Lizbeth Hedstrom

A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium

Cryptosporidium spp are well recognised as causes of diarrhoeal disease during waterborne epidemics and in immunocompromised hosts. Studies have also drawn attention to an underestimated global burden and suggest major gaps in optimum diagnosis, treatment, and immunisation. Cryptosporidiosis is increasingly identified as an important cause of morbidity and mortality worldwide. Studies in low-resource settings and high-income countries have confirmed the importance of cryptosporidium as a cause of diarrhoea and childhood malnutrition. Diagnostic tests for cryptosporidium infection are suboptimum, necessitating specialised tests that are often insensitive. Antigen-detection and PCR improve sensitivity, and multiplexed antigen detection and molecular assays are underused. Therapy has some effect in healthy hosts and no proven efficacy in patients with AIDS. Use of cryptosporidium genomes has helped to identify promising therapeutic targets, and drugs are in development, but methods to assess the efficacy in vitro and in animals are not well standardised. Partial immunity after exposure suggests the potential for successful vaccines, and several are in development; however, surrogates of protection are not well defined. Improved methods for propagation and genetic manipulation of the organism would be significant advances.

IMP Dehydrogenase: Structure, Mechanism and Inhibition

George Weber was among the first to recognize that extensive metabolic changes must underlie the unbridled proliferation of cancer cells 1. His molecular correlation hypothesis postulated that a defined set of key “pace-maker” enzymes are stringently linked to neoplastic transformation and progression, and that inhibition of these enzymes would provide an effective strategy for chemotherapy. Webers subsequent discovery that inosine 5′-monophosphate dehydrogenase (IMPDH) is amplified in tumors and rapidly proliferating tissues provided the foundation for drug design targeting this enzyme 2. Though yet to achieve much success in the cancer arena, IMPDH inhibitors are now widely used in immunosuppressive and antiviral chemotherapy, and IMPDH may also be a target for antimicrobial drugs. Clinical relevance aside, IMPDH is a fascinating enzyme. It traverses several conformations while catalyzing two different chemical transformations, utilizing unusual chemical strategies to promote each reaction. Monovalent cations such as K+ activate IMPDH, possibly by acting as a molecular lubricant to facilitate these conformational changes. The biology of IMPDH also displays some surprising twists. IMPDH binds nucleic acids and is associated with polyribosomes 3-6, though the physiological role of this interaction also has not yet been elucidated. Perhaps most intriguing is the discovery that mutations in IMPDH are associated with hereditary retinal disease 7-9. These mutations cluster to a subdomain that is not required for enzymatic activity, and the function of this subdomain is currently under debate. This article will review recent work on the biochemistry of IMPDH, integrating structure, function and inhibition. Earlier reviews on this topic include references 10-12. Several more focused reviews have addressed IMPDH as a drug target for immunosuppressive 13, cancer 14,15, antiviral 16 and antimicrobial chemotherapy 17, specific classes of IMPDH inhibitors 18, advances in structure and mechanism 19 and the role of IMPDH in retinal disease 20,21. The reader is also directed to a collection of papers from the 2000 meeting, Inosine monophosphate dehydrogenase: a major therapeutic target 22.

Azole Drugs Are Imported By Facilitated Diffusion in Candida albicans and Other Pathogenic Fungi

Despite the wealth of knowledge regarding the mechanisms of action and the mechanisms of resistance to azole antifungals, very little is known about how the azoles are imported into pathogenic fungal cells. Here the in-vitro accumulation and import of Fluconazole (FLC) was examined in the pathogenic fungus, Candida albicans. In energized cells, FLC accumulation correlates inversely with expression of ATP-dependent efflux pumps. In de-energized cells, all strains accumulate FLC, suggesting that FLC import is not ATP-dependent. The kinetics of import in de-energized cells displays saturation kinetics with a Km of 0.64 uM and Vmax of 0.0056 pmol/min/108 cells, demonstrating that FLC import proceeds via facilitated diffusion through a transporter rather than passive diffusion. Other azoles inhibit FLC import on a mole/mole basis, suggesting that all azoles utilize the same facilitated diffusion mechanism. An analysis of related compounds indicates that competition for azole import depends on an aromatic ring and an imidazole or triazole ring together in one molecule. Import of FLC by facilitated diffusion is observed in other fungi, including Cryptococcus neoformans, Saccharomyces cerevisiae, and Candida krusei, indicating that the mechanism of transport is conserved among fungal species. FLC import was shown to vary among Candida albicans resistant clinical isolates, suggesting that altered facilitated diffusion may be a previously uncharacterized mechanism of resistance to azole drugs.

