View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Springer - Publisher Connector Clinical Proteomics Journal Copyright ©Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN 1542-6416/04/01:45–67/$25.00 Molecular Targets New Targets for an Old Drug A Chemical Proteomics Approach to Unraveling the Molecular Mechanism of Action of Methotrexate Leticia M.Toledo-Sherman,*,1 Leroi Desouza,† Christopher M. Hosfield,† Linda Liao, Kelly Boutillier, Paul Taylor, Shane Climie, Linda McBroom-Cerajewski, and Michael F. Moran*,1 1MDS Proteomics Inc., 251 Attwell Drive,Toronto, ON M9W 7H4, Canada pathway architecture similar to that seen in sig- Abstract naling pathways and consistent with substrate Methotrexate has been a clinical agent used channeling. More importantly, we were able to in cancer, immunosuppression, rheumatoid capture protein targets that could potentially arthritis and other highly proliferative diseases provide new insights into the mechanism of for many years, yet its underlying molecular action of methotrexate in rheumatoid arthritis mechanism of action in these therapeutic areas and immunosuppression. The application of this is still unclear. We present a chemical proteomics approach to other drugs and drug candidates approach that uses ultra-sensitive mass spec- may facilitate the prediction of unknown and trometry coupled to an inverse protein-ligand secondary therapeutic target proteins and those docking computational technique to unravel related to the side effects and toxicity. These the mechanism of action of this drug. Using results demonstrate that this proteomics tech- methotrexate tethered to a solid support we nology could play an important role in drug were able to isolate a significant number of pro- discovery and development since it allows mon- teins. We effectively captured a large portion of itoring of the interactions between a drug and the de novo purine metabolome and propose a the protein content of a cell. *Author to whom all correspondence and reprint requests should be addressed: E-mail: [email protected], [email protected] †Authors contributed equally to this work. 45 46 ________________________________________________________________________ Toledo-Sherman et al. Key Words: Methotrexate; targets; mechanism of attached to solid supports in such a way that action; chemical proteomics; pharmaco-proteomics; when exposed to a biological sample its inter- metabolome; inverse docking; substrate channeling. acting protein partners can be captured and identified. Captured proteins are isolated by Introduction gel electrophoresis, readily analyzed by mass One of the most expensive, yet important spectroscopy and identified by searches against aspects of drug discovery and development is sequence databases. With this technique, we the clinical evaluation of emerging therapeu- are able to identify proteins that are interacting tics. Yet, it is at this stage that most drug can- directly with the drug probe or interacting didates fail to show efficacy or tolerable indirectly via protein–protein engagement with toxicities and are withdrawn (1). Furthermore, direct interactors. For the classification of direct a large number of drugs already in clinical use vs indirect interactions we have coupled our today have unknown or unclear molecular experimental methods with a computational mechanisms of action and toxicity. Precise technique termed inverse docking. This proce- knowledge of the mechanism of action of dure is used to dock a ligand against a library these drugs would facilitate critical decisions of protein structures. Recently, this inverse to be made regarding label and patent expan- docking procedure was shown to be effective sion, as well as the development of second- in identifying potential new targets of 4H- generation therapeutics. A promising aspect of tamoxifen and vitamin E (7), respectively. This the emerging field of proteomics is the devel- tool has also been used to identify potential opment of sensitive tools and methods for toxicity and side effect-related protein targets facilitating an understanding of key interac- of a small molecule (8). With the advent of tions between a drug and its targets at the structural genomics initiatives and given the molecular level (2), thus allowing a better richness of protein structures already present decision-making process so that only com- (9) in the protein database (http://www.rcsb. pounds with a high probability of showing the org/pdb), we expect that the three dimensional required efficacy profile and low toxicity are structures of many of the protein identified in considered for the clinic. our experiments would be readily accessible or Already, chemical proteomics, the capturing predictable