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M Lebl

Publications and source records attributed to M Lebl.

At least 37 records · Page 2Linked to original sources
PubMed1994

One bead, one chemical compound: use of the selectide process for anticancer drug discovery.

A technology for chemical synthesis and testing of libraries of millions of chemical entities has been developed for rapid molecular and cellular screening for drug leads. Each individual compound in the library is on a separate resin bead. Screening for binding activity can be conducted directly on the beads. Biological activity is assessed in solution phase assay by cleaving a portion of the compound from each bead. The molecular structure of the compound of interest is obtained by automated peptide sequencing from the bead of origin. We have applied this technology to anticancer drug discovery as well as to other pharmaceutical targets. For anticancer drug development, current molecular targets include B-cell lymphoma, the EGF receptor, and the HER2-neu receptor. Solution phase screening with dual cleavable libraries is being used for growth inhibition of human tumor cell lines. Initial in vitro leads have been identified in each of these areas of anticancer drug discovery.

Antibodies, Monoclonal↗
PubMed1993

Discovery of D-amino-acid-containing ligands with selectide technology.

When all L-amino acid (aa) random peptide libraries synthesized on solid-phase particles were screened (Selectide Technology), we identified several peptide ligands (YG_F_) that interacted specifically with an anti-beta-endorphin monoclonal antibody (clone 3E7) (single-letter aa symbols; symbols '_' indicate variable aa). Here, we report on the screening of three different D-aa-containing pentapeptide libraries (XxXxX, xXxXx and xxxxx, wherein X = L-aa, and x = D-aa) with the same antibody, in which several D-aa-containing ligands were identified. The binding affinities of many of these D-aa-containing ligands were as least two orders of magnitude lower than that of YGGFL, for which the Ki is 17.5 nM.

Amino Acid Sequence↗
PubMed1993

Discovery of biologically active peptides in random libraries: solution-phase testing after staged orthogonal release from resin beads.

To speed drug discovery, we developed an approach for identification of individual peptides with a desired biological activity from a library containing millions of peptides. The approach uses sequential orthogonal release of chemically synthesized peptides from insoluble beads, followed by testing in solution. In this system, each bead within a library of beads has one peptide sequence, but peptide molecules are attached to the bead with three types of chemical linkers, including two linkers cleavable at different pH optima. An uncleavable linker keeps some peptide attached to the bead for sequencing positives from the solution assay. Applicability of this discovery technique was documented by identifying ligands for a monoclonal antibody and for the human platelet fibrinogen receptor, glycoprotein IIb/IIIa.

Amino Acid Sequence↗
PubMed1993

The use of hydrogen-deuterium exchange to facilitate peptide sequencing by electrospray tandem mass spectrometry.

The utility of hydrogen-deuterium exchange for sequencing peptides by mass spectrometry is demonstrated. The number of exchangeable hydrogens in a peptide is readily obtained by electrospray analysis of the peptide dissolved in deuterated solvents. This information can be used, in conjunction with published computer algorithms for interpreting peptide mass spectra, to reduce significantly the number of candidate sequences that fit the experimental data. This information, when combined with fragment-ion information in the mass spectrum, greatly increases the reliability of sequence determination.

Amino Acid Sequence↗
PubMed1992

Synthesis and bioactivity studies of two isosteric acyclic analogues of melanin concentrating hormone.

Salmon melanin concentrating hormone (MCH) is a cyclic heptadecapeptide. MCH stimulates perinuclear aggregation of melanosomes within integumental melanocytes of teleost fishes resulting in skin blanching. MCH contains a disulfide bridge forming a 10-residue ring [sequence: see text]. It has been proposed that the ring is necessary for maintenance of potency. In order to test this proposal, we have synthesized two pseudo-isosteric analogues of MCH that cannot cyclize. They differed only in the polarity of the side chain group of positions 5 and 14. Serine was substituted for Cys5 and Cys14 in one peptide and L alpha-aminobutyrate (Abu) was the substitution at the two positions in the other peptide. Using a fish skin bioassay we determined that these analogues exhibit less than 1/10,000th the potency of the native hormone. These results suggest that the disulfide bridge is necessary to maintain the correct conformational and topographical features of the hormone for receptor binding and transmembrane signal transduction.

Amino Acid Sequence↗
PubMed1992

Conformationally biased analogs of oxytocin.

