Ann E. Eakin

Amplification and sequencing of genomic DNA fragments encoding cysteine proteases from protozoan parasites.

Cysteine protease gene fragments from three protozoan parasites Trypanosoma cruzi, Trypanosoma brucei, and Entamoeba histolytica were amplified by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotide primers. The primers used for the amplification were designed based upon amino acid sequences flanking the active site cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteases analyzed to date. The amplified DNA fragments, representing approximately 70% of the coding regions of the cysteine protease genes, were subcloned and sequenced. Sequence analysis and alignment showed significant sequence similarity to other members of the eukaryotic cysteine protease family (45% identical to chicken cathepsin L) and conservation of the cysteine, histidine, and asparagine residues which form the catalytic triad. These gene fragments provide molecular probes for further analysis of the structure and function of these important metabolic enzymes.

Atlantic cod cDNA encoding a psychrophilic chymotrypsinogen

A cDNA clone encoding a psychrophilic cod chymotrypsinogen has been isolated and characterized. The predicted amino acid sequence reveals a preproenzyme of 263 amino acids containing a unique 18 residue signal sequence. Amino acid sequence identity between the cod and mammalian chymotrypsinogens is 64-68%. Two highly conserved proline residues are substituted in cod chymotrypsin.

Investigation of the functional role of active site loop II in a hypoxanthine phosphoribosyltransferase

Hypoxanthine phosphoribosyltransferases (HPRTs) are of biomedical interest because defects in the enzyme from humans can result in gouty arthritis or Lesch-Nyhan syndrome, and in parasites these enzymes are potential targets for antiparasite chemotherapy. In HPRTs, a long flexible loop (active site loop II) closes over the active site during the enzyme catalyzed reaction. Functional roles for this loop have been proposed but have yet to be substantiated. For the present study, seven amino acids were deleted from loop II of the HPRT from Trypanosoma cruzi to probe the functional role of this active site loop in catalysis. The mutant enzyme (Deltaloop II) was expressed in bacteria, purified by affinity chromatography, and kinetic constants were determined for substrates of both forward (purine salvage) and reverse (pyrophosphorolysis) reactions catalyzed by the enzyme. Loop II deletion resulted in moderate (0.6-2.7-fold) changes in the Michaelis constants (K(m)s) for substrates other than pyrophosphate (PP(i)), for which there was a 5.8-fold increase. In contrast, k(cat) values were severely affected by loop deletion, with rates that were 240-840-fold below those for the wild-type enzyme. Together with previously reported structural data, these results are consistent with active site loop II participating in transition-state stabilization by precise positioning of the substrates for in line nucleophilic attack and in the liberation of PP(i) as a product of the salvage reaction.

Structure-based inhibitor design.

Time and costs associated with the discovery of new drugs have been significantly reduced by enzyme structure-based approaches to the discovery of new chemotherapeutic agents. However, fundamental components of the overall approach continue to rely on technologies which, by their nature, involve relatively random processes (i.e., combinatorial chemistry and high-throughput screening). Thus, the efficiency of the drug discovery process potentially could be further improved through better use of structural information. In this regard, three-dimensional structures of enzymes are now being solved at high resolution and/or in conformations that provide data that should be more useful for inhibitor design or discovery. Scientists are beginning to appreciate the importance of water as a possible competitor of inhibitors for binding to target enzymes. New computational algorithms are improving the efficiency of identifying flexible inhibitors from among the large numbers of compounds in chemical databases. Also, tools of molecular genetics together with structures of target enzymes are likely to be used more frequently in dealing with the development of resistance to novel chemotherapeutic agents. Instead of detailing success stories in structure-based drug discovery, the following article considers how future efforts to discover or design new drugs may increasingly rely on information about molecular targets and less on data acquired via approaches involving random methodologies.

