the Study of Arsenic Toxicology and Carcinogenesis

Jie Liu, National Institute of Environmental Health Sciences

Session 5. Metallotherapeutics and Disease

  1. The Antiviral Activity of Titanyl (TiO+2) Sulfate Toward Human Cytomegalovirus

Chad W. Schwietert, San Francisco State University

  1. Tachpyridine, A Novel Iron Chelator and a Potential Anti-Cancer Agent in the Treatment of Tumors with Mutated p53

Suzy V. Torti, Wake Forest University

  1. Anti-inflammatory Properties of M40403, a Low Molecular Weight Synthetic Superoxide Dismutase Mime

Daniela Salvemini, MetaPhore Pharmaceuticals

  1. Salen Mn Complexes, Combined SOD/Catalase Mimics with Broad Pharmacological Efficacy, are Protective in Models for Neurodegeneration

Susan Doctrow, Eukarion Inc.

  1. Peroxynitrite Decomposition Catalyzed by Water-Soluble Iron Porphyrins.

Roman Shimanovich, Princeton University

  1. Development of Materials for Storage, Release, and Detection of Nitric Oxide.

Andrew S. Borovik, University of Kansas

  1. Chemistry and Insulin Like Properties of Cobalt 2,6-pyridinedicarboxylate Complexes

Debbie C. Crans, Colorado State University

  1. What Is The Active Vanadium Pool In NIDDM Patients Dosed With Vanadyl Sulfate?

Gail Willsky, State University of New York at Buffalo

  1. Vanadium and Insulinomimesis: A Possible Role of Phosphatidylinositol 3-Kinase

Ashok K. Srivastava, University of Montreal

Other Topics

  1. NIH Support for Bioinorganic Chemistry

Peter C. Preusch, National Institutes of Health*

  1. In the Beginning… The Evolutionary Origin of Aerobic Metabolism

G. Charles Dismukes, Princeton University*



Case History and Recent Advances in Understanding Cisplatin

Stephen J. Lippard, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA, 02139.

-Diamminedichloroplatinum(II), or cisplatin, is in widespread use for the treatment of genitourinary, head and neck, and a variety of other cancers. The discovery of the anticancer properties of cisplatin arose from a completely serendipitous finding during experiments designed to probe the effects of electric fields on cell division. The trans isomer (trans-DDP) is inactive. Following administration by intravenous injection cisplatin is activated in cells by aquation to form cationic species that bind to DNA. The major adducts are 1,2-intrastrand cross-links, also formed by carboplatin, which has fewer dose-limiting side effects. The structural distortion in DNA that accompanies platination affects replication and transcription. In particular, the minor groove across from the cross-link is wide and shallow, creating a hydrophobic notch and surface that are recognized by various proteins. Included are high-mobility group (HMG) domain proteins. HMG-domain proteins bind with specificity to the major cisplatin-DNA adducts, forming a stable platinum-DNA-protein ternary complex. The X-ray structure of HMG domain A in HMG1, bound to a 16-mer DNA duplex containing a site specific intrastrand cisplatin 1,2-d(GpG) cross-link, reveals intercalation of a phenylalanine side chain into the hydrophobic cleft created in the minor groove across from the platinum adduct. Binding of HMG-domain proteins in this manner shields Pt adducts from nucleotide excision repair. TATA binding protein (TBP) similarly recognizes intrastrand cisplatin 1,2-d(GpG) cross-links by intercalation of phenylalanine side chains. Both hSSRP1, the first protein identified to bind with specificity to cisplatin-DNA adducts, and TBP are required for transcription. Cisplatin disruption of transcription may be the key event in its anticancer mechanism. The natural functions of HMG1 include facilitating the binding of steroid hormone receptors, such as estrogen or progesterone receptor (ER or PR), to their cognate DNA binding sites. HMG1 levels in human cancer cells bearing the appropriate hormone receptor, ER or PR, increase upon estrogen or progesterone treatment. In human MCF-7 breast cancer cells, which have both ER and PR, HMG1 was elevated after estrogen and progesterone treatment. Evsa-T is a breast cancer cell line that has PR but not ER. Estrogen treatment had no effect on HMG1 levels in Evsa-T cells. Progesterone doubled the HMG1 protein level in Evsa-T cells. Elevated HMG1 expression levels parallel increased sensitivity towards cisplatin. In MCF-7 cells, estrogen or progesterone treatment increased cisplatin sensitivity about 2-fold, according to LC50 values. A combination of estrogen and progesterone increased cisplatin sensitivity by a factor of 4; this additive effect suggests that these two hormones up-regulate HMG1 independently. These findings form the basis for a clinical trial to commence this year at the Dana Farber Cancer Institute. This work was supported by the NCI.