Four diastereomeric analogs of oxytocin containing substituted phenylalanine in position 2 were synthesized. This modified phenylalanine side chain contained one methyl group attached to the beta-carbon and the second one at the 2' position of the aromatic ring. All analogs were found to be inhibitors of uterotonic activity of oxytocin with pA2 values ranging from 6.0 to 8.3; the most potent one (pA2 = 8.3) contained dimethylphenylalanine of the D-erythro configuration.

Amino Acid Sequence↗
PubMed1991

High-performance liquid chromatography of peptides at reduced temperatures: separation of isomers.

Cholecystokinin analogues containing N-methyl amino acids were studied by reversed-phase high-performance liquid chromatography at reduced temperatures. A reduction in temperature to -17 degrees C led to lower efficiency, but at the same time separations of cis and trans isomers (and some impurities) were achieved. The velocity constants for cis-trans equilibria were calculated.

Amino Acid Sequence↗
PubMed1991

Collision-induced liquid secondary-ion mass spectra of oxytocin derivatives. A study of the influence of modifications in the disulfide bond on the adjacent peptide bond cleavage.

The influence of the inductive effect of modifications in the disulfide bridge in oxytocin analogs on the cleavage of the neighboring peptidic bond was studied. A correlation between the inductive constant of the group in the disulfide bridge and the particular fragment ion abundance was observed.

Amino Acid Sequence↗
PubMed1991

A monoclonal antibody applicable for determination of C-peptide of human proinsulin by RIA.

BALB/c, (BALB/c x B10.A)F1 and (BALB/c x B10)F1 hybrid mice were immunized with C-peptide of human proinsulin. The (BALB/c x B10.A)F1 hybrids were the best responders and yielded 3 hybridomas secreting specific monoclonal antibodies. One of them, C-PEP-01, bound the C-peptide with high affinity (Kas = 1.1 x 10(9) l/mol), cross-reacted fully with human proinsulin but not with insulin, glucagon or somatostatin and apparently recognized the regions of C-peptide comprising amino acid residues 8-13 and 25-31. A RIA system could be set up employing this monoclonal antibody suitable for estimation of C-peptide concentrations in a diagnostically useful range (1-50 ng/ml).

Amino Acid Sequence↗
PubMed1990

Conformationally restricted analogs of oxytocin; stabilization of inhibitory conformation.

Analogs of oxytocin containing tetrahydroisoquinoline carboxylic acid (Tic) of L or D configuration in position 2 were synthesized and their biological activities were tested. Both analogs showed negligible agonist activity in uterotonic, galactogogic, and pressor assays, but they are in vitro uterotonic inhibitors. In comparison with oxytocin analogs containing L- or D-phenylalanine in position 2, the analog with the D-configuration of the conformationally fixed aromatic residue has significantly increased inhibitory activity which suggests that the proper conformation for the interaction with the receptor, but not for its activation, was stabilized. 1H NMR and CD studies, supported by theoretical calculations, suggest that the conformational properties of the analog containing D-tetrahydroisoquinoline carboxylic acid are similar to those of [2-D-phenylalanine]oxytocin.

Animals↗
PubMed1989

Melanin concentrating hormone analogues: contraction of the cyclic structure. II. Antagonist activity.

Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, melanin concentrating hormone (MCH), is a cyclic hormone possessing both MCH-like (melanin granule aggregating effect) and melanocyte stimulating hormone (MSH)-like (melanin granule dispersing effect) activities. Nine ring-contracted analogues were synthesized and characterized for their melanotropic activity on the fish (Synbranchus marmoratus) and frog (Rana pipiens) bioassays. In most cases, these analogues were totally devoid of MCH-like agonist activity, demonstrating the essential role of the disulfide bridge between residues 5 and 14 of the hormone. [Ala5, Cys10]MCH, for example, was totally devoid of MCH-like activity. This analogue, like alpha-MSH, however, antagonized the melanosome aggregating actions of MCH on fish melanocytes. The antagonistic activity of the analogue, like that of alpha-MSH, was Ca2+-dependent. Evidence suggested that this antagonism of MCH activity was related to the intrinsic MSH-like activity of the analogue. These results suggest that MCH and alpha-MSH may be structurally and, therefore, evolutionarily related.

Animals↗
PubMed1989

Melanin concentrating hormone. III. Melanin concentrating hormone (MCH): the message sequence.

