Identification of Specific Inhibitors of IMP ... - ACS Publications

Chapter 14

Identification of Specific Inhibitors of IMP Dehydrogenase 1,*

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Downloaded by CORNELL UNIV on October 29, 2016 | http://pubs.acs.org Publication Date: March 3, 2003 | doi: 10.1021/bk-2003-0839.ch014

Frank R. Collart and Eliezer Huberman 1

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Biosciences Division and Biochip Technology Center, Argonne National Laboratory, 9700 South Cass Avenue, Building G 202, Argonne, IL 60439 *Corresponding author: email: [email protected]; telephone: 630-252-4859; fax: 630-252-5517

IMP dehydrogenase (IMPDH) is an important therapeutic target and I M P D H inhibitors are used in cancer chemotherapy and for immunosuppression. Although I M P D H inhibitors may have therapeutic potential as antimicrobial, antifungal or antiprotozoal agents, no specific I M P D H inhibitors have been identified for microbial organisms. The recent availability o f crystal structures of I M P D H from different organisms will facilitate the identification of these agents. We have developed a screening method for identifying I M P D H inhibitors that is applicable to any class of organism The system is amenable to high throughput systems for the screening of inhibitors generated by combinatorial chemistry or other methods and can be used to screen for inhibitors to I M P D H from any source for which a coding sequence is available. The addition of exogenous guanosine can be used as a method to identify inhibitory chemicals that specifically target I M P D H . This work was supported by the U . S . Department o f Energy, Office o f Health and Environmental Research, under Contract W-31-109-ENG-38.

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© 2 0 0 3 American Chemical Society

Pankiewicz and Goldstein; Inosine Monophosphate Dehydrogenase ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

283 IMPDH, an important therapeutic target and inhibitors of this enzyme have clinical importance in the treatment of neoplastic disease and viral infections and as immunosuppressive agents . These inhibitors may also be useful antimicrobial, antifungal or antiparasitic therapeutic agents, however, this application has not been widely investigated. The basis for the therapeutic utility of this enzyme stems form several unique features. IMPDH is a required enzyme for the de novo synthesis of purine nucleotides. Because of this essential function, allfree-livingorganisms contain the necessary genetic information to produce the IMPDH enzyme. From a therapeutic perspective, the only method to circumvent a block in IMPDH activity is via the salvage pathways. The utility of this enzyme as a therapeutic target is further enhanced by its unusual reaction mechanism that involves the sequential binding of IMP and NAD with the subsequent release of NADH and XMP . It is likely that this distinctiveness relative to other dehydrogenase enzymes contributes to the specificity of IMPDH inhibitors. 1

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A characteristic of IMPDH enzymes that has not been fully exploited from a therapeutic perspective is the biochemical diversity. Bacterial IMPDH enzymes show biochemical and kinetic characteristics that are different than the mammalian IMPDH enzymes (Table I). These differences include cofactorbinding affinity , sensitivity to inhibitors and cation requirements . These differences likely reflect an alteration in the reaction mechanism as a consequence of the variance of amino acid residues in the active site. Identification of these residues or combination of residues that impart this mammalian or microbial enzyme signature can be exploited for the development of agents that specifically target bacterial or protozoan IMPDH enzymes. 5

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Basis for the development new IMPDH inhibitors Progress in the identification IMPDH inhibitors has been leveraged by several developments in the scientific arena including: • The availability of genomic sequence information from many different organisms • The development of bacterial systems for expression of IMPDH enzymes •

The determination of the crystal structure for IMPDH from several sources including mammalian and bacterial enzymes

At the present time, IMPDH coding regions have been identified for greater than 50 different species with representatives from the eukaryotic, prokaryotic, and archaeal domains. The rapid accumulation of sequence information from various organisms provides a resource of new IMPDH enzymes to discern the full spectrum of diversity and will provide a basis for the development of specific IMPDH inhibitors that target selected organisms. The wealth of