^ Metal-Catalyzed Cleavage of Nucleic Acids

Cynthia J. Burrows, Ph.D., Department of Chemistry, University of Utah, Salt Lake City, Utah, USA, 84112-0850.

The sugar, the phosphate and the base moieties constitute the three chemical targets for metal-mediated reactions leading to scission of a DNA or RNA strand. Certain metal complexes, particularly those of lanthanides, can act as Lewis acids to facilitate phosphodiester hydrolysis; this occurs most readily for RNA in which the 2'-OH greatly enhances hydrolysis via intramolecular transesterification. Catalysts for efficient DNA hydrolysis have been a more difficult target, but some success in now seen in this area. In contrast, there are now a large number of redox-active transition metal complexes, particularly those capable of forming free or metal-bound electron-deficient oxygen species (metal-oxo, HO•, O2– •, etc.) that trigger DNA and RNA strand scission following hydrogen atom abstraction from the ribose moiety. Although metal complexes targeting phosphates or sugars typically have little intrinsic sequence or structure selectivity because all phosphates and all riboses roughly look alike, clever ligand design has led to cleavage agents that act upon specific primary, secondary or tertiary structures of nucleic acids. The third target–the heterocyclic bases–will form the major focus of this presentation. Guanine in particular presents a wealth of sites for metal binding and oxidation or alkylation. In principle, any metal complex that is sufficiently oxidizing to attack hydrogen atoms of the ribose moiety is also capable of abstracting an electron from G. Base oxidation does not directly yield strand scission, but rather creates a site that is sensitive to cleavage using either chemical reagents (such as HO or piperidine) or enzymes, principally those involved in DNA repair. Our laboratory has investigated a series of nickel(II) complexes that operate almost exclusively by one-electron oxidation of Gs. Ligand tuning provides complexes capable of reacting with O2 in the presence of a sacrificial reductant (usually sulfite) to generate Ni(III) species leading to oxidative damage at G. With redox-active ligands, the role of Ni(III) is to trigger formation of ligand radicals capable of generating covalent adducts.

Applications of nickel complexes to biomedical research include their use as (i) structural probes of DNA and RNA folding, (ii) sequence-specific cleavage agents when tethered to DNA recognition agents, (iii) site-specific tags of DNA or RNA allowing conjugation of fluorescein, biotin or other molecular probes, and (iv) models of DNA-based metal toxicity.

Leading references:

1. C. J. Burrows and J. G. Muller, “Oxidative Nucleobase Modifications Leading to Strand Scission,” Chem. Rev. 1998, 98, 1109-1152.

2. A. J. Stemmler and C. J. Burrows, “The Sal-XH Motif for Metal-mediated Oxidative DNA-Peptide Cross-linking,” J. Am. Chem. Soc. 1999, 121, 6956-6957.

3. S. E. Rokita and C. J. Burrows, “Probing Nucleic Acid Structure with Nickel and Cobalt-based Reagents,” Current Protocols in Nucleic Acid Chemistry, 2000, 2.4.1-2.4.7

Selective Inhibition of Human -Thrombin by Co(III) Schiff Base Complexes

Thomas J. Meade, Division of Biology and the Beckman Institute, California Institute of Technology, Pasadena, California, USA, 91125.

The use of metals in medicine has grown impressively in recent years as the result of a greatly advanced understanding of the structures of biologically active metal complexes and metal-containing proteins. A challenge for those in the inorganic and bioinorganic communities is the preparation of novel inorganic therapeutic agents that can be specifically coupled to a biologically active site by cooperative redox-binding ligation. We report on the selective inhibition of the serine protease human -thrombin by Co(III) Schiff base complexes. These complexes bind protein residues by ligand substitution at active sites and on enzyme surfaces in a random fashion. In order to increase inhibitor specificity and potency, a short peptide (-dFPR-) that is known to have a high affinity for the human -thrombin active site was covalently attached to the Co(III) complex. This work demonstrates that an active-site-directed peptide linked to a cobalt chelate can selectively inhibit thrombin and provides a platform for new inorganic drug candidates.

