Differential Splicing of - American Chemical Society

Chapter 6

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Differential Splicing of Pneumocystis carinii Inosine 5'-Monophosphate Dehydrogenase mRNA: Implications for Producing Catalytically Active Protein Dongjiu Ye, Jeniece Nott, and Sherry F. Queener* Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202

Pneumocystis carinii I M P D H was originally reported to be approximately 70 amino acids shorter at the amino terminus than I M P D H from other species , but recent work in our laboratory has demonstrated that four splicing variants of the protein are possible . Three of these variants, including one identical to the originally described mRNA, contain a stop codon in the 5' region of the pre-mRNA that results in loss of regions of the amino terminus thought to be important for enzyme activity. The fourth variant codes for a form of I M P D H that contains 529 amino acids and retains key amino terminal sequences (GeneBank Accession No. 196975) . In P. carinii isolated from infected rats, the splicing variant coding for the long form predominated and only the long form of the protein was detected; in organisms from culture, splicing variants coding for the short form of I M P D H predominated but only the long form of the protein was detected. The catalytic activity of this longer form of I M P D H from P. carinii was confirmed by a complementation study in bacteria lacking native I M P D H . P. carinii I M P D H 1

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© 2003 American Chemical Society

In Inosine Monophosphate Dehydrogenase; Pankiewicz, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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long form was expressed with a his-tag on the amino terminal end and purified by metal-affinity chromatography. The protein retains catalytic activity and, like the native protein in P. carinii, exists as a tetramer in solution. The his-tagged protein showed a K m for N A D of 24 ± 1 micromolar and a K m for I M P of 51 ± 8 micromolar, similar to forms of the enzyme from other species. Substrate inhibition was observed at millimolar concentrations of both N A D and I M P . The enzyme activity was strongly inhibited by mycophenolic acid (IC50 0.066 micromolar) and showed a different pattern of inhibition with other inhibitors than the mammalian enzyme, suggesting selective drug design is possible.

Pneumocystis carinii

Taxonomy Pneumocystis carinii is an pathogen whose taxonomic classification has been difficult since the organism was first described in 1909, when it was thought to be a life stage of trypanosomes . The organism was quickly recognized to be distinct from trypanosomes , but continued to be considered a protozoan on the basis first of morphology and later on the basis of susceptibility to antiprotozoal agents " . P. carinii is now considered to be more related to fungi than to protozoans, primarily on the basis of homologies in nucleic acids and proteins . 3

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Relation to human disease P. carinii caused only sporadic outbreaks of human disease before 1980, with most cases occuring among protein-malnourished children living in crowded conditions . The dramatic increase in P. carinii pneumonia that accompanied the onset of AIDS illustrated that P. carinii was primarily an opportunistic pathogen, proliferating and causing disease in those whose immune function was compromised. Other severely immunocompromised patients, such 8,11


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as those receiving immunosuppressive drugs for organ transplantation or other conditions are also susceptible to severe infections caused by this organism " . Before highly active anti-retroviral therapy (HAART) became widely used, P. carinii pneumonia was a leading cause of death in AIDS patients but five years after H A A R T became available, the situation had changed. For many patients H A A R T allows the immune system to remain sufficiently functional to protect patients from many of the more common opportunistic infections, including P. carinii pneumonia. The use of prophylaxis for P. carinii pneumonia is also wide-spread and has lowered the incidence of this disease. In spite of these advances, significant numbers of AIDS patients and organ transplant patients continue to suffer from serious infections caused by this organism ' . P. carinii infections are still a common finding in patients on first diagnosis with AIDS and the occurence of P. carinii pneumonia diminishes the prospects for long term survival. 12

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Therapy of P. carinii infections Therapy for P. carinii pneumonia has depended primarily upon older antimicrobial agents, such as pentamidine and the combination of trimethoprim with sulfamethoxazole. The use of pentamidine is often limited by toxicity unless it is administered in an aerosol form; this latter route is used more for prophylaxis than for therapy and is associated with more breakthrough infections than other routes . Alternative therapies for P. carinii pneumonia exist and there are several regimens for prophylaxis, but many of the drugs used are associated with significant toxicity and failures of therapy or prophylaxis are not uncommon " . The first choice for both prophylaxis and therapy of P. carinii pneumonia is trimethoprim/sulfamethoxazole in the same fixed combination used for bacterial infections. Whereas this therapy is very effective and well tolerated in nonAIDS patients, side effects occur frequently in HIV-infected individuals . Of more concern are recent reports that prophylaxis or therapy with trimethoprim/sulfamethoxazole may fail as drug-resistance mutations accumulate in dihydropteroate synthase, the target of sulfonamide drugs " . Questions have existed for some time about the role of trimethoprim in the combination when it is used against P. carinii, because the target enzyme dihydrofolate reductase in P. carinii is only weakly inhibited by trimethoprim . Moreover, trimethoprim alone shows little or no activity against the organism in culture or animal models . Thus, the extensive use of this combination for prophylaxis may have facilitated the development of drug resistance, since only one of the drugs in the combination seems to be active. If the drug-resistant forms of P. carinii spread, trimethoprim/ sulfamethoxazole, which is currently the primary clinical agent for 18,19

