Elizabeth A. Lunney

The Role of 4-phosphonodifluoromethyl- and 4-phosphono-phenylalanine in the selectivity and cellular uptake of SH2 domain ligands

Incorporation of 4-phosphonodifluoromethyl-phenylalanine (F2Pmp) and 4-phosphono-phenylalanine into SH2 targeted peptides and peptidomimetic ligands was found to effect binding affinity and selectivity of these ligands toward the Src and Abl SH2 domains. Furthermore, dipeptide analogs containing these phosphonate amino acids were used to produce prodrugs with excellent cellular delivery and reconversion rates.

A catalytic mechanism for caspase-1 and for bimodal inhibition of caspase-1 by activated aspartic ketones

We have evaluated 619 aspartic ketones with 9 different types of prime-side groups (acyloxymethyl, aryloxymethyl, arylthiomethyl, alkylthiomethyl, acylamino-oxymethyl, sulfonylaminomethyl, alpha-ketoamide, alpha-(1-phenyl-3-trifluoromethyl-pyrazol-5-yl)oxymethyl (PTP), and aliphatic ketones) as inhibitors of caspase-1. The inhibitory behaviors could be classified as reversible, inactivating, or bimodal (i.e. reversible inhibition followed by slow inactivation) based on the kinetically observed formation of reversible thiohemiketal complexes and conversion to an irreversible thioether adduct, and the mechanism of any given ketone was only poorly predictable on the basis of leaving group structure and chemistry. Among 201 bimodal inhibitors, the rate of conversion of the reversible thiohemiketal complex to the inactive thioether (k(i)) was strictly first-order, consistent with direct conversion of the thiohemiketal to the thioether with no intermediate collapse to free ketone and thiolate. We have examined 22 crystallographic structures of caspase-1 complexed as a thiohemiketal with the inhibitors from 8 different ketone classes, and found the Cys285S-C-C(alpha)-leaving group dihedral angle to be near either to 60 degrees or to 180 degrees. Only the 180 degrees conformation was permissive for SN2 displacement of the leaving group and, furthermore, positioned His237Ndelta to stabilize developing charge on the leaving group. Among these structures and 19 additional complexes, all showed a strong interaction between His237Ndelta and the ketone or thiohemiketal oxygen. We therefore propose a proteolytic mechanism for caspase-1 involving polarization of the scissile carbonyl by the His237 imidazolium group. During thiohemiketal/thioether conversion (but probably not during peptide hydrolysis), the leaving group is stabilized by the His237 imidazolium.

Design of peptidomimetic ligands for the pp60src SH2 domain

Abstract Potent ligands of the Src SH2 domain, discovered through structure-based drug design efforts, with the general structure Ac-pTyr-Glu-NRR′ are disclosed.

Novel phosphotyrosine mimetics in the design of peptide ligands for pp60src SH2 domain

Abstract We have designed and synthesized a series of phosphorylated penta- and tri-peptides of general structures Rx-Glu-Glu-Ile-Glu and Rx-Glu- D -Trp-NH2, where Rx represents a phosphotyrosine mimetic. These peptides show binding affinity to pp60src SH2 domain in the micromolar range. Data are presented that provide an account of their structure-activity relationships and specificity properties. We have designed and synthesized a series of phosphorylated penta- and tri-peptides of general structures Rx-Glu-Glu-Ile-Glu and Rx-Glu- D -Trp-NH2, where Rx represents a phosphotyrosine mimetic. These peptides show binding affinity to pp60arc SH2 domain in the micromolar range. Data are presented that provide an account of their structure-activity relationships and specificity properties.

Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor

With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfa nyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC50 of 200 nM with a therapeutic index of > 1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. Cmax and absolute bioavailability of 34S were higher and half-life and time above EC95 were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.