^ Metal Complexes and Drug Transport

David Piwnica-Worms, Program in Bioorganic Chemistry, Washington University School of Medicine, St. Louis, Missouri, USA, 63110.

Resistance to chemotherapeutic agents remains one of the major obstacles to successful systemic therapy of human cancer. The multidrug resistance (MDR1) P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP), members of the ATP-binding-cassette transporter family, are two of the best characterized of these resistance mechanisms. These transporters are conventionally thought to contribute to multidrug resistance by “exporting” drugs out of tumor cells in an energy-dependent manner and are relevant to a wide range of human cancers. Strategies designed to block expression or to circumvent this form of drug resistance are being actively sought by many academic and industrial laboratories in cancer research. To properly use these new target-selective drugs, highly sensitive assays for direct detection and functional characterization of the transport activity of Pgp and MRP in vivo would be desirable. We have synthesized and validated a variety of complexes incorporating gamma-emitting metal isotopes that are transported by MDR1 Pgp. These compounds include N4O2 Schiff-base phenolic [68Ga(III)]-complexes, hexakis(arylisonitrile)[99mTc(I)]-complexes, and N2O2P2 [99mTc(I)]-complexes as PET and SPECT imaging agents. We have extensively characterized these complexes using baculoviral expression of recombinant human MDR1 in host cells, human tumor xenografts in nude mice models, and mdr1a/1b(-/-) knockout mice. In addition, two commercial radiopharmaceuticals, [99mTc]Sestamibi and [99mTc]Tetrofosmin, have been validated as imaging probes of Pgp transport activity in patients and clinical studies are underway to functionally detect MDR in patients with advanced breast cancer.

The favorable cell membrane permeability properties of Schiff-base and amine phenolate metal complexes also have been exploited to generate novel antimalarials. These scaffolds are amenable to accommodating a variety of metals, including Al(III), Ga(III) and In(III), in addition to biocompatible metals relevant to malaria-host interactions such as Fe(III). Inhibiting both chloroquine-resistant and -sensitive strains, efficacy of these metal complexes correlates with their ability to inhibit heme polymerization, the same vital target as chloroquine, within the food vacuole of the causative organism, P. falciparum. An unusual selectivity profile has been discovered with complexes of the 3-MeO substituted ligand. This compound, equipotent as the Ga(III) or Fe(III) complex, is cytotoxic with an IC50 of <0.5 M, but selectively traverses the chloroquine resistance mechanism. Curiously, in a genetic cross of 21 independent recombinant progeny, susceptibility of this complex maps in perfect linkage with the chloroquine resistance phenotype suggesting that a locus for susceptibility to this compound is located on the same 36 kilobase segment of chromosome 7 previously identified as the chloroquine-resistance determinant. This metal complex offers an interesting template for the development of novel antimalarials that selectively target chloroquine resistance and in addition, may be useful as a probe of chloroquine resistance mechanisms in P. falciparum.

^ Metal Complexes as Receptor Ligands

Shubh Sharma, Palatin Technologies Inc., 175 May Street, Suite 500, Edison, New Jersey, USA, 08837.

Peptides constitute an excellent class of molecules for rapid drug discovery and lead optimization. This is promised by their high degree of structural diversity and conformational states. However, this very floppy structure eludes mapping a pharmacophore model for a biological target that is crucial to rapid translation of peptidic leads into peptidomimetics and small organic molecules with drug-like characteristics. Even the cyclized peptides and other conformationally restricted peptides and peptido-mimetics developed by the use of designer amino acids present these difficulties due to existence of multiple conformational states within these molecules. These difficulties are best exemplified by the fact that majority of attempts to convert peptide based agonists into small organic molecules usually result in the development of antagonists.

We are developing highly rigid and structurally well defined scaffolds obtained by complexing a metal-ion to a pre-designed linear peptide for their use in the process of drug development. These scaffolds are then decorated with suitable functional groups to induce biological affinity and specificity for a given biological receptor. The rigid structure of these complexes is predictable due to well defined geometry of the metal-ion co-ordination sphere. Our approach towards development of receptor specific Re[V]O complexed metallo-peptides will be presented. In particular, development of potent and receptor selective metallopeptide ligands for melanocortin receptors will be discussed

^ Metalloenzyme Drug Development Targets at Merck

John W. Kozarich, Merck Research Laboratories, PO Box 2000, Rahway, New Jersey, USA, 07065-0900.

