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P M Fischer

Publications and source records attributed to P M Fischer.

At least 19 recordsLinked to original sources
PubMed2005

Antiproliferative activity of olomoucine II, a novel 2,6,9-trisubstituted purine cyclin-dependent kinase inhibitor.

The study describes the protein kinase selectivity profile, as well as the binding mode of olomoucine II in the catalytic cleft of CDK2, as determined from cocrystal analysis. Apart from the main cell cycle-regulating kinase CDK2, olomoucine II exerts specificity for CDK7 and CDK9, with important functions in the regulation of RNA transcription. In vitro anticancer activity of the inhibitor in a panel of tumor cell lines shows a wide potency range with a slight preference for cells harboring a wild-type p53 gene. Cell-based assays confirmed activation of p53 protein levels and events leading to accumulation of p21(WAF1). Additionally, in olomoucine II-treated cells, Mdm2 was found to form a complex with the ribosomal protein L11, which inhibits Mdm2 ubiquitin ligase function. We conclude that perturbations in RNA synthesis may lead to activation of p53 and that this contributes to the antiproliferative potency of cyclindependent kinase inhibitors.

Antineoplastic Agents↗
PubMed2004

The design of drug candidate molecules as selective inhibitors of therapeutically relevant protein kinases.

The human genome encompasses some 2,000 proteins that utilize adenosine 5'-triphosphate (ATP) in one way or another and some 500 of these are protein-tyrosine and protein-serine/threonine kinases (PTKs & PSTKs). Substrate phosphorylation by these enzymes is nature's predominant molecular way of organizing cellular signal transduction and regulating biochemical processes in general. It is not surprising, therefore, that abnormal phosphorylation of cellular proteins is a hallmark of disease and that there has been a growing interest in the use of kinase inhibitors as drugs. In fact the search for such agents has recently culminated in the approval of the first kinase inhibitor drugs for medical use. Although it has been demonstrated exhaustively that potent and structurally diverse ATP-antagonistic small molecule kinase inhibitors can be found through mass screening and structure-guided design, the question of biochemical, cellular, and in vivo selectivity of such inhibitors remains much less clear. Here the medicinal chemistry of kinase inhibitors is reviewed critically with particular emphasis on target selectivity and specificity. Approaches based on chemical genomics, combinatorial target-guided ligand assembly, computational chemistry, and structural biology techniques, which aim at classifying both inhibitors and kinase targets, are given special emphasis. The various strategies in which differences in biochemical mechanism of kinase function can be exploited in order to attain selective inhibition are also discussed. Furthermore, recent developments in the design of inhibitors to selected individual validated therapeutic kinase targets, including cell cycle kinases and receptor PTKs, etc. are summarised.

Antineoplastic Agents↗
PubMed2003

The design, synthesis and application of stereochemical and directional peptide isomers: a critical review.

Physiological processes are regulated to a large extent by physical and chemical interactions between polypeptides. Although many small molecules have been discovered that can modulate such interactions and may be useful as drugs, the design of these agents purely from the knowledge of the details of a given protein-protein interaction, or through screening, remains difficult. Therefore, the peptidomimetic process, which aims at using peptides derived from either polypeptide binding partner directly, or after modification to improve affinity and physicochemical properties, continues to be attractive. The vast majority of naturally occurring polypeptides are composed of L-amino acids. Because natural proteins need to be metabolised, L-amino acid polypeptides are very prone to proteolytic degradation, a property that severely limits their therapeutic application. The proteolytic machinery is not well equipped to deal with D-amino acid polypeptides, however, and it is this finding above all else that has spurned research into stereochemical and directional manipulation of peptide chains. The expectation has been that systematic inversion of the stereochemistry at the peptide backbone alpha-carbon atoms, if accompanied by chain reversal, should yield proteolytically stable retro-inverso peptide isomers, whose side chain topology, in the extended conformation, corresponds closely to that of a native sequence, and whose biological activity emulates that of a parent polypeptide. The actual structural implications of modifying amino acid stereochemistry and peptide bond direction are reviewed critically here and the reasons for the lack of general success with this strategy are discussed. The application of polypeptides is particularly pertinent to synthetic vaccine design. Interestingly, the retro-inverso strategy has been more successful for immunological applications than elsewhere; recent finding are collated in this review. Partial rather than global retro-inversion holds much promise since the loss of crucial backbone hydrogen-bonding through peptide bond reversal can be avoided, while still permitting stabilisation of selected hydrolysis-prone peptide bonds. Generically applicable synthetic methods for such partially modified retro-inverso peptides are not as yet available; progress towards this goal is also summarised.

Amino Acid Sequence↗
PubMed2002

Highly potent p21(WAF1)-derived peptide inhibitors of CDK-mediated pRb phosphorylation: delineation and structural insight into their interactions with cyclin A.

