Modest Inhibitory Activity Research Articles

Synthesis of conformationally constrained potential inhibitors of mammalian metalloproteinases

The synthesis of carbocyclic molecules containing functional groups capable of interacting with appropriate binding sites in certain metalloproteinases is described. Preliminary biological evaluation reveals modest inhibitory activity for a thioether analog in the trisubstituted cyclopropane series.

Solution Phase Synthesis of Amide-Linked N-Acetyl Neuraminic Acid, α-Amino Acid, and Sugar Amino Acid Conjugates1

Peracetylated N-acetylneuraminic acid (NeuAc) was efficiently coupled to esters of glycine, alanine, and serine using BOP and HOBT in the presence of DIEA. Deprotection of the esters readied the NeuAc-α-amino acid conjugates for further elaboration. Coupling of the NeuAc-gly adduct with β-O-methoxy neuraminic acid methyl ester afforded a selectively protected glycine linked sialic acid dimer. 2-Amino-3,4-di-O-benzyl-(1→6)-anhydroglucose and alanine benzyl ester were also efficiently coupled to the bis adduct giving novel trimeric analogs. Elimination of the anomeric acetate from the NeuAc-gly dimer followed by global deprotection provided a novel saccharopeptide with modest clostridial sialidase inhibitory activity.

Development of two monoclonal antibodies against Plasmodium falciparum sporozoite surface protein 2 and mapping of B-cell epitopes.

The Plasmodium yoelii sporozoite surface protein 2 (PySSP2) is the target of protective cellular immunity. Cytotoxic T cells specific for the Plasmodium falciparum analog PfSSP2, also known as thrombospondin-related anonymous protein (TRAP), are induced in human volunteers immunized with irradiated sporozoites. PfSSP2 is an important candidate antigen for a multicomponent malaria vaccine. We generated and characterized three monoclonal antibodies (MAbs) specific for PfSSP2/TRAP. The MAbs PfSSP2.1 (immunoglobulin G1 [IgG1]), PfSSP2.2 (IgG2a), and PfSSP2.3 (IgM) were species specific and identified three distinct B-cell epitopes containing sequences DRYI, CHPSDGKC, and TRPHGR, respectively. PfSSP2.1 partially inhibited P. falciparum liver-stage parasite development in human hepatocyte cultures (42 and 86% in two experiments at 100 microg/ml). Mice immunized with vaccinia virus expressing full-length PfSSP2 protein produced antibodies to (DRYIPYSP)3, and humans living in malaria-endemic areas (Indonesia and Kenya), who have lifelong exposure and partial clinical immunity to malaria, had antibodies to both (DRYIPYSP)3 and (CHPSDGKCN)2. Mice immunized with multiple antigen peptides MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSDGKCN)3P2P30 in TiterMax developed antibodies to sporozoites that partially inhibited sporozoite invasion of human hepatoma cells (39 to 71% at a serum dilution of 1:50 in three different experiments). The modest inhibitory activities of the MAbs and the polyclonal antibodies to PfSSP2/TRAP epitopes do not suggest that a single-component vaccine designed to induce antibodies against PfSSP2/TRAP will be protective. Nonetheless, the MAbs directed against PfSSP2, and the peptides recognized by these MAbs, will be essential reagents in the development of PfSSP2/TRAP as a component of a multivalent P. falciparum human malaria vaccine.

Structure-activity relationships for the 8-alkylpterins: a new class of mechanism-based substrates for dihydrofolate reductase (DHFR).

The substrate activity with both human and chicken dihydrofolate reductases (DHFR) has been examined for a series of 8-alkylpterins, 6-methyl-8-alkylpterins, and 7-methyl-8-propylpterin. All the 8-alkylpterins exhibited substrate activity with Vmax/[E]o values ranging from 1.0 to 5.4 and 2.6 to 14.8 s-1 for chicken and human DHFRs, respectively, with activity varying in the order 8-methyl > 8-allyl > 8-isopropyl > or = 8-ethyl > or = 8-propyl for both enzymes. Km values were found to range from 6.2 to 47 and 14 to 261 microM for chicken and human DHFRs, respectively, with the strength of binding varying in the order 8-propyl > 8-isopropyl > 8-ally > 8-methyl > 8-ethyl for both enzymes. Addition of a 6-methyl substituent affected the activity of the 8-alkylpterins significantly. While 6,8-dimethylpterin was a much better substrate than 8-methylpterin, the 6-methyl-8-propyl, 6-methyl-8-allyl, and 6-methyl-8-isopropyl compounds showed no substrate activity and 6-methyl-8-ethylpterin showed very weak activity with chicken enzyme only. 7-Methyl-8-propylpterin showed no substrate activity. Thermodynamic dissociation constants (Kd) for the compounds in binary complex with both human and chicken DHFRs ranged from 23 to 351 and 15 to 127 microM, respectively. Trends for the KdS were consistent with the kinetic data in suggesting stronger binding for 8-alkylpterins with larger 8-substituents. Comparison of Kd values with corresponding Km values suggested both strong cooperativity (6,8-dimethylpterin) and antagonism (6-methyl-8-isopropylpterin) with NADPH in binding to DHFR. Kd values of 20 and 10 microM for the ternary complexes of 7-methyl-8-propylpterin with human and chicken enzyme, respectively, suggest modest inhibitory activity. Application of molecular graphics modeling of ligands in the DHFR binding site has provided insight in interpreting the structure-activity relationships. The finding that different binding orientations are possible for ligands with small (8-methyl) or larger (e.g., 8-propyl) 8-substituents helps to explain the 6-methyl substituent effect and the transition from weak binding and high activity to tight binding and low activity as a function of ring-substituent pattern.

