Design and biological activity of (S)-4-(5-([1-(3-chlorobenzyl)-2-oxopyrrolidin-3-ylamino]methyl)imidazol-1-ylmethyl)benzonitrile, a 3-aminopyrrolidinone farnesyltransferase inhibitor with excellent cell potency.

The synthesis, structure-activity relationships, and biological properties of a novel series of imidazole-containing inhibitors of farnesyltransferase are described. Starting from a 3-aminopyrrolidinone core, a systematic series of modifications provided 5h, a non-thiol, non-peptide farnesyltransferase inhibitor with excellent bioavailability in dogs. Compound 5h was found to have an unusually favorable ratio of cell potency to intrinsic potency, compared with other known FTIs. It exhibited excellent potency against a range of tumor cell lines in vitro and showed full efficacy in the K-rasB transgenic mouse model.

Evaluation of amino acid-based linkers in potent macrocyclic inhibitors of farnesyl-protein transferase.

A series of amino acid-based linkers was used to investigate the effects of various substituents upon the potency, pharmacokinetic properties, and conformation of macrocyclic farnesyl-protein transferase inhibitors (FTIs). As a result of the studies described herein, highly potent FTIs with improved pharmacokinetic profiles have been identified.

Comparison of knowledge-based and distance geometry approaches for generation of molecular conformations.

A knowledge-based approach for generating conformations of molecules has been developed. The method described here provides a good sampling of the molecule's conformational space by restricting the generated conformations to those consistent with the reference database. The present approach, internally named et for enumerate torsions, differs from previous database-mining approaches by employing a library of much larger substructures while treating open chains, rings, and combinations of chains and rings in the same manner. In addition to knowledge in the form of observed torsion angles, some knowledge from the medicinal chemist is captured in the form of which substructures are identified. The knowledge-based approach is compared to Blaney et al.'s distance geometry (DG) algorithm for sampling the conformational space of molecules. The structures of 113 protein-bound molecules, determined by X-ray crystallography, were used to compare the methods. The present knowledge-based approach (i) generates conformations closer to the experimentally determined conformation, (ii) generates them sooner, and (iii) is significantly faster than the DG method.

3,8-Diazabicyclo[3.2.1]octan-2-one peptide mimetics: synthesis of a conformationally restricted inhibitor of farnesyltransferase.

A new synthesis of the 3,8-diazabicyclo[3.2.1]octan-2-one framework is described. Transannular enolate alkylation of piperazinone derivatives provides a flexible route to highly constrained bicyclic peptidomimetic synthons with substitution at the Calpha position. The chemistry was used to produce a conformationally constrained farnesyltransferase inhibitor, which aided the elucidation of enzyme-bound conformation.

Imidazole-containing diarylether and diarylsulfone inhibitors of farnesyl-protein transferase.

The design and syntheses of non-thiol inhibitors of farnesyl-protein transferase are described. Optimization of cysteine-substituted diarylethers led to highly potent imidazole-containing diarylethers and diarylsulfones. Polar diaryl linkers dramatically improved potency and gave highly cell active compounds.

Conformation of a novel tetrapeptide inhibitor NH2-D-Trp-D-Met-Phe(pCl)-Gla-NH2 bound to farnesyl-protein transferase.

Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the C-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to display antitumor activity in both tissue culture and animal models, and thus represent a potential therapeutic strategy for the treatment of human cancers. A synthetic tetrapeptide library, which included an expanded set of 68 L-, D- and noncoded amino acids, has been screened for inhibitors of FPTase activity. The tetrapeptide, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was shown to be competitive with the isoprenyl cosubstrate, farnesyl diphosphate (FPP) but not with the peptide substrate, the C-terminal tetrapeptide of the Ras protein. The FPTase-bound conformation of the inhibitor, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect (TRNOE) experiments. Ligand competition experiments identified the NOEs that originate from the active-site conformation of the inhibitor. Structures were calculated using a combination of distance geometry and restrained energy minimization. The peptide backbone is shown to adopt a reverse-turn conformation most closely approximating a type II' beta-turn. The resolved conformation of the inhibitor represents a distinctly different structural motif from that determined for Ras-competitive inhibitors. Knowledge of the bound conformation of this novel inhibitor provides a template and future direction for the design of new classes of FPTase antagonists.

Non-thiol 3-aminomethylbenzamide inhibitors of farnesyl-protein transferase.

The design and syntheses of non-thiol inhibitors of farnesyl-protein transferase are described. Substitutions on an imidazolylmethyl-AMBA-methionine template gave a highly potent and cell-active inhibitor.

Dorsal horn spatial representation of simple cutaneous stimuli.