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113 this disease, will cease to be effective. Given the slow growth of the organism and the length of time it has taken for the first drug-resistant forms to be detected, the argument might be made that failure of the primary drug is a long way off, but no one can predict how rapidly drug-resistant forms may spread, given the new information on transmission of the organism. Although it was originally thought that humans carried cysts of P. carinii in their lungs and infections arose from organisms that might have been picked up long before the illness, current evidence suggests that P. carinii can be passed from human to human in certain settings, probably by the airborne route ' . If this is the case, it might be expected that drug-resistance forms would spread more rapidly than if independent mutations were occurring in individual patients. In order to maintain effective control of P. carinii infections in humans, new drugs must be developed to fill the void left by the failure of established agents. Several lines of study show promise, including the development of new generation inhibitors of dihydrofolate reductase that are selective for the P. carinii enzyme " and the development of chemical relatives of pentamidine with more selectivity and fewer side effects . New drug targets also need to be identified in P. carinii. One exploitable target may be inosine 5monophosphate dehydrogenase (IMPDH, E C 1.1.1.205) \


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Studies of purine metabolism in P. carinii +

I M P D H catalyzes the NAD -dependent conversion of inosine 5'monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). I M P D H has been extensively studied as a drug target because the reaction catalyzed by this enzyme is a required and often rate-limiting step in de novo synthesis of guanine nucleotides (Figure 1). For example, the immunosuppressant mycophenolate mofetil, the anticancer drug tiazofurin, and the antiviral agent ribavirin are all inhibitors of I M P D H " . The potential of I M P D H to be a drug target in P. carinii was suggested by the fact that growth of the organism in short term culture was blocked by low concentrations of mycophenolic acid or tiazofurin (Figure 2). The level of activity shown in the dose responses for these compounds places them among the most potent agents tested in the 24-well plate culture model, which has been widely used to demonstrate antipneumocystis activity " . B y way of comparison, trimethoprim/sulfamethoxazole at 170/990 pM, respectively, produce inhibition similar to that shown by 1 fiM mycophenolic acid. One way some organisms offset inhibition of I M P D H is to salvage xanthine, guanine or guanosine. These metabolites enter the pathway after the I M P D H reaction and thus can drive synthesis of guanine nucleotides in the absence of significant I M P D H activity. Limited studies exist on metabolism in P. carinii 37

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ATP

GTP

guanylate reductase


ADP

GDP

GMP XMP

synthetase"

•GMP

Guanine

Xanthine

purine nucleoside purine nucleoside phosphorylase phosphorylase

Inosine

Adenosine

Guanosine

adenosine deaminase

Figure 1. Purine metabolic pathways. A generalized sequence of metabolic reactions is shown, because the pathways and enzymes involved in P. carinii have not been elucidated. The process ofde novo synthesis, not shown in the figure, would produce IMP. Salvage pathways are mediated by APRT, HGPRT, purine nucleoside phosphorylase and adenosine kinase. A P R T , adenine phosphoribosyl transferase; HGPRT, hypoxanthine-guano sine phosphoribosyl transferase.


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Days of exposure to drug Figure 2. Inhibition of P. carinii growth in short term cultures by IMPDH inhibitors. These studies were performed using the short term 24-well plate culture system designed for testing drug susceptibility ofF. carinii, where the organism is grown on monolayers of human embryonic lung cells. Data shown are means ± SEM for quadruplicate samples. Error bars on the curve for 1 pM MPA (mycophenolic acid) are small and are within the range covered by the symbol.


116 and no studies have focused on purine metabolism. Early reports suggested that P A B A , glucose, pyruvate, amino acids and uridine were taken up by P. carinii, but that hypoxanthine and thymidine were not . This result was interpreted as evidence that the organisms did not proliferate when they were isolated from rat lung and suspended in medium. A n alternate explanation is that these organisms accurately reflected the naturally low state of purine and pyrimidine salvage in P. carinii The critical enzyme for salvage of xanthine and guanine is H G P R T (hypoxanthine, guanosine phosphoribosyltransferase, E C 2.4.2.8), which still has not been identified in P. carinii. The culture data (Figure 2) indicated that inhibition of I M P D H alone was sufficient to impair growth of P. carinii, consistent with the suggestion that salvage pathways in this organism may be of minor importance. In fact, adding excess guanine to the growth media of P. carinii in short term culture did not overcome the inhibition produced by mycophenolic acid (Figure 3); guanosine and xanthine also failed to reverse the growth inhibition. In separate studies, direct uptake of guanine, guanosine and xanthine could not be demonstrated with P. carinii isolated from infected rat lungs (data not shown). These results were consistent with the earlier studies by other laboratories that failed to show salvage of purines by P. carinif*. To date the evidence suggests that salvage of purines in P. carinii is weak or nonexistent. A low rate of purine salvage would tend to make the effects of inhibition of I M P D H more devastating to P carinii than to other organisms, where salvage of significant amounts of purines could potentially bypass the blockade of I M P D H and allow synthesis of G M P from xanthine, guanine, or guanosine. That such a situation exists is suggested by the results of the culture studies which show profound growth inhibition at micromolar or submicromolar concentrations of I M P D H inhibitors. I M P D H thus seems to satisfy one criterion for being a potential drug target in P. carinii, namely that it seems essential to the organism


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Cloning IMPDH from P. carinii

c D N A library from cultured P. carinii The difficulties inherent in acquiring sufficient supplies of P. carinii for biochemical studies drove us to explore recombinant methods to produce I M P D H from this organism. The original cloning was performed several years ago using degenerate primers designed to highly conserved regions of known I M P D H proteins. The most highly purified organisms available at that time 1


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