James R. Appleman

The therapeutic potential of agents acting via purine receptors

A host of physiological processes associated with the cardiovascular (CV) system, central nervous system (CNS), and a variety of other organ systems and tissues are regulated by agents, primarily adenosine (ado) and adenosine triphosphate (ATP), that act via cell-surface purine receptors. These receptors have therefore been the focus of a variety of programmes directed at the discovery and development of new therapeutic agents, most notably for the treatment of disorders of the CV system. Currently, only a handful of agents, including ado, theophylline, dipyridamole, and ticlopidine, are approved for clinical use. A variety of new agents intended for use in CV disease, disorders of the CNS, such as Parkinsons disease, treatment of pain, inflammatory disorders, and diverse other pathophysiological conditions are in clinical development. Historically, ado receptors have been the primary target. Recent research efforts have begun to examine alternative strategies including agents that modulate endogenous levels of extracellular ado and agents that act via P(2) receptors.

Mutations of human dihydrofolate reductase causing decreased inhibition by methotrexate.

Oligonucleotide-directed mutagenesis has been used by many laboratories to obtain information regarding the significance of the structural features of various enzymes. When an amino acid residue, or group of residues, is deleted from the enzyme structure, or is replaced by other residues by directed mutagenesis, inferences can be drawn regarding the relation of that portion of the structure to the catalytic activity of the enzyme. This approach has been applied extensively to the study of dihydrofolate reductase (DHFR) by Benkovic and his collaborators.

Kinetic investigation of methotrexate resistant human dihydrofolate reductase (hDHFR) mutants at Phe31.

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (H2folate) to tetrahydrofolate (H4folate). This enzyme is necessary for maintaining adequate levels of H4folate and its derivatives, which are essential for the biosynthesis of purines, thymidylate and several amino acids. This enzyme is therefore a target for anticancer drugs such as methotrexate (MTX). Cancer patients receiving high dose MTX as part of their therapy frequently develop clinical resistance and molecular mechanisms of such resistance are of interest.

Structure-based Design of Pyridone-Aminal eFT508 Targeting Dysregulated Translation by Selective Mitogen-activated Protein Kinase Interacting Kinases 1 and 2 (MNK1/2) Inhibition.

Dysregulated translation of mRNA plays a major role in tumorigenesis. Mitogen-activated protein kinase interacting kinases (MNK)1/2 are key regulators of mRNA translation integrating signals from oncogenic and immune signaling pathways through phosphorylation of eIF4E and other mRNA binding proteins. Modulation of these key effector proteins regulates mRNA, which controls tumor/stromal cell signaling. Compound 23 (eFT508), an exquisitely selective, potent dual MNK1/2 inhibitor, was designed to assess the potential for control of oncogene signaling at the level of mRNA translation. The crystal structure-guided design leverages stereoelectronic interactions unique to MNK culminating in a novel pyridone-aminal structure described for the first time in the kinase literature. Compound 23 has potent in vivo antitumor activity in models of diffuse large cell B-cell lymphoma and solid tumors, suggesting that controlling dysregulated translation has real therapeutic potential. Compound 23 is currently being evaluated in Phase 2 clinical trials in solid tumors and lymphoma. Compound 23 is the first highly selective dual MNK inhibitor targeting dysregulated translation being assessed clinically.

Design, Synthesis, and Structure-Activity Relationships of the First Highly Potent, Selective, and Bio Available Adenosine 5′-Monophosphate Deaminase Inhibitors

Structure-activity studies have been performed to optimize the potency of this novel series of AMPDA inhibitors. Conformational rigidification of the N-3 sidechain resulted in substantial effect on the potency. Addition of the hydrophobic groups provided further benefit. The most potent compound identified, 4g (Ki = 3 nM), bears little structural resemblance to AMP and exhibits a remarkable improvement (10(3) and 10(5)) in binding affinity relative to the original lead and AMP, respectively. The application of prodrug strategy achieved a large improvement (benzyl ester 5d) in oral bioavailability, resulting in compounds that should be useful in evaluating the role of AMPDA in normo- and pathophysiological states.

Use of 10-Propargyl-5,8-Dídeazafolate and Directed Mutagenesis to Probe the Catalytic Mechanism of Thymidylate Synthase

Thymidylate synthase (TS) catalyzes the conversion of dUMP and CH2H4folate to dTMP and folate by methyl group and hydride transfer. The enzyme has been well characterized from many sources including a 2.1 A resolution X-ray structure of E. coli TS (TS).1 With the exception of TS from protozoans the enzyme consists of two identical subunits with a dimeric molecular weight of approximately 60,000. Although an active site is present on both subunits, there is debate as to whether the active sites function independently, if catalytic activity alternates between subunits, or to what degree of coordination exists between subunits. It is believed that substrate binding and product release are ordered with dUMP binding first, initially to a single subunit, followed by CH2H4folate binding to the same subunit occupied by dUMP.2 Recently, however, it was suggested that the dUMP binding site is accessible in the TS-CH2H4folate-polyglutamate binary complex. 3 These results suggest that TS complexed with the polyglutamated form of CH2H4folate may represent an intermediate along the TS reaction pathway.

Methotrexate-Insensitive Mutants of Human Dihydrofolate Reductase (hDHFR) Constructed by Site Directed Mutagenesis at Phenylalanine 34

Dihydrofolate reductase (DHFR) catalyzes NADPH-dependent reduction of 7,8-dihydrofolate (H2folate) into 5,6,7,8-tetrahydrofolate (H4folate), which is essential for the synthesis of thymidylate and hence the synthesis of DNA. Since rapidly growing cells can be killed by inhibition of hDHFR by methotrexate (MTX), this and other inhibitory drugs are used in the chemotherapy of cancer. A serious problem in such chemotherapy with MTX is development of MTX-resistance in tumor cells. Spontaneous mutation in the DHFR gene is suspected to be one of the causes for such resistance.

AN IMPROVED STRATEGY FOR THE DETERMINATION OF THE ROLE IN CATALYSIS OF AMINO ACID RESIDUES IN THYMIDYLATE SYNTHASE

Publisher Summary Enzymes are unparalleled as catalysts with respect to both rate acceleration and specificity. This chapter discusses strategy that are employed to address how enzymes work and what is the physical arrangement (three-dimensional structure) of a particular enzyme of interest and how does this arrangement, including positioning of specific functional groups, catalyze the particular chemical reaction that is the enzymes raison d’etat for thymidylate synthase (TS) from Escherichia coli. This strategy is substantially better than less rigorous methods similarly based upon site-directed mutagenesis and analysis of mutant enzymes. The chapter presents results that demonstrate how this strategy works in practice for determining the catalytic function of TS residues.