Metalloproteases and peptidases constitute a diverse family of enzymes many of which have therapeutic potential. Two metallopeptidases of potential importance as antimicrobial targets, carbapenemase and peptide deformylase will be discussed. The efforts at Merck to design potent inhibitors of these enzymes that rely on coordination to the metal-containing catalytic center will be presented. In addition, SAR, high resolution X-ray structures of complexes and in vitro and in vivo efficacy data will also be presented.

^ Nitric Oxide Synthase

Thomas L. Poulos, C.S. Raman, and Huiying Li, Department of Molecular Biology and Biochemistry, University of California, Irvine, California, USA, 92697-3900.

Nitric oxide synthase (NOS) catalyzes the conversion of arginine to citrulline and nitric oxide. The reaction proceeds in two steps. The first step is a classic P450-like monooxygenation where one O2 derived O atom is added to the substrate in a reaction requiring two electrons. The second step is more unique to NOS, since only one electron and O2 are required. NOS requires the essential co-factor tetrahydrobiopterin (H4B) which now is thought to be a source of electrons during the O2 activation part of the NOS catalytic cycle. NOS is a homodimeric protein consisting of a heme, FMN, and FAD domains. The flow of electrons is from NADPH-to-FAD-to-FMN-to-heme. There are three human NOS isoforms: eNOS (endothelial NOS, cardiovascular system, iNOS (inducible NOS, immune system), and nNOS (neuronal system). Both eNOS and nNOS are regulated by calmodulin, and the NO produced by these isoforms is targeted to guanylate cyclase (GC). The binding of NO to GC activates GC leading to production of the ultimate signaling molecule, cGMP. iNOS has calmodulin bound as permanent subunit and does not operate via GC but instead, iNOS produced NO is used as a cytotoxic molecule to help ward off infection. There is considerable interest in developing isoform-selective inhibitors. For example, one would like to block the cytoxic effects of NO produced by iNOS or ischemic injury due, in part, to NO produced by nNOS while leaving eNOS alone owing to its critical role in maintaining vascular tone. Toward this end we have solved the crystal structure of the heme domain for all three isoforms. eNOS crystals diffract best which has enabled us to solve the structure of several eNOS-inhibitor complexes. Some of these have provided important insights into the catalytic mechanism as well as what features of the substrate are most critical for binding and activity. In some cases there have been surprises, where a compound thought to be targeted to the active site also is an antagonist of H4B binding. It has become clear that using structure-based drug design to develop isoform-selective inhibitors will be a challenging problem since the structure of the three isoforms are so similar including active site details. Therefore, it is likely that selective inhibitors may need to be targeted to regions of the protein outside of the active site where sequence variations are larger. Some of the eNOS structures solved provide hints as to how this might be accomplished. Supported by NIH Grant GM57353H 4 B

^ Molecular Modeling Of Metalloenzymes: Current Status And Future Prospects

Kenneth M. Merz Jr., Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, USA, 16802.

In this presentation we will give a brief overview of how molecular modeling has been accurately and fruitfully applied to the study of metalloenzymes. Using zinc metalloenzymes as our paradigm, we will trace the use of molecular mechanics (MM), combined quantum mechanics(QM)/MM and fully QM studies of zinc containing enzymes. Particular focus will be given to the electrostatic representation of the metal containing active site in metalloenzymes using these diverse methodologies. Finally, the future prospects and impact of using ever more sophisticated modeling tools to study metalloenzymes will be discussed.

^ Modeling Matrix Metalloproteases: A Case Study

Benjamin Burke, Agouron Pharmaceuticals, Inc., San Diego, California, USA, 92121-1111.

Matrix metalloproteases are a family of enzymes that play important roles in controlling the integrity of extracellular matrix. The therapeutic applications proposed for inhibitors of this family include cancer, arthritis and ophthalmological diseases. The active site of this family of enzymes contains a zinc critical for activity. This zinc is liganded by inhibitors containing a variety of chelating groups. The manner in which the different ligands interact with the zinc will be discussed. We have developed a molecular model of the zinc cluster and have used it in studies to predict ligand affinity within certain chelating group classes. Our model is described along with the results of the study.