Alpha-Mercaptoacrylic Acid Derivatives as Novel Selective Calpain Inhibitors

Calpain is a family of cytosolic cysteine proteases activated by calcium (1–5). There are two major isoforms, µ- and m-calpain, requiring low and high micromolar calcium for in vitro activity, respectively. Both µ- and m-calpain have a large catalytic subunit (80 kDa) and a small regulatory subunit (29 kDa). Both subunits contain EF-hand calcium-binding structures which control the proteolytic activity of the enzyme. Calpains are implicated in a number of pathological conditions, including stroke, myocardial ischemia and cataract, where intracellular calcium levels are elevated markedly (6). In cerebral ischemia, synaptic glutamate (excitotoxin) buildup leads to excessive activation of ionotropic N-methyl-D-aspartate NMDA- and AMPA/kainate-receptors (7–8). The resultant calcium influx triggers calpain activation. Calpain then degrades cytoskeletal proteins which leads to the loss of cell integrity and cell death (9). Several peptide calpain inhibitors (e.g., calpain inhibitor I (acetyl-Leu-Leu-Nle-H) and MDL28170 (carbobenzoxy-Val-Phe-H)) were found to have neuroprotective effects in various models of excitotoxicity and/or ischemia (10-12). However, these agents are nonselective and cross-inhibit other cysteine proteases. Thus, it is highly desirable to develop selective and potent calpain inhibitors.

Nonpeptidic HIV protease inhibitors: 6-Alkyl-5, 6-dihydropyran-2-ones possessing achiral 3-(4-amino/carboxamide-2-t-butyl, 5-methylphenyl thio) moiety: Antiviral activities and pharmacokinetic properties

Dihydropyran-2-ones possessing amino and carboxamide functionalities on 3-SPh (2-tert-butyl, 5-methyl) ring were synthesized and evaluated for their antiviral activities. Both the enantiomers of inhibitor 15 were synthesized. The in vitro resistance profile, inhibitory activities against cytochrome P450 isozymes and pharmacokinetic properties of inhibitor 15S will be discussed.

A topliss tree analysis of the HIV-protease inhibitory activity of 6-phenyl-4-hydroxy-pyran-2-ones

Abstract In a study of 4-hydroxy-pyran-2-ones as possible inhibitors of HIV protease, a series of compounds were synthesized following the Topliss operational scheme for substitution on a phenyl group at the 6 position of the pyrone. In addition, a number of compounds with polar substituents were made.

Design, synthesis and solution structure of a renin inhibitor Structural constraints from NOE, and homonuclear and heteronuclear coupling constants combined with distance geometry calculations

A macrocyclic renin inhibitor was designed using molecular modeling and a model of human renin. The synthesized molecule displayed poor binding affinity. To investigate the reasons for the observed inactivity, the structure of the compound has been studied by NMR spectroscopy and distance geometry. Structural constraints for distance geometry calculations were derived from nuclear Overhauser effects and homonuclear and heteronuclear three bond coupling constants. Homonuclear coupling constants were measured directly from the resolution‐enhanced proton spectra and heteronuclear coupling constants were measured from the natural abundance 15N‐ and 13C‐edited TOCSY experiments. One φ angle was determined uniquely by this method and two were reduced to two possible values each. By using a statistical analysis of 400 structures generated with distance geometry, two families of structures were found to be consistent with the NMR data. The solution structures so derived were different from the originally designed structure, including an internal hydrogen bond. This provides a possible explanation for the lack of effectiveness of this compound.

6-Phenyl-6-alkylamido-5,6-dihydro-2H-pyran-2-ones: novel HIV protease inhibitors

Abstract Publications from our laboratories have recently described a series of 3-thioaryl substituted-4-hydroxy-pyrones 1 as HIV protease inhibitors. The current work examines the analogous 5,6-dihydro-2H-pyran-2-ones with 6,6-substitutions focusing on the use of 1°, 2°, and 3° alkyl amides of various chain lengths to fill the S1 through S3 enzyme pockets.