The tumour suppressor protein p21(WAF1) plays a central role in regulating eukaryotic cell-cycle progression. Through its association with G1- and S-phase CDK complexes it regulates activation of the retinoblastoma protein (pRb) and E2F transcription factors. Recognition of CDK/cyclin complexes by p21 occurs, at least in part, through a protein-protein interaction with a binding groove on the cyclin subunit. The same groove has been shown to be involved in the recruitment of macromolecular CDK substrates, including pRb and E2F. Blocking of this recruitment site therefore prevents recognition and subsequent phosphorylation of CDK substrates and offers a therapeutic approach towards restoration of p21-like tumour suppression. Starting from the C-terminal cyclin-binding domain of p21 we have identified the minimal and optimized bioactive (152)HAKRRLIF(159) peptide sequence with respect to CDK protein kinase inhibition where pRb is the substrate. The phosphorylation of histone H1, however, which does not contain a recognizable cyclin-binding motif, was unaffected. Detailed structure-activity relationship investigations revealed that the determinants within this sequence are residues Arg(155), Leu(157) and Phe(159) and more completely define the composition of the cyclin-binding motif. A marked increase in potency was obtained upon replacement of the native Ser(153) with an Ala residue in the context of short synthetic peptide inhibitors and significantly, this mutation resulted in comparable affinity with CDK2/cyclin A as does the full-length recombinant p21 (which has CDK2 and cyclin A binding sites). Peptides derived from various proteins known to interact with cyclins were compared for potency and selectivity. A molecular model of the complex between the cyclin groove and the HAKRRLIF peptide was constructed. This model accounts for the observed peptide structure-activity relationships, including the potency enhancement of the LIF sequence occupying the hydrophobic pocket. Furthermore, it provides generic insights into molecular interactions governing cyclin groove recognition and lays the foundation for the development of peptidomimetic inhibitors of CDKs.

Amino Acid Motifs↗
PubMed2001

Synthesis, biological activity, and solution structures of a cyclic dodecapeptide from the EGF-2 domain of blood coagulation factor VII.

The cyclic dodecapeptide, disulfide-cyclo-[H-Cys-Val-Asn-Glu-Asn-Gly-Gly-Cys(Acm)-Glu-Gln-Tyr-Cys-OH], which corresponds to the 91-102 sequence of the second epidermal growth factor domain of human blood coagulation factor VII, was synthesized using solid-phase procedures. It was shown to be an inhibitor at the key step in the induction of coagulation by the extrinsic pathway, i.e. the factor VII/tissue factor-catalyzed activation of coagulation factor X. The solution structure of this peptide was investigated by NMR spectroscopy and was computer-modeled via molecular mechanics. Structures were calculated based on 112 distance and nine dihedral angle constraints. The resulting backbone structures were classified into two structural subsets: one which exhibited a twisted '8'-shaped folding and another describing an open, circular 'O' outline. The local backbone structures of segments Asn3-Glu4-Asn5, Gly7-Cys8 and Gln10-Tyr11 were well preserved among the two subsets. Apart from the unrestrained N- and C-termini, Gly6 and Glu9 sites exhibited marked local disorder between the two subsets, suggesting localized flexible hinges likely to govern tertiary structure interconversion between the two subsets. Two transient hydrogen bonds were identified from pH chemical shift titrations by matching the pKa values of NH and carboxylate groups, which supported the occurrence of the '8' structure, and agreed with temperature coefficients of peptidyl NH resonances. Structure-function relationships of the peptide were discussed in terms of the likely physiological function of the disulfide-bonded loop in factor VII which the peptide represents.

Amides↗
PubMed2001

Recent advances and new directions in the discovery and development of cyclin-dependent kinase inhibitors.

The eukaryotic cell division cycle is coordinated by cyclin-dependent protein kinases (CDKs) and cyclin subunits specific for the different phases of the cycle. These complexes phosphorylate target substrates, including the retinoblastoma susceptibility gene product (pRb) and related proteins. Cellular neoplastic transformations are accompanied by loss of regulation of cell cycle checkpoints, frequently through aberrant expression of CDKs and cyclins, as well as loss or mutation of their negative regulators. Consequently, one strategy in the development of mechanism-based anticancer therapeutics has been to halt malignant cellular proliferation through inhibition of the enzymatic activity of CDKs. The development of inhibitors selective for the ATP binding sites of particular protein kinases is a comparatively recent medicinal chemistry endeavor. Advances relevant to CDK inhibition are reviewed critically and alternative approaches to CDK inhibition, as well as applications of CDK inhibitors to therapeutic areas other than oncology, are also discussed.

