Stereoselectivity and affinity in molecular pharmacology. IV. A search for eudismic-affinity correlations among angiotension II analogues

In order to establish the applicability of eudismic analysis (correlation between potency and the ratio of isomer potencies), the extensive data reported on the biological activities of angiotensin II analogues were examined. For a series of 13 heptapeptides epimeric at the N-terminal residue, apparent correlations were found for pressor activity in vivo, but not for 10 of them on the basis of their myotropic activity under in vitro conditions. The analogues belongning to sub-groups displaying correlations have side chains of sufficiently different nature so that their grouping is questionable. 3 octapeptides epimeric at the second residue displayed an excellent correlation for their pressor activity. The demonstrated differential metabolic susceptibility of these epimers, however, casts further doubt on the usefulness of the data as a measure of their potency. The lack of a correlation under in vitro conditions would indicate that eudismic-affinity correlations (EAC) may be limited to epimers which differ less in their properties than these angiotensin analogues. Nevertheless, until better receptor affinity estimates become available, the observed correlations may be useful in advancing our knowledge of the angiotensin II receptor in particular, and of stereoselective molecular recognition in general.     

Synthesis and biological activity of seventeen analogues of human insulin

We synthesized seventeen analogues of human insulin, applying the principle of stepwise, selective formation of the disulphide bonds. Most of these analogues only differ from human insulin in the replacement of a single amino acid in positions 2, 5, 6, 7, 8 and 11 of the A chain and 5, 7, 13 and 16 of the B-chain. The influence of these modifications on the physicochemical properties of the analogues is discussed. Eight analogues could be crystallized. All the analogues produce the same biological effects as insulin, but differ markedly in their potency. In isolated fat cells in vitro, [HisA8]insulin showed a relative potency of 2.46 in stimulating glucose oxidation (human insulin = 1), whereas [D-CysA6,A11]insulin had a potency of only 0.00027. Very low potency was observed when IleA2 or the half-cystines A6, A7, A11 or B7 were modified. Replacement of the invariant GlnA5 by alanine only reduced potency slightly. All the analogues are full agonists. The effects of the analogues on glucose oxidation and lipolysis are correlated, supporting the view that they are mediated by a common receptor on the fat-cell membrane. Hypoglycaemic potencies in the rat were similar to potencies in vitro. As expected, no correlation was demonstrable between antiserum binding--measured in the radioimmunoassay--and biological activity. Several results of this investigation are difficult to reconcile with the current view regarding the structure-activity relationship of insulin which appears to require further refinement.     

Antiviral potency of adenosine analogues: correlation with inhibition of S-adenosylhomocysteine hydrolase

For a series of adenosine analogues a close correlation (r = 0.986) was found between their antiviral potency (against vesicular stomatitis virus) and their inhibitory effects (Ki/Km) on S-adenosylhomocysteine (AdoHcy) hydrolase; thus, in order of increasing inhibitory potency for both virus replication and AdoHcy hydrolase activity: (S)-9-(2,3-dihydroxypropyl)adenine less than (RS)-3-adenin-9-yl-2-hydroxypropanoic acid (isobutyl ester) less than carbocyclic 3-deazaadenosine less than neplanocin A. Our findings point to AdoHcy hydrolase as the target for the broad-spectrum antiviral activity of these adenosine analogues.     

Mutation spectra in Salmonella of analogues of MX: implications of chemical structure for mutational mechanisms