A model of lamina III-IV dorsal horn cell receptive fields (RFs) has been developed to visualize the spatial patterns of cells activated by light touch stimuli. Low-threshold mechanoreceptive fields (RFs) of 551 dorsal horn neurons recorded in anesthetized cats were characterized by location of RF center in cylindrical coordinates, area, length/width ratio, and orientation of long axis. Best-fitting ellipses overlapped actual RFs by 90%. Exponentially smoothed mean and variance surfaces were estimated for these five variables, on a grid of 40 points mediolaterally by 20/segment rostrocaudally in dorsal horn segments L4-S1. The variations of model RF location, area, and length/width ratio with map location were all similar to previous observations. When elliptical RFs were simulated at the locations of the original cells, the RFs of real and simulated cells overlapped by 64%. The densities of cell representations of skin points on the hindlimb were represented as pseudocolor contour plots on dorsal view maps, and segmental representations were plotted on the standard views of the leg. Overlap of modeled and real segmental representations was at the 84% level. Simulated and observed RFs had similar relations between area and length/width ratio and location on the hindlimb: r(A) = 0.52; r(L/W) = 0.56. Although the representation of simple stimuli was orderly, and there was clearly only one somatotopic map of the skin, the representation of a single point often was not a single cluster of active neurons. When two-point stimuli were simulated, there usually was no fractionation of response zones or addition of new zones. Variation of stimulus size (area of skin contacted) produced less variation of representation size (number of cells responding) than movement of stimuli from one location to another. We conclude that stimulus features are preserved poorly in their dorsal horn spatial representation and that discrimination mechanisms that depend on detection of such features in the spatial representation would be unreliable.

On the futility of blind search: an algorithmic view of "no free lunch".

The paper is in three parts. First, we use simple adversary arguments to redevelop and explore some of the no-free-lunch (NFL) theorems and perhaps extend them a little. Second, we clarify the relationship of NFL theorems to algorithm theory and complexity classes such as NP. We claim that NFL is weaker in the sense that the constraints implied by the conjectures of traditional algorithm theory on what an evolutionary algorithm may be expected to accomplish are far more severe than those implied by NFL. Third, we take a brief look at how natural evolution relates to computation and optimization. We suggest that the evolution of complex systems exhibiting high degrees of orderliness is not equivalent in difficulty to optimizing hard (in the complexity sense) problems, and that the optimism in genetic algorithms (GAs) as universal optimizers is not justified by natural evolution. This is an informal tutorial paper--most of the information presented is not formally proven, and is either "common knowledge" or formally proven elsewhere. Some of the claims are intuitions based on experience with algorithms, and in a more formal setting should be classified as conjectures.

NMR studies of novel inhibitors bound to farnesyl-protein transferase.

Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the carboxyl-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for the membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to inhibit ras-dependent cell transformation and thus represent a potential therapeutic strategy for the treatment of human cancers. The FPTase-bound conformation of a tetrapeptide inhibitor, CVWM, and a novel pseudopeptide inhibitor, L-739,787, have been determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect experiments. Ligand competition experiments identified the NOEs that originate from the active-site conformation. Structures were calculated with the combination of distance geometry and restrained energy minimization. Both peptide backbones are shown to adopt nonideal reverse-turn conformations most closely approximating a type III beta-turn. These results provide a basis for understanding the spatial arrangements necessary for inhibitor binding and selectivity and may aid in the design of therapeutic agents.

Conformationally constrained o-tolylpiperazine camphorsulfonamide oxytocin antagonists. Structural modifications that provide high receptor affinity and suggest a bioactive conformation.

A series of new o-tolylpiperazine camphorsulfonamide OT antagonists is described. Analogs containing conformationally constrained 1-acylamino-2-propyl substituents at the camphor C2 endo position exhibit high affinity for OT and AVP-V1a receptors or high affinity and selectivity for OT receptors, depending on functionalities present in the acyl group. Determination of the preferred conformation of potency-enhancing 1-acylamino-2-propyl substituents using molecular mechanics energy calculations and X-ray crystallography, along with topological similarities to a conformationally constrained cyclic hexapeptide OT antagonist, suggests a receptor-bound conformation for this series of non-peptide OT antagonists.

Effect of template secondary structure on the inhibition of HIV-1 reverse transcriptase by a pyridinone non-nucleoside inhibitor.

The importance of RNA secondary structure on HIV-1 reverse transcriptase catalyzed polymerization and on the potency of the pyridin-2-one inhibitor 3-(4,7-dichlorobenzoxazol-2-ylmethylamino)-5-ethyl-6-meth ylpyridin-2(1H)-one, L-697,661, were investigated by employing heteromeric primer-template systems. Our data revealed that a stem-loop hairpin secondary structure in the RNA template could lead to strong hindrance of reverse transcription in the reaction catalyzed by HIV-1 reverse transcriptase resulting in the build up of intermediate-length (pause) polymerization products. The presence of L-697,661 greatly enhanced the accumulation of the pause products suggesting that the rate of enzyme translocation from the pause product might be more potently inhibited than polymerization up to the pause site. Model experiments using a synthetic RNA template containing a stem-loop hairpin revealed that the inhibitory potency of L-697, 661 increased 2-fold upon polymerization to within four bases of the secondary structure. Inhibitor potency was enhanced over 6-fold when primer-extension proceeded through the duplex region of the stem-loop.