3-Deoxy-D-manno-octulosonate-8-phosphate synthase catalyzes the C-O bond cleavage of phosphoenolpyruvate

The mechanism of 3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P) synthase was investigated. When [18O]-PEP specifically labeled in the enolic oxygen is a substrate for KDO8P synthase, the 18O is recovered in Pi. This indicates that the KDO8P synthase reaction proceeds with C-O bond cleavage of PEP similar to that observed in the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase catalyzed condensation of PEP and erythrose-4-phosphate (1). No evidence for a covalent enzyme-PEP intermediate could be obtained. No [32P]-Pi exchange into PEP nor scrambling of bridge 18O to non-bridging positions in [18O]-PEP was observed in the presence or absence of arabinose-5-phosphate or its analog ribose-5-phosphate. Bromopyruvate inactivated KDO8P synthase in a time dependent process. It is likely that bromopyruvate reacts with a functional group at the PEP binding site since PEP, but not arabinose-5-phosphate, protects against inactivation.

Using supramolecular hydrogels to discover the interactions between proteins and molecular nanofibers of small molecules

Here we report the first example of the use of supramolecular hydrogels to discover the protein targets of aggregates of small molecules.

Isolation and Characterization of Mycophenolic Acid-resistant Mutants of Inosine-5!-monophosphate Dehydrogenase*

Mycophenolic acid (MPA) is a potent and specific inhibitor of mammalian inosine-monophosphate dehydrogenases (IMPDH); most microbial IMPDHs are not sensitive to MPA. MPA-resistant mutants of human IMPDH type II were isolated in order to identify the structural features that determine the species selectivity of MPA. Three mutant IMPDHs were identified with decreased affinity for MPA. The mutation of Gln277 → Arg causes a 9-fold increase in the Ki of MPA, a 5-6-fold increase in the Km values for IMP and NAD, and a 3-fold decrease in kcat relative to wild type. The mutation of Ala462 → Thr causes a 3-fold increase in the Ki for MPA, a 2.5-fold increase in the Km for NAD, and a 1.5-fold increase in kcat. The combination of these two mutations does not increase the Ki for MPA, but does increase the Km for NAD 3-fold relative to Q277R and restores kcat to wild type levels. Q277R/A462T is the first human IMPDH mutant with increased Ki for MPA and wild type activity. The third mutant IMPDH contains two mutations, Phe465 → Ser and Asp470 → Gly. Ki for MPA is increased 3-fold in this mutant enzyme, and Km for IMP is also increased 3-fold, while the Km for NAD and kcat are unchanged. Thus increases in the Ki for MPA do not correlate with changes in Km for either IMP or NAD, nor to changes in kcat. All four of these mutations are in regions of the IMPDH that differ in mammalian and microbial enzymes, and thus can be structural determinants of MPA selectivity.