Implications of selective type II IMP dehydrogenase (IMPDH) inhibition by the 6-ethoxycarbonyl-3,3-disubstituted-1,5-diazabicyclo[3.1.0]hexane-2,4-diones on tumor cell death

It was shown previously that three 1,5-diazabicyclo[3.1.0]hexane-2,4-diones selectively inhibited human Type II IMP dehydrogenase (IMPDH) from Tmolt4 cell leukemia [Barnes et al., Biochemistry 2000;39:13641-50]. The agents acted as competitive inhibitors of this isoform, yet when tested against human Type I at concentrations ranging from 0.5 to 500 microM, Type I was not inhibited. This study focuses on the antineoplastic activity and cellular effects of one of these agents and two new derivatives containing ethoxycarbonyl substitution at position C6. Agents were studied for antiproliferative activity in human Tmolt4 leukemia (EC(50) 3.3 to 9.2 microM) and alterations in the levels of enzymes involved with cellular metabolism, including DNA and RNA syntheses due to IMPDH inhibition. Results reported here demonstrate that 6-ethoxycarbonyl-3,3-disubstituted-1,5-diazabicyclo[3.1.0]hexane-2,4-diones are effective inhibitors of DNA synthesis (30-66% inhibition) due to reductions in dGTP pool levels. Collectively, the three agents proved to be selective inhibitors of human IMPDH Type II activity (K(i) 11-33 microM), leading to cytotoxicity in a number of suspended and solid tumor lines, notably MCF-7 (EC(50) 0.7 to 6.0 microM). In addition, negative cytotoxic actions of these agents on WI-38 cell growth, a normal rapidly growing human line, suggest that specific targeting of Type II IMPDH would help to eliminate cell killing in lines where Type I predominates. Furthermore, effects of agents on DNA synthesis and cell death could be reversed by the addition of exogenous guanosine to the medium. Results from in vitro studies suggest that the 6-ethoxycarbonyl-3,3-disubstituted-1,5-diazabicyclo[3.1.0]hexane-2,4-diones may be used as effective isozyme-selective chemotherapeutic agents.

LIMITED PROTEOLYSIS OF A TRYPANOSOMAL HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE YIELDS CRYSTALS THAT DIFFRACT X-RAYS TO NEAR ATOMIC RESOLUTION

Two crystal forms of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi were grown and characterized. Proteolytic modification at the C-terminus of the recombinant enzyme yielded monoclinic crystals that diffract X-rays to higher resolution than the original, trigonal crystal form. Data from the monoclinic crystal form enabled determination of the crystal structure for the trypanosomal HPRT to 1.4 A resolution.

Mechanism of action of the antitumor agents 6‐benzoyl‐3,3‐disubstituted‐1,5‐diazabicyclo[3.1.0]hexane‐2,4‐diones: Potent inhibitors of human type II inosine 5′‐monophosphate dehydrogenase

The observation that expression of the IMPDH gene is tightly linked with cellular proliferation and transformation has led to an interest in developing inhibitors that deplete intracellular guanine nucleotide pools. IMPDH exists as 2 isoforms, one of which is induced in tumor cells, type II and thus has led to new interest in this target for the design of isoform‐selective anticancer chemotherapeutic agents. Several classes of IMPDH inhibitor are now in use or under development; however, only the 1,5‐diazabicyclo[3.1.0]hexane‐2,4‐diones show selectivity for the type II isoform. In the current study, we further evaluated chemical modification of this class to determine the necessary components for selective type II IMPDH inhibition. The 6‐benzoyl‐3,3‐disubstituted‐1,5‐diazabicyclo[3.1.0]hexane‐2,4‐diones were effective cytotoxic agents in human leukemias, lymphomas, breast, glioma and HeLa‐S 3 suspended uterine carcinoma screens with ED50 values 0.3 to 12 μM. The agents acted as antimetabolites by inhibiting de novo purine biosynthesis at the key regulatory enzyme IMPDH, resulting in suppression of DNA synthesis and dGTP pool levels within 60 min. Furthermore, the derivatives were specific for the type II isoform as opposed to type I, acting in a competitive manner with Ki values of 5.1 to 63 μM. Addition of the 6‐benzoyl moiety to the bicyclic parent ring structure afforded the most potent agent in the novel class of 1,5‐diazabicyclo[3.1.0]hexane‐2,4‐diones that selectively inhibits type II IMPDH activity.