Functionalized depsipeptides, substrates and inhibitors of β-lactamases and DD-peptidases

A series of derivatives of phenyl phenylacetylglycinates (aryl phenaceturates) with a carboxylate substituent meta to the oxygen of the phenoxide leaving group and a functionalized methylene group in the ortho- or para-position have been synthesized. These molecules possess a latent o- or p-quinone methide electrophile which could be unmasked during enzymic turnover and could react with an active site nucleophile. This chemistry does seem to occur in solution where a common hydrolysis product, independent of the benzylic leaving group, presumably o- or p-hydroxymethylphenol, was observed. These depsipeptides are substrates of class A and C β-lactamases, particularly of the latter, comparable with the parent m-carboxyphenyl phenaceturate. They also have modest inhibitory activity against these enzymes and against the serine DD-peptidase of Streptomyces R61. The inhibition of a class C β-lactamase was turnover dependent, as expected of mechanism-based inhibitor, but the small leaving group dependence of the inhibition suggested that the quinone methide, if it was in fact responsible for the inhibition, was generated in solution subsequent to release of the product phenol from the active site.

8‐Azaguanine 2′,3′‐Dideoxyribonucleosides: Glycosylation of the 5‐amino‐7‐methoxy‐3H‐1,2,3‐triazolo[4,5‐d]pyrimidinyl anion with 2,3‐dideoxy‐D‐glycero‐pentofuranosyl chloride

AbstractThe synthesis of the regioisomeric 8‐azaguanine N7‐, N8‐, and N9‐(β‐D‐2′,3′‐dideoxyribonucleosides) (1, 2, and 3, respectively) and of the diamino derivative 13 is described. The anion of 5‐amino‐7‐methoxy‐3H‐1,2,3‐triazolo[4,5‐d]pyrimidine (5) was glycosylated with 5‐O‐[(tert‐butyl)dimethylsilyl]‐2,3‐dideoxy‐D‐glycero‐pentofuranosyl chloride (6; anomeric mixture), yielding the regioisomeric 2′,3′‐dideoxyribofuranosides as anomeric mixtures 7a/10a, 8a/11a, and 9a/12a. They were desilylated with Bu4NF in THF affording the 5‐amino‐7‐methoxy‐nucleosides 7b–12b. Treatment with aqueous NaOH gave the 8‐azaguanine β‐D‐2′,3′‐dideoxynucleosides 1–3 and their α‐D‐anomers 14–16. The reaction of 7b with NH3/MeOH yielded the diamino compound 13. The N‐glycosylic bond of 8‐aza‐2′,3′‐dideoxyguanosine (1) is four‐times more stable against acid than that of 2′,3′‐dideoxyguanosine. Compounds 1, 2, and 13 were converted to their 5′‐triphosphates 17–19 which showed only modest inhibitory activity against HIV‐reverse transcriptase.

Effect of calcium binding protein modulators on myofibrillar MgATPase activity and cGMP‐inhibitable phosphodiesterase activity from human cardiac muscle