284 genomic information has also provided the incentive for the development of bacterial systems for expression of IMPDH enzymes. IMPDH enzymes from more that 20 different organisms have been expressed in Escherichia coli. A number of expression clones developed at the Robotic Molecular Biology Facility at Argonne National Laboratory are listed in Table II. Many of these clones are expressed as tagged fusion proteins to facilitate downstream protein assay and purification procedures. For most bacterially expressed IMPDH enzymes, addition of an N-terminal fusion tag does appear to significantly affect enzyme activity. These expression clone resources have contributed to characterization of the biochemical and kinetic properties and provided an enzyme resource for crystallization screens and the subsequent determination of the crystal structures for several IMPDH enzymes. The availability of IMPDH crystal structuresfromeukaryotic ' and bacterial ' organisms provides detailed molecular information about the catalytic site and insight into the reaction mechanism. This information suggests there are a number of areas that can be attractive target regions (Table III) for therapeutic development. 8 9

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IMPDH inhibitor screen Developments in combinatorial chemistry allow the rapid and economical synthesis of hundreds to thousands of discrete compounds. These compounds are typically arrayed in moderate-sized libraries of small organic molecules designed to provide targeted or directed diversity. However, there is not a published method to systematically screen the various chemical agents for utility as IMPDH inhibitors. Development of such an assay would enhance and accelerate the discovery of therapeutically useful inhibitors.

Description of the method. We have developed an efficient method to identify specific inhibitors of IMPDH. The method includes a prokaryotic or eukaryotic host organism that is a guanosine auxotroph. Our present system used the E. coli H712 strain n

originally described by Nijkamp, et al. This strain does not produce a function IMPDH enzyme and requires the addition of a guanine nucleotide precursor(s) to the culture medium for cell growth. For the screening method, the H712 strain is transformed with an IMPDH expression vector capable of producing a functional IMPDH enzyme. The present system uses a broad host range vector pJFl 18EH, constructed by Fiirste et al. This expression system uses ampicillin resistance as a selectable marker and permits the regulated expression of foreign coding sequences in an E. coli host. The pJF118EH vector has the properties of inducible expression such that in the absence of an inducer the expression of the cloned foreign is low. Induction of the foreign gene is initiated by the addition of IPTG to the bacterial culture medium. H712 bacteria transformed with the 12


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Table I. Differential inhibitor sensitivity of human and S. pyogenes IMPDH Human

Streptococcus K/ICsofrM)

Ratio Human/Strep

1

Inhibitor

MPA Mizoribine-P

0.01 0.01

150

S

1

15,000 50

J

Ribavirin-P

0.4

6

15

|

TAD

0.19

10-20

80

1

Table II. Expression of Recombinant IMPDH in E. coli Organism

Eukaryotic

Haemophilus influenzae Pseudomonas aeruginosa

Enzyme Truncated enzyme Enzyme Enzyme Enzyme Enzyme Enzyme

Bacillus siearothemophilus

PCR fragment

Human Human Arabidopsis thaliana Trypanosoma bruceii Streptococcus pyogenes

Bacteria

Mycobacterium tuberculosis Enzyme

Archaea

Enzyme Activity

Product

Pyrococcus furiosus

Enzyme

Halobacterium kalobium

PCR fragment

m+

1

-

Table HI. Molecular target regions amenable for IMPDH inhibitors. Target region I

IMP binding site

Cofactor binding site Cation requirement CBS dimmer domain Tetrameric contacts

Therapeutic relevance 1 Several inhibitors of bacterial and mammalian 1 enzymes are currently available (e.g.ribavirinand mizoribine) Inhibitors that occupy this site display a wide 1 variance in effectiveness for mammalian and bacterial IMPDH enzymes. Some IMPDH exhibit a reduce requirement for monovalent cations. Domain function is unknown but contains a cleft with binding potential. The tetrameric form of the enzyme is essential for attainment of the catalytic pocket environment