Animals↗
PubMed2000

A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA: affinity, stoichiometry, and thermodynamics.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and control of cell proliferation, and its activity can be modulated by interaction with p21(Waf1/Cip1) [Cox, L. S., (1997) Trends Cell Biol. 7, 493-497]. This protein-protein interaction provides a particularly good model target for designing therapeutic agents to treat proliferative disorders such as cancer. In this study, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 has been investigated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141-160) peptide and PCNA has been determined to be 1.14 x 10(7) M(-)(1), corresponding to a K(d) of 87.7 nM. Measurement of the interaction of truncation and substitution analogues based on the p21 (141-160) sequence with PCNA revealed that the N-terminal part (residues 141-152) of the above peptide is the minimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153-160), though, inhibited significantly the ability of the peptides to compete with the full-length p21 (141-160) for binding to PCNA. Alanine mutation of Met 147 or Asp 149 completely abolished or significantly decreased, respectively, the level of the PCNA binding and the inhibition of SV40 DNA replication. Comparison of the data obtained by the competitive PCNA binding assay and the ITC measurements demonstrated the usefulness of this assay for screening for compounds that could modulate the PCNA-p21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNA and interruption of the PCNA-p21 (141-160) complex. As a result of this screening, we have identified a 16-residue peptide (consensus motif 1 peptide) with the following sequence: SAVLQKKITDYFHPKK. Consensus motif 1 peptide and p21 (141-160) have similar affinities for binding PCNA and abilities to inhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessing p21-mimetic strategies for cancer treatment.

Amino Acid Sequence↗
PubMed2000

Structure-activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin.

Peptides derived from the third alpha-helix of the homeodomain (residues 43-58; Penetratin) of Antennapedia, a Drosophila homeoprotein, were prepared by simultaneous multiple synthesis. Sets of N- and C-terminally truncated peptides, as well as a series of alanine substitution analogues, were studied. Cell penetration assays using human cell cultures with these peptides revealed that the C-terminal segment 52Arg-Arg-Met-Lys-Trp-Lys-Lys58 of the parent sequence was necessary and sufficient for efficient cell membrane translocation. Individual Ala substitutions of the peptide's basic residues led to markedly decreased cell internalization ability, whereas replacement of hydrophobic residues was tolerated surprisingly well. Subcellular localization was seen to be affected by substitutions, with analogues being addressed preferentially to the cytosol or to the nucleus. Conformational constriction of the Penetratin sequence through placement and oxidation of flanking cysteine residues afforded a cyclic disulfide peptide which had lost most of its membrane translocation capacity.

Antennapedia Homeodomain Protein↗
PubMed2000

Inhibitors of cyclin-dependent kinases as anti-cancer therapeutics.

Initiation, progression, and completion of the cell cycle are regulated by various cyclin-dependent kinases (CDKs), which are thus critical for cell growth. Tumour development is closely associated with genetic alteration and deregulation of CDKs and their regulators, suggesting that inhibitors of CDKs may be useful anti-cancer therapeutics. Indeed, early results suggest that transformed and normal cells differ in their requirement for e.g. cyclin/CDK2 and that it may be possible to develop novel antineoplastic agents devoid of the general host toxicity observed with conventional cystostatic drugs. Numerous active-site inhibitors of CDKs have been studied; the main limitation with these ATP antagonists is kinase specificity for CDKs. However, screening of compound collections, as well as rational design based on enzyme-ligand complex crystal structures, are now yielding pre-clinical candidates, particularly certain purine and flavonoid analogues, with impressive potency and selectivity. Natural CDK inhibitors (CKIs), e.g. the tumour suppressor gene products p16(INK4), p21(WAF1), and p27(KIP1), form the starting point for the design of mechanism-based CDK inhibitors. A number of these small proteins have been dissected and inhibitory lead peptides amenable to peptidomimetic development have been identified. Conversion of these peptides into pharmaceutically useful molecules is greatly aided by the recent elucidation of CKI/CDK crystal and solution structures. Additional interaction sites on CDKs being exploited for the purposes of inhibitor design include: phosphorylation/dephosphorylation sites, macromolecular substrate binding site, CKS regulatory subunit binding sites, cyclin-binding site, cellular localisation domain, and destruction box. Finally, progress has recently been made in the application of antisense technology in order to target CDK activity.

Amino Acid Sequence↗
PubMed1999

Linear analogues derived from the first EGF-like domain of human blood coagulation factor VII: enhanced inhibition of FVIIa/TF complex activity by backbone modification through aspartimide formation.