Human immunodeficiency virus type 1 protease inhibitors: evaluation of resistance engendered by amino acid substitutions in the enzyme's substrate binding site.

The human immunodeficiency virus type 1 (HIV-1) protease is a homodimeric aspartyl endopeptidase that is required for virus replication. A number of specific, active-site inhibitors for this enzyme have been described. Many of the inhibitors exhibit significant differences in activity against the HIV-1 and HIV type 2 (HIV-2) enzymes. An initial study was conducted to ascertain the HIV-1 protease's potential to lose sensitivity to several test inhibitors while retaining full enzymatic activity. The substrate binding sites of the HIV-1 and HIV-2 enzymes are almost fully conserved, except for four amino acid residues at positions 32, 47, 76, and 82. Accordingly, recombinant mutant type 1 proteases were constructed that contained the cognate type 2 residue at each of these four positions. The substitution at position 32 resulted in a significant adverse effect on inhibitor potency. However, this substitution also mediated a noted increase in the Km of the substrate. Individual substitutions at the remaining three positions, as well as a combination of all four substitutions, had very little effect on enzyme activity or inhibitor susceptibility. Hence, the four studied active site residues are insufficient to be responsible for differences in inhibitor sensitivity between the HIV-1 and HIV-2 proteases and are unlikely to contribute to the generation of inhibitor-resistant mutant HIV-1 protease.

1-((7,7-Dimethyl-2(S)-(2(S)-amino-4-(methylsulfonyl)butyramido)bicyclo [2.2.1]-heptan-1(S)-yl)methyl)sulfonyl)-4-(2-methylphenyl)piperaz ine (L-368,899): an orally bioavailable, non-peptide oxytocin antagonist with potential utility for managing preterm labor.

Modifications to the previously reported spiroindenylpiperidine camphor-sulfonamide oxytocin (OT) antagonist L-366,509 have produced a new series of o-tolylpiperazine (TP) camphor-sulfonamides. A number of analogues in the TP series that incorporate a modified or unmodified L-methionine sulfone amide at the C2 endo position on the camphor ring exhibit high affinity for OT receptors (IC50 = 1.3-15 nM) and good selectivity for binding to OT versus arginine vasopressin V1a and V2 receptors. Several of these analogues were additionally characterized as potent antagonists of OT-stimulated contractions of the isolated and/or in situ rat uterus. Compound 7 (L-368,899) exhibited the best overall profile of OT receptor affinity (IC50 = 8.9 nM, rat uterus; 26 nM, human uterus), potency for inhibition of OT-stimulated contractions of the isolated rat uterus (pA2 = 8.9) and in situ rat uterus (AD50 = 0.35 mg/kg after intravenous (i.v.) administration and 7.0 mg/kg after intraduodenal administration), aqueous solubility (3.7 mg/mL at pH 5.0), and oral bioavailability in several species (35% (rat), 25% (dog), and 21% (chimpanzee) as estimated from radioreceptor determination of drug levels in plasma after oral and i.v. dosing). On the basis of these favorable properties, 7 has begun clinical testing for use as an oral and i.v. tocolytic agent. Molecular modeling alignment studies have provided support for the hypothesis that the TP camphor-sulfonamide portion of the non-peptide structures may serve as a mimetic of the important D-AA2-Ile3 dipeptide (AA = aromatic amino acid) found in many potent OT antagonists from the cyclic hexapeptide and OT analogue structural classes.

Carboxylic acid replacement structure-activity relationships in suosan type sweeteners. A sweet taste antagonist. 1.

N-(4-Cyanophenyl)-N'-(2-carboxyethyl)urea (2), an analogue of suosan [1,N-(4-nitrophenyl)-N'-(2-carboxyethyl)urea], is a known high-potency sweetener derived from beta-alanine. Sulfonic and phosphonic acid analogues of 2 were prepared to develop structure-activity relationships through modification of the carboxylic acid region of this family of sweeteners. Neither of the carboxylic acid replacements resulted in sweet analogues. However, we found that N-(4-cyanophenyl)-N'-[(sodiosulfo)methyl]urea (7) is an antagonist of the sweet taste response. The bitter taste response to caffeine, quinine, and naringin was also antagonized. Antagonist 7 was found to inhibit the sweet taste perception of a variety of sweeteners. Antagonist 7 had no effect on the sour or salty taste response.