A Screening Pipeline for Antiparasitic Agents Targeting Cryptosporidium Inosine Monophosphate Dehydrogenase

Background The protozoan parasite Cryptosporidium parvum is responsible for significant disease burden among children in developing countries. In addition Cryptosporidiosis can result in chronic and life-threatening enteritis in AIDS patients, and the currently available drugs lack efficacy in treating these severe conditions. The discovery and development of novel anti-cryptosporidial therapeutics has been hampered by the poor experimental tractability of this pathogen. While the genome sequencing effort has identified several intriguing new targets including a unique inosine monophosphate dehydrogenase (IMPDH), pursuing these targets and testing inhibitors has been frustratingly difficult. Methodology and Principal Findings Here we have developed a pipeline of tools to accelerate the in vivo screening of inhibitors of C. parvum IMPDH. We have genetically engineered the related parasite Toxoplasma gondii to serve as a model of C. parvum infection as the first screen. This assay provides crucial target validation and a large signal window that is currently not possible in assays involving C. parvum. To further develop compounds that pass this first filter, we established a fluorescence-based assay of host cell proliferation, and a C. parvum growth assay that utilizes automated high-content imaging analysis for enhanced throughput. Conclusions and Significance We have used these assays to evaluate C. parvum IMPDH inhibitors emerging from our ongoing medicinal chemistry effort and have identified a subset of 1,2,3-triazole ethers that exhibit excellent in vivo selectivity in the T. gondii model and improved anti-cryptosporidial activity.

Inhibitor mediated protein degradation.

The discovery of drugs that cause the degradation of their target proteins has been largely serendipitous. Here we report that the tert-butyl carbamate-protected arginine (Boc(3)Arg) moiety provides a general strategy for the design of degradation-inducing inhibitors. The covalent inactivators ethacrynic acid and thiobenzofurazan cause the specific degradation of glutathione-S-transferase when linked to Boc(3)Arg. Similarly, the degradation of dihydrofolate reductase is induced when cells are treated with the noncovalent inhibitor trimethoprim linked to Boc(3)Arg. Degradation is rapid and robust, with 30%-80% of these abundant target proteins consumed within 1.3-5 hr. The proteasome is required for Boc(3)Arg-mediated degradation, but ATP is not necessary and the ubiquitin pathways do not appear to be involved. These results suggest that the Boc(3)Arg moiety may provide a general strategy to construct inhibitors that induce targeted protein degradation.

Glutathione (GSH)-decorated magnetic nanoparticles for binding glutathione-S-transferase (GST) fusion protein and manipulating live cells

Iron oxide-based magnetic nanoparticles (MNP) surface-decorated with glutathione (GSH) via a dopamine anchor bind to human α1-glutathione S-transferase (GST) with high affinity and specificity and are able to separate GST fusion proteins from cell lysates. Both the purified GST and the protein of interest (POI) preserve their innate properties. The conjugate of MNP and the GST fusion protein also enables magnetic manipulation of cells.

Validation of IMP dehydrogenase inhibitors in a mouse model of cryptosporidiosis

ABSTRACT Cryptosporidium parasites are a major cause of diarrhea and malnutrition in the developing world, a frequent cause of waterborne disease in the developed world, and a potential bioterrorism agent. Currently, available treatment is limited, and Cryptosporidium drug discovery remains largely unsuccessful. As a result, the pharmacokinetic properties required for in vivo efficacy have not been established. We have been engaged in a Cryptosporidium drug discovery program targeting IMP dehydrogenase (CpIMPDH). Here, we report the activity of eight potent and selective inhibitors of CpIMPDH in the interleukin-12 (IL-12) knockout mouse model, which mimics acute human cryptosporidiosis. Two compounds displayed significant antiparasitic activity, validating CpIMPDH as a drug target. The best compound, P131 (250 mg/kg of body weight/day), performed equivalently to paromomycin (2,000 mg/kg/day) when administered in a single dose and better than paromomycin when administered in three daily doses. One compound, A110, appeared to promote Cryptosporidium infection. The pharmacokinetic, uptake, and permeability properties of the eight compounds were measured. P131 had the lowest systemic distribution but accumulated to high concentrations within intestinal cells. A110 had the highest systemic distribution. These observations suggest that systemic distribution is not required, and may be a liability, for in vivo antiparasitic activity. Intriguingly, A110 caused specific alterations in fecal microbiota that were not observed with P131 or vehicle alone. Such changes may explain how A110 promotes parasitemia. Collectively, these observations suggest a blueprint for the development of anticryptosporidial therapy.