We determined the mutation spectra in Salmonella of four chlorinated butenoic acid analogues (BA-1 through BA-4) of the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and compared the results with those generated previously by us for MX and a related compound, MCF. We then considered relationships between the properties of mutagenic potency and mutational specificity for these six chlorinated butenoic acid analogues. In TA98, the three most potent mutagens, BA-3, BA-4, MX, and the organic extract, all induced large percentages of complex frameshifts (33-67%), which distinguish these agents from any other class of compound studied previously. In TA100, which has only GC sites for mutation recovery, >71% of the mutations induced by all of the agents were GC-->TA transversions. The availability of both GC and TA sites for mutation in TA104 resulted in greater distinctions in mutational specificity than in TA100. MX targeted GC sites almost exclusively (98%); the structurally similar BA-4 and BA-2 produced mutations at similar frequencies at both GC and AT sites; and the structurally similar BA-3 and BA-1 induced most mutations at AT sites (69%). Thus, large variations in structural properties influencing relative mutagenic potency appeared to be distinct from the more localized similar structural features influencing mutagenic specificity in TA104. Among a set of physicochemical properties examined for the six butenoic acids, a significant correlation was found between pK(a) and mutagenic potency in TA100, even when the unionized fraction of the activity dose was considered. In addition, a correlation in CLOGP for BA-1 to BA-4 suggested a role for bioavailability in determining mutagenic potency. These results illustrate the potential value of structural analyses for exploring the relationship between chemical structure and mutational mechanisms. To our knowledge, this is the first study in which such analyses have been applied to structural analogues for which both mutagenic potency and mutation spectra date were available.     

Determination of binding affinities of retinoids to retinoic acid-binding protein and serum albumin

Binding affinities of retinoic acid and its synthetic analogues to intracellular retinoic acid-binding protein, which is a possible candidate for mediating their biological function, and to serum albumin, the plasma transport protein, were evaluated. A quantitative method involving elimination of interfering serum albumin by immunoprecipitation was developed to measure the binding efficiency of these retinoids, some of which are active in modifying epithelial differentiation and preventing tumorigenesis. Two cyclopentenyl analogues of retinoic acid and 13-cis-retinoic acid showed, like retinoic acid, a binding efficiency of 100% for the cellular binding protein. With the phenyl, dichlorophenyl and trimethylmethoxyphenyl analogues of retinoic acid, the binding efficiency increased as the substituents on the aromatic ring increased; thus the trimethylmethoxyphenyl analogue binds almost as efficiently as retinoic acid itself. However, the trimethylmethoxyphenyl analogue with a sulphur atom on the side chain has a much decreased binding affinity. The correlation noticed between the binding efficiency of these retinoids and their biological activity in differentiation and/or in the control of tumorigenesis particularly enhances the confidence in the present method of determining the relative binding efficiencies. None of the vitamins, hormones and cofactors tested, showed appreciable affinity for the retinoic acid-binding site. Studies on binding of retinoic acid and its analogues to serum albumin indicate that no correlation exists between binding affinity for albumin and their biological potency.     

Halogenated tryptophan derivatives disrupt essential transamination mechanisms in bloodstream form Trypanosoma brucei

Amino acid metabolism within Trypanosoma brucei, the causative agent of human African trypanosomiasis, is critical for parasite survival and virulence. Of these metabolic processes, the transamination of aromatic amino acids is one of the most important. In this study, a series of halogenated tryptophan analogues were investigated for their anti-parasitic potency. Several of these analogues showed significant trypanocidal activity. Metabolomics analysis of compound-treated parasites revealed key differences occurring within aromatic amino acid metabolism, particularly within the widely reported and essential transamination processes of this parasite.     

A spectroscopic study of the binding of N-7-substituted cap analogues to human protein synthesis initiation factor 4E

The binding of N-7-substituted cap analogues to eIF-4E from human erythrocytes is described. Data presented here indicate that there is a correlation between the tightness of binding of these cap analogues to eIF-4E and their potency as inhibitors of protein synthesis. This result indicates that the inhibitory activity of the cap analogues is strictly a function of the affinity of the analogue for eIF-4E under equilibrium conditions. The pH dependence of binding of the cap analogues to eIF-4E indicates that the enolate form of the cap is preferred, as originally postulated by Rhoads et al. [(1983) Biochemistry 22, 6084-6088]. Data indicate that there are differences in the mode of binding of alkyl-substituted and aryl-substituted cap analogues to eIF-4E arising from favorable interactions of the phenyl ring with the guanosine moiety. These differences may explain the enhanced recognition of the aryl-substituted cap analogues by eIF-4E.     