^ Fundamentals of Receptor-Based Metalloradiopharmaceuticals

Shuang Liu, Medical Imaging Division, DuPont Pharmaceuticals Company, North Billerica, Massachusetts, USA, 01862.

Development of a receptor-based target-specific radiopharmaceutical is a long and complicated process, and requires a good understanding of fundamentals in biology, chemistry and nuclear medicine. Significant progress has been made for the last several years in the development of peptide target-specific radiopharmaceuticals. 99mTc-labeled small peptides have become a class of imaging agents for the diagnosis of various diseases while 90Y-labeled somatostatin analogs are under investigation for tumor radiotherapy. This talk will focus on some fundamental aspects in the design and development of target-specific radiopharmaceuticals based on small peptides, the technetium chemistry, technetium cores and bifuctional coupling agents, quality control and characterization of 99mTc-labeled small peptides. It will also present some of our recent results on the isomerism and solution dynamics of 90Y-labeled DTPA- and DOTA-biomolecule conjugates.

^ Radiolabeled Somatostatin Analogs for Targeted Radiotherapy of Cancer

Carolyn J. Anderson, Washington University School of Medicine, St. Louis, Missouri, USA, 63110.

Targeted radiotherapy is the administration of a radiolabeled molecule designed to deliver a therapeutic dose of radiation to malignant tumors with high specificity, while maintaining low accumulation in non-target tissues. Radionuclides used to label radiopharmaceuticals for cancer therapy generally decay by particle emission, i.e. , -, + or Auger electrons. Metal radionuclides for labeling therapeutic radiopharmaceuticals include 90Y, 111In, 177Lu, 67Cu and 64Cu. Analogs of the hormone somatostatin have been labeled with metal radionuclides, and they target somatostatin-receptor-positive tumors as both diagnostic and targeted radiotherapy agents. Indium-111-DTPA-octreotide and 90Y-DOTA-Tyr3-octreotide are currently in clinical trials as targeted radiotherapy agents for patients with neuroendocrine tumors at various institutions in the U.S. and Europe.

Copper-64 (half-life = 12.7 h; 17.4% ; 39% ) has applications in diagnostic imaging with positron emission tomography (PET) and targeted radiotherapy. At Washington University School of Medicine, we developed several 64Cu-labeled somatostatin analogs and have demonstrated their potential as PET imaging and targeted radiotherapy agents. A clinical PET imaging trial showed that 64Cu-TETA-octreotide imaged neuroendocrine tumors in patients as effectively as 111In-DTPA-octreotide (a FDA approved imaging agent) and conventional gamma scintigraphy. In two patients, more tumors were visualized with 64Cu-TETA-octreotide. Pre-clinical targeted radiotherapy studies have been carried out with 64Cu-TETA-Tyr3-octreotate in a tumor-bearing rat model, and complete tumor regressions occurred in all treated rats. The therapeutic effectiveness of 64Cu-labeled somatostatin analogs may be related to the fact that in cells treated with 64Cu-TETA-octreotide, 64Cu was found to localize in the cell nucleus, which may increase the probability of tumor cell death. Taken together, these data suggest that 64Cu-labeled somatostatin analogs may be effective as targeted radiotherapy agents in patients with somatostatin-receptor-positive tumors.

^ Biophysical Tuning and Targeting of Gadolinium(III) Chelates for MRI

Randall B. Lauffer, EPIX Medical, Inc., Cambridge, Massachusetts, USA, 02142.

The development of new magnetic resonance imaging (MRI) contrast agents is an active area related to bioinorganic chemistry. Most of the current emphasis is in the design of new gadolinium(III) chelates that localize in a particular area of the body and cause large enhancements in the 1/T1 relaxation rate of water protons. In addition to the synthesis of new chelates that bind to protein targets, active areas of investigation include the dependence of relaxivity on the rigidity of binding as well as the inner sphere water residence time and electron spin relaxation behavior. The development and properties of AngioMARK (MS-325), an albumin-targeted agent in advanced clinical trials for blood vessel imaging, will serve to illustrate these areas. In addition, a fibrin-targeted Gd-based agent is also under development which would allow the direct detection of blood clots as a bright spot on MR images

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