by homology modeling from of a select proteome with a chemical agent or related protein structures. Proteins for which drug, has shown potential in accelerating drug inverse docking proposes binding modes rep- discovery and development (3,4). Recently, a resenting low energy complexes, as scored by a chemical proteomics approach was used to consensus scoring technique (10) combining identify the targets of the widely used quino- five different score functions, are deemed hits lines and offered an insight into the mechanism and worth considering as direct binders of the of action of these drugs (5). We are interested in ligand. developing such methods and have access To demonstrate the potential of combining to mass spectrometry instrumentation with these two technologies, we selected the widely ultra-sensitivity (6). One of these methods is a used drug methotrexate (MTX). Methotrexate is pharmaco-proteomics approach to understand- a folate antimetabolite that has been used exten- ing the interaction between a drug and its tar- sively for the treatment of highly proliferative gets. We have developed an affinity-based diseases such as rapidly growing tumors, acute chemical proteomics platform that uses small leukemia, rheumatoid arthritis, psoriasis, pneu- molecules (e.g., a drugs or drug candidates) mocystis carinii caused-pneumonia associated Clinical Proteomics _______________________________________________________________ Volume 1, 2004 Chemical Proteomics for Target Discovery _______________________________________________________ 47 with AIDS, and other chronic inflammatory The main problem with classical antifolates disorders. Methotrexate has recognized effi- is that accumulation of polyglutamated meta- cacy as an anti-inflammatory (11), immuno- bolites causes drug resistance in cells. Several suppressive (12) and anticancer agent (13). mechanisms of resistance (22) defective trans- In inflammation and immunosuppression, port through cell membranes, amplification of despite its wide use and several theories DHFR, reduced expression of FPGS and behind its efficacy, the underlying molecular upregulation of γ-GH have been identified as mechanism of action of MTX remains unclear. the underlying basis for the development of In cancer, the mechanism of action of MTX has resistance to antifolates. Because of the increased been understood as occurring because of cyto- resistance with current antifolate drugs, there toxicity originating from the accumulation is a need for new antifolate targets. The devel- of the corresponding polyglutamated MTX opment of clinical diagnostic markers for metabolites in cells (14). Methotrexate is taken antifolate drug resistant tumors would also be into cells by reduced folate carrier (RFC) beneficial in deciding which therapies to protein, where it is polyglutamated by folyl- choose for those tumors. Of equal importance polyglutamate synthetase (FPGS). Upon poly- to clinical applications, is the understanding glutamation, MTX binds to dihydrofolate of the molecular mechanism of action and reductase (DHFR) interrupting the conver- toxicity of existing and emerging antifolates sion of dihydrofolate to the activated N5,N10- therapeutics. methylene-tetrahydrofolate. N5,N10-methylene- In order to address some of these issues we tetrahydrofolate is the main methylene donor set out to investigate the underlying molecular in de novo purine biosynthesis, it provides mechanism of action of MTX. Structurally, the the methyl group in the conversion of MTX-DHFR (23) (Fig. 1A) pair is one the best deoxyuridine-5’-monophosphate (dUMP) to understood ligand-protein systems and at the deoxythymidine-5′-monophosphate (dTMP) time of writing a search of the protein data- for DNA synthesis and for many trans- bank for the keyword MTX resulted in 66 methylation processes. The polyglutamated entries. Most of these entries are of MTX or metabolite is subject to back glutamyl hydrol- derivatives in complexes with DHFR from dif- ysis by γ-GH and efflux from cells. ferent species and DHFR mutants, but struc- The three main targets of antifolate drugs tures for TS (1AXW)(24) also exist. The crystal in the clinic are DHFR, thymidylate syn- structure of GART in complex with a molecule thase (TS), and glycinamide ribonucleotide of glycinamideribonucleotide (GAR) and a transformylase (GART) (15). Several newer folate analog is also available (1CDE) (25). In generation classical (polyglutamates) and all these structures the aminopterin and the nonclassical (nonpolyglutamates) antifolate α-carboxylate groups of the molecule are drugs are now under clinical evaluation with buried inside the binding site and make key promising results (16). It is well known now hydrogen bond interactions with