A comparative study of the permeation of dihexadecyl phosphate vesicles by various carboxylic acids and some of their tetrazole analogues

The evaluation of the effect that perturbers may have on a membrane model is described. The model was made from dihexadecyl phosphate bilayers. The perturbers used were carboxylic acids and some of their tetrazole analogues. The results show a good correlation between the permeation properties of the carboxylic acids and tetrazole analogues. Moreover, this study reveals that membrane-perturbing effects are mainly under the control of conformational parameters and dependent on the carbon chain length of the permeants.     

Comparative biological activities of potent analogues of alpha-melanotropin. Effect of nonaromatic and para substituted aromatic amino acids at position 7

Several alpha-melanotropin (alpha-MSH) analogues with para substituted aromatic and nonaromatic amino acids in the 7-position of the hormone were prepared and their melanotropic activities determined in the frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays. D and L-Phe(p-NO2), D- and L-Tyr, D- and L-Ala, and Gly were substituted in the 7-position. The use of substituted D or L-aromatic amino acids in the 7th position of the central Ac-[Nle4]-alpha-MSH4-11-NH2 fragment resulted in a loss in potency relative to the corresponding phenylalanine-containing analogue. The loss in potency cannot be due entirely to steric hindrance at the melanophore receptor, since nonaromatic amino acids substituted in the 7th position of this octapeptide fragment also generally led to a loss in biological activity. We reported previously that replacement of phenylalanine-7 by its D enantiomer led to a marked increase in potency in each fragment analogue tested. Analogues containing other D amino acids in the 7th position also were more potent than their L amino acid-containing analogues with one exception: Ac-[Nle4, Ala7]-alpha-MSH4-11-NH2 was more potent than Ac-[Nle4, D-Ala7]-alpha-MSH4-11-NH2 in the frog skin bioassay. Replacement of phenylalanine-7 by glycine resulted in a large decrease in potency in both bioassays, illustrating the importance of the side chain group, in this position of alpha-MSH, to biological potency of the hormone.     

Functionalized amino acid anticonvulsants: synthesis and pharmacological evaluation of conformationally restricted analogues

Proven conformationally restricted analogues of anticonvulsant functionalized amino acids (FAAs) were prepared using short-range cyclizations and evaluated in pharmacological assays providing new information concerning the structural requirements for FAA function.     

Biological and biochemical properties of human rasH genes mutated at codon 61

Using site-directed mutagenesis, we have introduced mutations encoding 17 different amino acids at codon 61 of the human rasH gene. Fifteen of these substitutions increased rasH transforming activity. The remaining two mutants, encoding proline and glutamic acid, displayed transforming activities similar to the normal gene. Overall, these mutants vary over 1000-fold in transforming potency. Increased levels of p21 expression were required for transformation by weakly transforming mutants. The mutant proteins were unaltered in guanine nucleotide binding properties. However, all 17 different mutant proteins displayed equivalently reduced rates of GTP hydrolysis, 8- to 10-fold lower than the normal protein. There was no quantitative correlation between reduction in GTPase activity and transformation, indicating that reduced GTP hydrolysis is not sufficient to activate ras transforming potential.     

Methionine enkephalin and isosteric analogues. Part II.: Receptor conformation of methionine enkephalin

Biological activities are reported for two different types of analogues of methionine enkephalin. Cyclic analogues, bridged between the amino- and carboxy- terminals of the parent peptide, are inactive. In contrast, significant levels of activity are displayed by linear isosterically modified analogues in which the Tyr1-Gly2 peptide bond is replaced by either -CH2NH- or -CH2CH2-. Similar replacements of the Gly2-Gly3 peptide bond yield compounds with much reduced potency. These modifications serve as useful probes of the receptor conformation. Based on these findings, a model is proposed for interaction between enkephalin and its receptor.     

Evidence for the pharmacological relevance of benzodiazepine receptors to anticonvulsant activity

Each of a series of benzodiazepines was found to be effective in preventing convulsions evoked by intermittent photic stimulation of epileptic chickens. There was a high correlation between the anticonvulsant potencies (mean effective dosages) and the affinity of the agents for the putative benzodiazepine receptor as measured by displacement of [3H]diazepam from binding sites on chicken synaptosomal membranes. This correlation in a genetic model of epilepsy provides further evidence that benzodiazepines exert their anticonvulsant effects by interacting with the benzodiazepine receptor.     

