TMC-95A, B, C, and D, novel proteasome inhibitors produced by Apiospora montagnei Sacc. TC 1093. Taxonomy, production, isolation, and biological activities

In our course of screening for novel proteasome inhibitors, TMC-95A and its diastereomers, TMC-95B to D, were isolated from the fermentation broth of Apiospora montagnei Sacc. TC 1093. TMC-95A inhibited the chymotrypsin-like (ChT-L), trypsin-like (T-L), and peptidylglutamyl-peptide hydrolyzing (PGPH) activities of 20S proteasome with IC50 values of 5.4nM, 200nM, and 60nM, respectively. TMC-95B inhibited these activities to the same extent as TMC-95A, while the inhibitory activities of TMC-95C and D were 20 to 150 times weaker than that of TMC-95A and B. TMC-95A did not inhibit m-calpain, cathepsin L, and trypsin at 30 microM, suggesting its high selectivity for proteasome. Taxonomy of the producing strain is also described.     

TMC-171A,B,C and TMC-154, novel polyketide antibiotics produced by Gliocladium sp. TC 1304 and TC 1282

Four new antibiotics, TMC-171A (2), B (3), C (4) and TMC-154 (5) have been isolated from the fermentation of fungal strains Gliocladium sp. TC 1304 and TC 1282, respectively. Spectroscopic and degradation studies have shown that TMC-171s and TMC-154 were new members of the TMC-151 class of antibiotics, unique polyketides modified with a D-mannose and a D-mannitol or a D-arabitol. These compounds showed moderate cytotoxicity to various tumor cell lines.     

TMC-264, a novel inhibitor of STAT6 activation produced by Phoma sp. TC 1674

A novel inhibitor of STAT6 activation, named as TMC-264 (1), was discovered from the fermentation broth of Phoma sp. TC 1674. Based on spectroscopic analyses, TMC-264 was found to be a novel tricyclic polyketide with chloro-1H-dibenzo[b,d]pyran-4,6-dione. TMC-264 suppressed expression of IL-4 driven luciferase and germline Cepsilon mRNA with IC50 values of 0.3 microM and 0.4 microM, respectively. TMC-264 exhibited a potent inhibitory activity against tyrosine phosphorylation of STAT6 with an IC50 value of 1.6 microM, whereas TMC-264 weakly inhibited tyrosine phosphorylation of STAT5 with an IC50 value of 16 microM, but did not inhibit the phosphorylation of STAT1 up to 40 microM. TMC-264 blocked formation of the complexes between phosphorylated STAT6 and STAT6 oligonucleotides in a dose dependent manner, while TMC-264 did not affect the formation of phosphorylated STAT1/STAT1 oligonucleotides complexes. These results suggested that TMC-264 selectively inhibited IL-4 signaling by interfering both of phosphorylation of STAT6 and binding of the phosphorylated STAT6 to the recognition sequence.     

TMC-86A, B and TMC-96, new proteasome inhibitors from Streptomyces sp. TC 1084 and Saccharothrix sp. TC 1094. I. Taxonomy, fermentation, isolation, and biological activities

TMC-86A, B and TMC-96, new 20S proteasome inhibitors with an epoxy-beta-aminoketone moiety, were isolated from the fermentation broth of Streptomyces sp. TC 1084 and Saccharothrix sp. TC 1094, respectively. TMC-86A, B and TMC-96 inhibited the chymotrypsin-like and peptidylglutamyl-peptide hydrolyzing activities of 20S proteasome with the following IC50 values: TMC-86A, 5.1 microM and 3.7microM; TMC-86B, 1.1 microM and 31 microM; TMC-96, 2.9 microM and 3.5 microM, respectively. TMC-86A, B and TMC-96 exhibited the weak inhibitory activity against the trypsin-like activity of 20S proteasome with IC50 values of 51 microM, 250 microM, and 36 microM, respectively. They did not inhibit m-calpain, cathepsin L, and trypsin at 100 microM, suggesting their high specificity for proteasome. Taxonomy of the producing strains is also described.     

TMC-69, a new antitumor antibiotic with Cdc25A inhibitory activity, produced by Chrysosporium sp. TC1068. Taxonomy, fermentation and biological activities

A new antibiotic designated TMC-69 has been isolated from the fermentation broth of a fungal strain Chrysosporium sp. TC 1068. TMC-69 exhibited moderate in vitro cytotoxic activity. TMC-69-6H, a derivative of TMC-69 prepared by hydrogenation, possessed more potent in vitro cytotoxicity than TMC-69, and exhibited in vivo antitumor activity against murine P388 leukemia and B16 melanoma. TMC-69-6H was found to specifically inhibit Cdc25A and B phosphatases.     