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pJF118EH vector containing the human or S. pyogenes IMPDH coding regions produce function enzyme and no longer require the addition of guanosine to the culture medium for growth. Furthermore, the EMPDH enzymes produced in the E. coli host retain the biochemical and kinetic characteristics of the source (i.e., either human or S. pyogenes). This system allows an evaluation of the effect of various agents on the growth of the recombinant bacteria and the identification of putative IMPDH inhibitors. However, there are chemicals that inhibit host cell proliferation regardless of whether or not the host is expressing a prokaryotic or eukaryotic IMPDH enzyme. To identify agents specifically targeting IMPDH, the inhibitor profile for a particular agent is compared to the same profile obtained when a guanine nucleotide precursor such as guanine or guanosine is added to the culture medium. Guanosine is the preferred agent due to its high solubility in aqueous solutions. These guanine nucleotide precursors are able to circumvent the block on IMPDH activity imposed by IMPDH inhibitors and can be used to exclude nonspecific growth inhibitory or toxic compounds. Agents specifically targeting IMPDH will exhibit a decrease ability to inhibit cell proliferation in the presence of guanine/guanosine while the growth inhibitory effects of nonspecific or toxic agents will not be ameliorated by guanine/guanosine.

Plate assay system for candidate inhibitors. A plate assay system containing various amounts of several different IMPDH inhibitors were added to 7mm filter disks and the disks placed on a lawn of H712 bacteria containing either the human or S. pyogenes IMPDH expression plasmid and IPTG for induction of enzyme expression. Inhibitors were selected on the basis of reports in the literature regarding specificity for inhibition of human IMPDH. Both mycophenolic acid (MPA) and ribavirin are clinically useful and MPA is known to inhibit human, but not bacterial IMPDH . Some inhibitors, ribavirin, tiazofurin, and mizoribine, require cellular activation for utility as IMPDH inhibitors. For several of the inhibitors, a clear area was observed around the filters corresponding to the degree of inhibition (Figure 1A). Various 6

inhibition patterns were observed for bacteria containing the human or S. pyogenes IMPDH expression vectors that ranged from no inhibition, to inhibition of human IMPDH, to inhibition of both forms of IMPDH. Furthermore all of the growth inhibitory chemicals showed a differential between the low and high does. The inhibition profiles obtained with this panel of IMPDH inhibitors demonstrate the IMPDH enzyme produced in bacteria retains the biochemical and kinetic characteristics of the source (i.e. human or S. pyogenes). The results also illustrate the utility of this approach for identification of the inhibitory spectrum of IMPDH inhibitors.



287 This method also has the capability to identity clinically useful features of the potential IMPDH inhibitors. Clinically useful agents must be transported into the target organism and may require activation for therapeutic effectiveness. In this screening procedure, the use of a host organism that mimics the characteristics of the eventual therapeutic target can provide useful information regarding clinically useful properties of potential therapeutic agents. The results presented in Figure 1A demonstrate ribavirin and mizoribine are transported into the bacterial cells and are activated by the host system to a form, which is capable of inhibiting IMPDH. Thus, this method inherently excludes compounds that might be impermeable to the host organism. Alternatively, because many inhibitory compounds require activation by chemical modification, this method can be applied to determine the competency of a host organism for metabolic activation of IMPDH inhibitors. To demonstrate that the observed growth inhibition was specific for IMPDH, some of the inhibitor filter disks were placed on a lawn of H712 bacteria on plated supplemented with guanosine. On these plates, the exogenous guanosine reduced the inhibitory effect of the mycophenolic acid (Figure IB). The ability of guanosine to ameliorate the growth inhibition indicates the specificity of these chemicals for IMPDH.