Antimycobacterial and H1-antihistaminic activity of 2-substituted piperidine derivatives

2-Substituted derivatives of the antihistaminic agents Bamipine, Diphenylpyraline and of their 1-phenyl analogues were tested for their antimycobacterial and H(1)-antagonistic activities. They are strong H1-receptor antagonists and also inhibit the growth of mycobacterials with a maximum MIC of 6.25 microg/mL against Mycobacterium tuberculosis H(37)Rv. H1-receptor antagonistic potency was slightly decreased by substitution in ring position 2 and distinctly diminished by N-aryl substitution. The antimycobacterial potency of Diphenylpyraline was in general increased by substitution in ring position 2, whereas only a few Bamipine derivatives showed markedly improved activity. A correlation between the two activities was not detected for those compounds.     

Design,synthesis and biological evaluation of amino acids-oleanolic acid conjugates as influenza virus inhibitors

Viral entry inhibitors are of great importance in current efforts to develop a new generation of anti-influenza drugs. Inspired by the discovery of a series of pentacyclic triterpene derivatives as entry inhibitors targeting the HA protein of influenza virus, we designed and synthesized 32 oleanolic acid (OA) analogues in this study by conjugating different amino acids to the 28-COOH of OA. The antiviral activity of these compounds was evaluated in vitro. Some of these compounds revealed impressive anti-influenza potencies against influenza A/WSN/33 (H1N1) virus. Among them, compound 15a exhibited robust potency and broad antiviral spectrum with IC₅₀ values at the low-micromolar level against four different influenza strains. Hemagglutination inhibition (HI) assay and docking experiment indicated that these OA analogues may act in the same way as their parent compound by interrupting the interaction between HA protein of influenza virus and the host cell sialic acid receptor via binding to HA, thus blocking viral entry.     

Ceruletide, ceruletide analogues and cholecystokinin octapeptide (CCK-8): effects on isolated intestinal preparations and gallbladders of guinea pigs and mice

The smooth muscle stimulatory effects of cholecystokinin octapeptide (CCK-8), ceruletide (CER), ten analogues of CER, and carbachol were studied in isolated organs of the guinea pig and the mouse (stomach, ileum, duodenum, colon and gallbladder). On a molar basis, CCK-8 and CER had in all organs except stomach greater potency (lower EC50) than carbachol. The effectiveness (Emax) of CCK-8 and CER was in the gut less than that of carbachol, in the guinea pig gallbladder equal with and in the mouse gallbladder superior to that of carbachol. The alteration of peptide structure was virtually without influence on effectiveness; however, it greatly modified the potency and the organ selectivity of the effect. There was no clear-cut correlation between the potency to stimulate smooth muscle and to alter the behavior of the mouse.     

Probing the steric requirements of the γ-aminobutyric acid aminotransferase active site with fluorinated analogues of vigabatrin

We have synthesized three analogues of 4-amino-5-fluorohexanoic acids as potential inactivators of γ-aminobutyric acid aminotransferase (GABA-AT), which were designed to combine the potency of their shorter chain analogue, 4-amino-5-fluoropentanoic acid (AFPA), with the greater enzyme selectivity of the antiepileptic vigabatrin (Sabril®). Unexpectedly, these compounds failed to inactivate or inhibit the enzyme, even at high concentrations. On the basis of molecular modeling studies, we propose that the GABA-AT active site has an accessory binding pocket that accommodates the vinyl group of vigabatrin and the fluoromethyl group of AFPA, but is too narrow to support the extra width of the distal methyl group in the synthesized analogues.     