TMC-256A1 and C1, new inhibitors of IL-4 signal transduction produced by Aspergillus niger var niger TC 1629

New inhibitors of IL-4 signal transduction, designated as TMC-256A1 and C1, were discovered together with TMC-256B1, a previously known dihydronaphthopyrone, from the fermentation broth of Aspergillus niger var niger TC 1629 by using an IL-4 driven reporter gene assay. Based on spectroscopic analyses, TMC-256A1 and C1 were found to be new members of the naphthopyrone antibiotics. TMC-256A1, B1 and C1 inhibited the IL-4 driven luciferase activity with IC50 values of 25 microM, 30 microM and 1.7 microM, respectively in this assay system. Furthermore, these compounds inhibited the expression of germline C epsilon mRNA with IC50 values of 6.6 microM , 34 microM and 0.31 microM, respectively.     

TMC-66, a new endothelin converting enzyme inhibitor produced by Streptomyces sp. A5008.

A new endothelin converting enzyme (ECE) inhibitor, TMC-66 was isolated from the fermentation broth of Streptomyces sp. A5008. The structure of TMC-66 was elucidated by spectroscopic analyses to be a new member of benzo[a]naphthacenequinone class of antibiotics. TMC-66 had a highly selective inhibitory activity for ECE with an IC50 value of 2.9 microM. Taxonomy of the producing strain is also described.     

Effectiveness of nonpeptide clinical inhibitor TMC-114 on HIV-1 protease with highly drug resistant mutations D30N, I50V, and L90M

The potent new antiviral inhibitor TMC-114 (UIC-94017) of HIV-1 protease (PR) has been studied with three PR variants containing single mutations D30N, I50V, and L90M, which provide resistance to the major clinical inhibitors. The inhibition constants (K(i)) of TMC-114 for mutants PR(D30N), PR(I50V), and PR(L90M) were 30-, 9-, and 0.14-fold, respectively, relative to wild-type PR. The molecular basis for the inhibition was analyzed using high-resolution (1.22-1.45 A) crystal structures of PR mutant complexes with TMC-114. In PR(D30N), the inhibitor has a water-mediated interaction with the side chain of Asn30 rather than the direct interaction observed in PR, which is consistent with the relative inhibition. Similarly, in PR(I50V) the inhibitor loses favorable hydrophobic interactions with the side chain of Val50. TMC-114 has additional van der Waals contacts in PR(L90M) structure compared to the PR structure, leading to a tighter binding of the inhibitor. The observed changes in PR structure and activity are discussed in relation to the potential for development of resistant mutants on exposure to TMC-114.     

TMC-114 (Tibotec)

Tibotec (formerly Tibotec-Virco) is developing TMC-114 as a potential treatment for HIV-1 infection. In February 2001, TMC-126 was revealed as the series prototype, from which TMC-114 was developed. Because of its improved antiviral and superior pharmacokinetic properties, TMC-114 was selected for clinical development. Phase II trials of TMC-114 are underway.     

Cyclic cholecystokinin analogues that are highly selective for rat and guinea pig central cholecystokinin receptors.

Cholecystokinin (CCK) analogues (JMV310, JMV320, JMV328, and JMV332), obtained by side chain to side chain cyclization of a lysine residue in position 28 with a lysine residue in position 31, were found to be highly selective for the brain CCK receptor (CCK-B receptor), both in guinea pig and rat. In these analogues, the C-terminal tetrapeptide region of the molecule, which is the crucial determinant for binding to CCK-B receptors, has been constrained by cyclization. These analogues were highly potent in inhibiting binding of labeled CCK-8 to rat and guinea pig brain membranes (apparent affinity in the nanomolar range) but were poorly efficacious in inhibiting binding of labeled CCK-8 to rat or guinea pig pancreatic acini. In agreement with their low affinity for the pancreatic receptor, these CCK analogues were not very potent in stimulating amylase secretion. These cyclic CCK analogues were demonstrated to be highly selective for the brain CCK receptors.     