Melanin concentrating hormone (MCH) is a heptadecapeptide synthesized by the hypothalamus and secreted by the neurohypophysis of the teleost pituitary gland. MCH stimulates melanosome aggregation within teleost melanocytes but also exhibits MSH-like (melanosome dispersing) activity on tetrapod (frog and lizard) melanocytes. We have synthesized a number of MCH analogues to determine the essential features of the primary structure necessary to stimulate either melanosome aggregation or dispersion in fish or tetrapod melanocytes, respectively. An analysis of the potencies and actions of these analogues on vertebrate melanocytes is provided and demonstrates that the two activities have different structural requirements.

Amino Acid Sequence↗
PubMed1989

Melanocyte stimulating hormone and melanin concentration hormone may be structurally and evolutionarily related.

Two melanotropic peptides, melanin concentration hormone (MCH) and alpha-melanocyte stimulating hormone (alpha-MSH), exert opposing actions on melanosome (melanin granule) movements within teleost pigment cells, melanocytes (melanophores). MCH stimulates melanosome aggregation to the cell center whereas alpha-MSH stimulates pigment organelle dispersion out into the dendritic processes of the melanocytes. The actions of alpha-MSH are dependent upon extracellular calcium (Ca2+), whereas those of MCH are actually enhanced in the absence of the cation. At high concentrations (10(-5)-10(-8) M) MCH also exhibits MSH-like activity (autoantagonism), an effect which is abolished in the absence of Ca2+. Therefore, MCH exhibits MCH-like as well as MSH-like activity depending on the presence or absence of extracellular Ca2+. An analogue of MCH, [Ala5, Cys10]MCH, has been synthesized which is totally devoid of MCH activity but still exhibits MSH-like activity. These results suggest that the two melanotropic peptides share some component of structural similarity and may be evolutionarily related.

Amino Acid Sequence↗
PubMed1989

Analogs of oxytocin containing a pseudopeptide Leu-Gly bond of cis and trans configuration.

Analogs of deamino-oxytocin wherein the Leu-Gly peptide bond has been replaced by a tetrazole moiety or by a double bond of trans configuration were synthesized and their biological activities evaluated. Trans double bond was found to be the most appropriate substitution for the amide bond (uterotonic activity 24% of the deamino-oxytocin). In the case of all three analogs low but prolonged galactogogic activity was found and the ratio of uterotonic in vitro and in vivo activity was surprisingly high (ranging from 4.5 to 20).

Animals↗
PubMed1988

Melanin concentrating hormone analogues: contraction of the cyclic structure. 1. Agonist activity.

Melanin concentrating hormone (MCH) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, which is synthesized in the hypothalamus and secreted by the neurohypophysis of teleost fishes. This hormone exhibits both MCH-like as well as alpha-MSH (alpha-melanocyte stimulating hormone) like activity. We have examined the role of the disulfide bond for the two contrasting melanotropic activities of MCH. Nine analogues of the parent peptide were synthesized and characterized for biological activity. The disulfide ring was contracted from the 5-14 to the 7-14, 8-14, and 10-14 residues with concomitant substitution of alanine for Cys at position 5 in each of the heptadecapeptides. Similar substitutions were made in a series of MCH analogues. In addition, the following cyclic peptides also were synthesized: [Cys7]MCH, [Cys8]MCH, and [Cys10]MCH. The fish-skin bioassay is sensitive to MCH at a concentration of 10(-12) M. All ring-contracted analogues were inactive at 10(-6) M or lower concentrations; less than 1/1,000,000 compared to MCH (1.0) except [Ala5,Cys8]MCH (0.0008; 1/1250), [Cys10]MCH (0.000 09; 1/10,000), and [Cys8]MCH (0.000 001; 1/1,000,000). In the frog-skin bioassay, [Ala5,Cys10]MCH, although lacking MCH-like activity in the fish-skin bioassay, was equipotent to MCH in its alpha-MSH-like component of activity. Most other analogues were either inactive or much less active than MCH in stimulating melanosome dispersion. These results demonstrate that the disulfide bond between positions 5 and 14 is essential for the MCH-like activity since contraction of the ring generally leads to inactive peptides. Contraction of the disulfide bridge does not, however, have as great an effect on the MSH-like activity of MCH.

Animals↗