AbstractCardiac muscle force development and shortening result from the interaction between actin and myosin within the myofibrillar contractile unit. This interaction is dependent upon intracellular Ca2+ and is controlled by the troponin‐tropomyosin regulatory proteins situated along the length of the actin thin filament. Enhancing the Ca2+ sensitivity of cardiac contractile protein interactions has been proposed as a novel mechanism for some positive inotropic agents. These myofibrillar Ca2+ sensitizers have variable effects on myofibrillar MgATPase activity among nonhuman animal species, and different effects between myofibrils from failed nonhuman hearts are evident. In this study, we have evaluated several myofibrillar calcium sensitizers, at maximal testable concentrations, for their effects on the calcium‐activated MgATPase of myofibrils prepared from normal human hearts and from hearts of patients with congestive heart failure. Bepridil (100 μM) increased MgATPase at pCA 9.0 (basal activity) and pCa 6.5 (an intermediate concentration of Ca2+ which stimulated activity 42–56% of maximal), with no effect at pCa 5.0 (maximal Ca2+ activity) in both normal and failing hearts. APP 201–533 (300 μM) increased MgATPase at pCa 6.5 in all hearts, but increased MgATPase at pCa 5.0 only in normal hearts. Pimobendan (100 μM) increased MgATPase at pCa 6.5 only in normal hearts and increased activity at pCa 5.0 only in failing hearts. In contrast, trifluoperazine (100 μM) reduced MgATPase at pCa 5.0 and pCa 9.0 in all hearts and at pCa 6.5 in failing hearts. The stimulating effects of bepridil, APP 201–533, and pimobendan (approximately 7–17%) were less than those observed in studies with nonhuman cardiac myofibrils (typically 30–40%). These data suggest that some myofibrillar Ca2+ sensitizers can have either modest stimulatory or inhibitory activity on human cardiac myofibrillar MgATPase activity. While these (Ca+‐sensitizing) effects on MgATPase activity are statistically significant, the biological relevance with respect to enhancing cardiac contractility is equivocal. Furthermore, both pimobendan and APP 201–533 inhibited cAMP‐phosphodiesterase III purified from failing human hearts with IC50 values of 0.35 and 2.0 μM, respectively. Since other inotropic mechanisms, such as cAMP‐phosphodiesterase inhibition, are also evident at much lower concentrations with some of these agents, the relevance of myofibrillar Ca2+ sensitization as the predominant inotropic mechanism of action of these compounds is questionable. © 1993 Wiley‐Liss, Inc.

Potentiation of the somatogenic and lactogenic activity of human growth hormone with monoclonal antibodies

Monoclonal antibodies of certain epitope specificity have been shown to produce a marked dose-dependent enhancement of the somatogenic and lactogenic activity of human GH (hGH). Two antibodies (EB1 and EB2), binding to distinct antigenic determinants and expressed on both hGH and human chorionic somato-mammotrophin (hCS), significantly enhanced the hGH-stimulated uptake of 35S-labelled sulphate into cartilage. Similarly, these antibodies enhanced the lactogenic activity of both hGH and hCS in the pigeon crop sac test. Two hGH specific monoclonal antibodies (QA68 and NA71), defining a further two epitopes, exhibited only modest enhancing or inhibitory activity in these assays, whereas the binding of certain combinations of monoclonal antibodies resulted in either reversal of enhancement or inhibition of hormone activity. Univalent antibody fragments derived from EB1 were as enhancing as the intact antibody indicating that bivalency dependent mechanisms were not involved in the phenomenon. Enhancing monoclonal antibodies were relatively poor inhibitors of 125I-labelled hGH binding to liver microsomal receptors, which is in contrast with their previously described property of potent suppression of hGH interaction with lymphoid cell receptors. It is tentatively concluded that 'restriction' of hormone binding to particular hGH receptors, relevant to somatic growth or lactogenic activity, may play a role in the enhancement phenomenon of hGH in vivo.

ChemInform Abstract: AGENTS WITH POTENTIAL SPECIFICITY AGAINST MELANOTIC MELANOMA

AbstractAuf den im Formelschema skizzierten Wegen werden die neuen Aminosäuren (V) und (XII) dargestellt.

Agents with potential specificity against melanotic melanoma.

Agents were designed to exploit the high tyrosinase activity in melanotic melanoma relative to normal tissues. If specific tyrosinase activation of these agents occurred, the production of toxic metabolites in the melanoma cells would produce selective cell kill. Synthesis and antitumor activities of three new amino acids, 1a [beta-[(p-hydroxyphenyl)amino]alanine hydrochloride], 1b [N delta-(p-hydroxyphenyl)ornithine hydrochloride], and 1c [N delta-(m-hydroxyphenyl)ornithine dihydrochloride], were described. Compounds 1a and 1b were approximately 2-fold more active against the B-16 melanotic melanoma than the amelanotic melanoma cell line in vitro. Compound 1b was approximately 2-fold more potent than compound 1a against either cll line and was 8-fold more potent than L-glutamic acid gamma-(4-hydroxyanilide), a natural product isolated from mushroom. No significant growth inhibitory activity was found for the m-hydroxy analogue 1c at 100 micrometers, the highest concentration tested. Similarly, compound 1b exhibited better activity against P-388 (ED50 = 9.5 x 10(-6) M) than 1a (ED50 = 3.2 x 10(-5) M) and was about 30-fold more potent than 1c. Against human epidermoid carcinoma of the nasopharynx (KB), these agents showed modest inhibitory activity with ED50 values in the range of 1.2 to 3 x 10(-4) M. No in vivo activity against P-388 and B-16 at doses up to 150-200 mg/kg was observed. The biological results suggest that a nonspecific oxidation rather than a specific tyrosinase activation is involved in the biological action of these new compounds.