Activity of novel 4-PIOL analogues at human alpha 1 beta 2 gamma 2S GABA(A) receptors--correlation with hydrophobicity

A series of novel 5-(4-piperidyl)-3-isoxazolol (4-PIOL) analogues where the 4-position of the 3-isoxazolol ring was substituted with groups of different size, flexibility, and lipophilicity have been characterised. Their activity as agonists and/or antagonists on human alpha(1)beta(2)gamma(2S) GABA(A) receptors expressed in Xenopus oocytes was studied using two-electrode voltage clamp electrophysiology. Methyl- and ethyl-substituted 4-PIOL analogues were characterised as partial agonists since weak agonist responses could be potentiated with lorazepam and inhibited by the competitive antagonist 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyradizinum bromide (SR95531). All larger substituents in the 4-position of the 3-isoxazolol ring of 4-PIOL converted the compounds into pure competitive antagonists. Additionally, for GABA, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), piperidine-4-sulphonic acid (P4S), and 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL), a negative linear correlation was found between the agonist efficacy of the compound and the ability of lorazepam to potentiate EC(95) responses. Furthermore, a positive linear correlation between the lipophilicity of the substituents in the 4-position of the 3-isoxazolol ring of 4-PIOL and the antagonist affinity was found. These data suggest that the GABA(A) receptor contains a hydrophobic binding pocket at the GABA recognition site and that the binding of the 4-PIOL analogues is largely determined by the transfer from the aqueous phase to the hydrophobic pocket.     

Development of conformationally constrained linear peptides exhibiting a high affinity and pronounced selectivity for delta opioid receptors

A series of linear conformationally constrained opioid peptides was designed in an attempt to develop highly selective and potent agonists for the delta opioid receptors. These enkephalin analogues corresponding to the general formula Tyr-D-X(OY)-Gly-Phe-Leu-Thr(OZ) were obtained by incorporating bulky residues (X = Ser or Thr; Y = tert-butyl or benzyl; Z = tert-butyl) into the sequence of the previously reported delta specific agonists DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr). In binding studies based on displacement of mu and delta opioid receptor selective radiolabeled ligands from rat brain membranes, the two constrained hexapeptides, Tyr-D-Ser(O-t-Bu)-Gly-Phe-Leu-Thr (1, DSTBULET) (KI(mu) = 374 nM, Kr(delta) = 6.14 nM, KI(delta)/KI(mu) = 0.016) and in particular Tyr-D-Ser(O-t-Bu)-Gly-Phe-Leu-Thr(O-t-Bu) (7, BUBU) (KI(mu) = 475 nM, KI(delta) = 4.68 nM, KI(delta)/KI(mu) = 0.010) were shown to be among the most potent and selective delta probes reported to date. A roughly similar pattern of selectivity was obtained with the guinea pig ileum and mouse vas deferens bioassays. In addition, the analgesic potency (hot-plate test) of these peptides intracerebroventricularly administered in mice was shown to be significantly related to their mu-receptor affinity.     

Development of conformationally restricted analogues of bradykinin and somatostatin using constrained amino acids and different types of cyclization

The structure-based design of peptide drugs requires the knowledge of the bioactive conformation. Studies on this receptor-bound 3D structure require linear or cyclic analogues with strongly reduced flexibility, but high biological activity, since only analogues with retained potency have preserved the bioactive conformation. Constrained amino acids containing double bonds or bulky substituents at the N(alpha)-, C(alpha)- and C(beta)-atom as well as at the aromatic ring atom were successfully applied to obtain potent and stable analogues of bradykinin and somatostatin, which due to their restricted conformation were suitable objects for conformational studies. Besides the generation of constrained cyclic analogues with improved biological and pharmacological properties, cyclic peptides were used as convenient models for the study of turn formations. Cyclization of the linear peptide bradykinin was performed by linking the N-terminus and the C-terminus, and in both bradykinin and somatostatin by cyclization using the amino acid side chains and by backbone cyclization. The later requires the introduction of N(alpha)-functionalised amino acids for ring closure which can be performed either through incorporation of N(alpha)-functionalised amino acids or dipeptide building units. Conformational analysis of a cyclic bradykinin analogue by means of NMR-studies together with molecular dynamics simulation led to a quasicyclic 3D structure with two turns and together with other 3D structures provided a pharmacophore model of bradykinin antagonists.     

Structural determinants of affinity for the phencyclidine binding site of the N-methyl-D-aspartate receptor complex: discovery of a rigid phencyclidine analogue of high binding affinity

To learn more about the binding conformation of phencyclidine (PCP) and to arrive at analogues of higher affinity, which may serve as noncompetitive N-methyl-D-aspartate receptor antagonists, eight optically pure PCP analogues were designed with the aid of computer. These compounds represent conformationally constrained versions of PCP in which the motion of the phenyl ring is frozen, thus allowing a determination of the orientation of the phenyl ring relevant to binding. The analogues were synthesized by a Diels-Alder strategy and tested in a radioligand binding assay to evaluate their affinity for the PCP binding site of the N-methyl-D-aspartate receptor complex. One of the analogues was found to bind with nanomolar affinity (IC50 = 19 nM) and to be 73-fold more potent in binding than its enantiomer. These results, which further elucidate the structural determinants of high affinity binding, should aid both in the design of higher affinity molecular probes of the PCP binding site and in the discovery of potential neuroprotective agents.     