High throughput screening. The microbial system is amenable to high throughput systems for the screening of inhibitors generated by combinatorial chemistry or other methods. To examine this approach we used microwell plates and a Biomek workstation to implement automated protocols to assess the effect of various inhibitors on bacterial growth. Inhibitors were added to an arrayed culture of recombinant H712 bacteria and the effect on bacterial growth assessed after an overnight incubation by measurement of absorbance at 600 nm. The inhibition pattern observed for H712 bacteria containing the human IMPDH expression plasmid (Figure 2) is similar to that observed for the plate assay. All of the tested IMPDH inhibitors decrease the growth of the recombinant strain as a function of inhibitor concentration with MPA being the most effective inhibitor of bacterial growth. A parallel analysis using H712 bacteria containing the S. pyogenes IMPDH expression plasmid (Figure 3) illustrated the difference between the bacterial and mammalian IMPDH enzymes. The growth of the variant containing the S. pyogenes enzyme is not inhibited by MPA consistent with results observed with the purified bacterial enzyme . Bacterial growth is inhibited by treatment with ribavirin and mizoribine consistent with results obtained using the purified enzyme (Table I). In contrast to the results obtained using purified IMPDH enzymes, the in vivo screening method shows ribavirin is a more effective inhibitor of the bacteria expression the human and S, pyogenes IMPDH enzymes than mizoribine. This effect is likely represents a differential ability related to transport or metabolic activation of these agents. 5



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Figure 1. Assays of candidate inhibitors. (A) Different amounts (1 and 5 jjg) of several different IMPDH inhibitors were added to 7mm filter disks and the disks placed on a lawn ofH712 bacteria containing either the human or S. pyogenes IMPDH expression plasmid and IPTG for induction of enzyme expression. A control disk containing the solvent but no inhibitor was placed in the center of the dish. The inhibitors used were MPA, ribavirin (Rb), tiazofurin (Tz), and mizoribine (Mz). After incubation at 37 °C overnight, the plates were examined for inhibition of bacterial cell growth. (B) IMPDH inhibitors were added to 7mm filter disks placed on a lawn ofH712 bacteria containing the human IMPDH expression plasmid. The growth medium of the indicated plate was supplemented with 50 fjg/ml of guanosine. After incubation at 37 °C overnight, the plates were examinedfor inhibition of bacterial cell growth. The restoration of bacterial growth on the plates containing exogenous guanosine indicates the specificity of this chemicalfor IMPDH.



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Figure 2. Inhibitor response of H712 bacteria containing the human IMPDH expression plasmid. Inhibitors were added to the initial well to a final concentration of 2.5 mM. The final well of the dilution series contained an inhibitor concentration of 5 JJM. The absorbance (at 600 nm) of the plate cultures were determined after incubation at 37 °C overnight



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Figure 3. Inhibitor response of H712 bacteria containing the S. pyogenes IMPDH expression plasmid. Inhibitors were added to the initial well to a final concentration of 2.5 mM. The final well of the dilution series contained an inhibitor concentration of 5 juM. The absorbance (at 600 nm) of the plate cultures were determined after incubation at37°C overnight


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Summary The microbial screening system of the present invention has a number of advantages for the identification of specific IMPDH inhibitors: 1.


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5.

The microbial system is amenable to high throughput systems for the screening of inhibitors generated by combinatorial chemistry or other methods This method can be used to screen for inhibitors to IMPDH from any source for which a coding sequence is available. At the present time, more than 50 different IMPDH coding sequencesfromthe eukaryotic, bacterial and archaeal domains are available in the DNA sequence databases The addition of exogenous guanosine can be used as a method to verify inhibitory chemicals that specifically target IMPDH and to distinguish from other causes of host cell inhibition. The method provides metabolic information such as the ability of the inhibitors to enter the cell and to be activated directly or after their cellular metabolism This method can be used to identify chemicals capable of differentially inhibiting IMPDHfromorganisms that are genetically dissimilar.

This method can be applied for the identification of specific IMPDH inhibitors, which could be used to as the basis for the design of pharmaceuticals. Additional human IMPDH inhibitors could be identified for use as chemotherapeutic agents for the treatment of neoplasm's and as immunosuppressive agent. This method is also useful for the identification of agents selective for various IMPDH isoforms (e.g. human Type I and II). This method also has the potential for identification of inhibitors of bacterial, fungal, protozoan or viral IMPDH that do not inhibit the human or other mammalian enzymes and may be effective therapeutic agents.

Acknowledgment The submitted manuscript has been created by the University of Chicago as Operator of Argonne National Laboratory ("Argonne") under Contract No. W31-109-ENG-38 with the U.S. Department of Energy. The U.S. Government retains for itself, and others acting on its behalf, a paid-up, nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the Government.


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