A comparison of structure-activity relationships within alpha-MSH on melanophores of Anolis carolinensis and Rana pipiens

We have compared the structure-function relationship of the tridecapeptide alpha-melanocyte stimulating hormone (alpha-MSH) on the melanophores of the lizard Anolis carolinensis and the frog Rana pipiens by determining the melanosome-dispersing potency of 15 shorter peptide sequences and 8 substituted alpha-MSH analogues. Major differences were found between the lizard and the frog in their response to alpha-MSH peptide fragments and analogues. In Anolis, the sequence Ser-Tyr-Ser- is not as important for the pigmentary response as in Rana since alpha-MSH-(4-13) was nearly as potent (89%) as alpha-MSH-(1-13) (100%), whereas in Rana alpha-MSH-(4-13) potency was reduced to 7.5%. In addition, loss of potency due to removal of residues Pro and Val was more marked in Rana (alpha-MSH-(1-11) = 0.1%) than in Anolis (alpha-MSH-(1-11) = 1%), suggesting that this C-terminal sequence is necessary for pigmentary activity in the frog melanophore. These results together with those of other peptide fragments and analogues have led us to define the minimal pigmentary sequence of alpha-MSH as alpha-MSH-(4-12) in Anolis in contrast to alpha-MSH-(1-13) in Rana. This suggests that Anolis and Rana alpha-MSH receptors recognise different message amino acids of the alpha-MSH peptide sequence even though the final response (melanosome dispersion) is the same.     

Designing synthetic superagonists of C3a anaphylatoxin

An extensive structure-activity study of synthetic analogues of the C3a anaphylatoxin was conducted. Our goal was to map C3a-C3a receptor interactions by designing synthetic analogue molecules having maximal biologic potency. Nonspecific binding of the polycationic C3a to polyanionic molecules on cellular surfaces often obscures specific binding to the receptor. Less cationic synthetic C3a analogues would be useful tools in identifying and characterizing the various cell types having C3a receptors. These factors should also be useful as pharmacologic probes for mechanism studies, as high-affinity ligands for target cell identification, and for receptor isolation. Attachment of amino-terminal hydrophobic groups such as Fmoc to C3a analogues [as orginally introduced by Gerardy-Schahn et al. (1988) Biochem. J. 255, 209] markedly enhanced the potency of synthetic C3a peptides. The enhancement effect on potency from introducing hydrophobic groups to C3a analogues was interpreted as possibly being nonspecific. Our systematic search for an optimal peptide length, composition, and N-terminal hydrophobic unit resulted in several superpotent C3a analogues having 200-1500% the potency of natural C3a. One particularly potent C3a peptide was designed by incorporating two tryptophanyl residues at the N-terminal end of a 15-residue C3a analogue. The superpotent peptide W-W-G-K-K-Y-R-A-S-K-L-G-L-A-R has several residues differing (underlined) from the sequence corresponding to positions 63-77 in human C3a, a region that contains the essential functional site of the molecule. This 15-residue model peptide exhibited the greatest biological potency of all peptides tested, being 12-15 times more active than natural C3a. Since an optimal distance was found to exist between the N-terminal hydrophobic unit (W-W) and the C-terminal primary binding site (LGLAR), we concluded that the hydrophobic unit interacts specifically with a secondary binding site on the C3a receptor. The presence of both a primary (effector) and secondary (hydrophobic) binding site on these linear synthetic ligands, which can interact cooperatively with the C3a receptor, presumably accounts for the high relative potency of the analogues. Our design of superpotent analogues of C3a demonstrates the feasibility for constructing small synthetic peptides to mimic natural biologic factors that depend on secondary or tertiary structure for their activity. These synthetic peptide studies demonstrate that a linear array of amino acids (e.g., W-W) can successfully substitute for a conformation-dependent binding site on a bioactive factor.     

Synthesis and immunobiological activity of base substituted 2-amino-3-(purin-9-yl)propanoic acid derivatives

2-Amino-3-(purin-9-yl)propanoic acids substituted at position 6 of the purine base moiety by dimethylamino, cyclopropylamino, pyrrolidin-1-yl, hydroxy, and sulfanyl group as well as their 2-aminopurine analogues were prepared from corresponding 9-(2,2-diethoxyethyl)purines and 2-aminopurines, respectively, by the Strecker synthesis. 2-Aminopropanoic acid derivatives were tested for their immunostimulatory and immunomodulatory potency. Some of these compounds significantly enhanced secretion of chemokines RANTES and MIP-1alpha, the most potent was 2-amino-6-sulfanylpurine derivative. Most of these compounds also augmented NO biosynthesis triggered primarily by IFN-gamma.