Bisubstrate ketone analogues as serotonin N-acetyltransferase inhibitors.

Serotonin N-acetyltransferase, also called the melatonin rhythm enzyme, is thought to play an important regulatory role in circadian rhythm in animals and people. A series of analogues were synthesized in which indole and coenzyme A were linked via ketone tethers as designed inhibitors of this enzyme. These compounds were tested against purified serotonin N-acetyltransferase. The parent ketone compound was found to be as potent as an amide linked compound studied previously, suggesting that there are no key hydrogen bonds to the nitrogen atom of the corresponding substrate necessary for tight inhibition. Reduction of the parent ketone afforded the diastereomeric carbinol mixture which showed reduced inhibitory potency, arguing against tetrahedral analogue mimicry as an important inhibitory theme. Several conformationally constrained ketone analogues were synthesized and investigated, and the results indicated that directing the orientation of the two substrates within the bisubstrate system could be used to maximize enzyme inhibition.     

QSAR analysis of Delta(8)-THC analogues: relationship of side-chain conformation to cannabinoid receptor affinity and pharmacological potency

A novel quantitative structure-activity relationship (QSAR) for the side-chain region of Delta(8)-tetrahydrocannabinol (Delta(8)-THC) analogues is reported. A series of 36 side-chain-substituted Delta(8)-THCs with a wide range of pharmacological potency and CB1 receptor affinity was investigated using computational molecular modeling and QSAR analyses. The conformational mobility of each compound's side chain was characterized using a quenched molecular dynamics approach. The QSAR techniques included a modified active analogue approach (MAA), multiple linear regression analyses (MLR), and comparative molecular field analysis (CoMFA) studies. All three approaches yielded consistent results. The MAA approach applied to a set of alkene/alkyne pairs identified the most active conformers as those with conformational mobility constrained within an approximately 8 A radius. MLR analyses (restricted to 15 hydrocarbon side-chain analogues) identified two variables describing side-chain length and terminus position that were able to fit the pharmacological data for receptor affinity with a correlation coefficient for pK(D) of 0.82. While chain length was found to be directly related to receptor affinity, the angle made by the side chain from its attachment point to its terminus (angle defined by C3-C1'-side-chain terminus carbon, see Figure 1) was found to be inversely related to affinity. These results suggest that increased side-chain length and increased side-chain ability to wrap around the ring system are predicted to increase affinity. Therefore, the side chain's conformational mobility must not restrict the chain straight away from the ring system but must allow the chain to wrap back around toward the ring system. Finally, the CoMFA analyses involved all 36 analogues; they also provided data to support the hypothesis that for optimum affinity and potency the side chain must have conformational freedom that allows its terminus to fold back and come into proximity with the phenolic ring.     

Synthesis and electrophysiological characterization of cyclic morphiceptin analogues

A challenge in opioid peptide chemistry and pharmacology is the possibility to develop novel peptides with peripheral selectivity. An enzymatically stable opioid peptide could involve an antidiarrheal effect. For this reason, we constrained the highly selective and potent tetrapeptide morphiceptin with a 6-atom bridge, resulting in a cyclic amide and an ester analogue, 2 and 3, respectively. Taking advantage of the functional coupling of the opioid receptor with the heteromultimeric G-protein-coupled inwardly rectifying K+ (GIRK1/GIRK2) channel, either the wild-type mu-, kappa-, delta- or a mutated mu-opioid receptor (hMORS329A) was functionally co-expressed with GIRK1/GIRK2 channels and a regulator of G-protein signaling (RGS4) in Xenopus laevis oocytes. The two-microelectrode voltage clamp technique was used to measure the opioid receptor activated GIRK1/GIRK2 channel responses. Both cyclic analogues were equally potent via the wild-type mu-opioid receptor hMORwt (EC(50) value 976.5 +/- 41.7 for 2 and 1017.7 +/- 60.7 for 3), while the EC(50) value for Tyr-Pro-Phe-D-Pro-NH(2) measured 59.3 +/- 4.8 nM. These three agonists displayed a four to five times decreased potency via hMORS329A as compared to the wild type. Interestingly, no effect on kappa- and delta-opioid receptors was observed. The intramolecular bridge created by cyclization of morphiceptin prevents dipeptidyl peptidase IV from interacting with these analogues. We conclude that constraining morphiceptin with a 6-atom bridge resulted in enzymatically stable peptidomimetics that are exclusively active on mu-opioid receptors. These analogues provide an interesting template in the promising approach for the design of potential antidiarrheal agents.