Coagulation factor VII bound to its cofactor tissue factor is the physiological initiator of blood coagulation. The interaction between factor VII and tissue factor involves all four of the structural modules found in factor VII, with the most significant contribution coming from the first EGF-like domain. In this study, the synthesis and biological activity of several analogues derived from the first EGF-like domain of FVII comprising the sequence 45-83 are reported on. The six cysteine residues found in the native protein were replaced by Abu. The peptides were isolated from a multicomponent mixture following standard Fmoc solid phase synthesis. Purification and characterisation of the heterogeneous product showed that aspartimide formation was a major side-reaction, occurring predominantly at the Asp46-Gly47 and Asn57-Gly58 dipeptides. Although relatively common in peptide synthesis, the extent to which this side-reaction had taken place was considered surprising. Reported herein are the analytical methods used to isolate and characterise several of the modified products. Also, the inhibitory effect of these peptides on the formation and enzymatic activity of the factor VIIa/tissue factor complex have been compared. Surprisingly, the peptide containing an iso-Asp residue at position 57 possessed 66-fold higher inhibitory activity compared with the original target peptide. A possible explanation for this increase in observed activity is presented.

Amino Acid Sequence↗
PubMed1998

A peptide sequence from mouse tissue factor inhibits human tissue factor dependent factor X activation.

Synthetic peptides based on the putative factor X recognition site of human (Thr-Leu-Tyr-Tyr-Trp-Lys-Ser-Ser-Ser-Ser), rabbit (Thr-Leu-Tyr-Tyr-Trp-Arg-Ala-Ser-Ser-Thr), and murine tissue factor (Ile-Ile-Thr-Tyr-Arg-Lys-Gly-Ser-Ser-Thr) were dose-dependent inhibitors of human tissue factor/factor VIIa catalyzed factor X activation with IC50 values of 220, 17, and 33 microM, respectively. The mouse results were highly surprising given the low homology between the human and mouse sequence (40%) and that mouse tissue factor, in contrast with rabbit tissue factor, does not support the procoagulant activity of human factor VIIa on factor X. The inhibitory mechanism of the murine peptide was noncompetitive with respect to factor X but competitive with respect to tissue factor, indicating the peptide competes with tissue factor (or the tissue factor/factor VIIa complex) for binding to factor X. The peptide could be N-terminally truncated by two Ile without loss of inhibitory activity or changed inhibitory mechanism. Substitution of two Gly for the two Ile, which increased solubility, decreased IC50 to 17 microM whereas scrambling the peptide made it inactive.

Amino Acid Sequence↗
PubMed1998

Synthesis and activity of dimeric bradykinin antagonists containing diaminodicarboxylic acid bridge residues.

Enhancement of a ligand's interaction with a receptor through presenting the ligand in multimeric form is a topic of general interest. Thus dimerization of single-chain bradykinin antagonist peptides has previously been shown to be beneficial in terms of potency and duration of action. While crosslinking polypeptides at terminal positions using suitable dicarboxylic acids and diamines is comparatively straight-forward synthetically, internal dimerizations are usually achieved through oxidation or double S-alkylations of cysteine residues, resulting in metabolically unfavourable disulphide and thioether cross-links. Using suitably modified standard solid-phase peptide synthesis protocols, dimeric bradykinin antagonist peptides [H-(D-Arg)-Arg-Pro-Hyp-Gly-Phe]2-X-[(D-Phe)-Leu-Arg-OH]2 were synthesized where X corresponds to a L,L-2,7-diaminosuberic or L,L-2,9-diaminosebacic acid residue, respectively. The biological activity of these peptides was comparable to that of conventional dimeric bradykinin antagonists cross-linked through cystine or bis(succinimido)alkyl bridges.

Animals↗
PubMed1997

Design of low molecular weight hematoregulatory agents from the structure-activity relationship of a dimeric pentapeptide.

We report herein, a new class of simple hematoregulatory semipeptides, formally derived from the cystine-dimerized peptide pGlu-Glu-Asp-Cys-Lys-OH, where the disulfide bond has been replaced by an isosteric dicarba bridge. The structure-activity relationship (SAR) of a series of analogues incorporating replacements at positions 1 and 2 of peptide 1 led to the design of active conformationally constrained cyclic peptides (12, 13). Ring closure was achieved by cyclization of the N-terminal amino groups at position 2 of peptide 2 using pyrazine-2,3-dicarboxylic acid. Subsequent excision of the putative C-terminal scaffold domain from the active cyclic peptides resulted in the discovery of a new class of low molecular weight hematoregulatory agents exemplified by compound 16. This semipeptide analogue, comprising two D-Ser residues connected via amide bonds to the acid groups of pyrazine-2,3-dicarboxylic acid, had comparable biological activity to the lead peptide 1. The stereochemical requirements for the observed biological activity of these novel compounds were examined. Furthermore, the hematopoietic synergistic activity induced by compound 16 in stromal cell cultures was blocked by an antibody known to neutralize the hematoregulatory effect of 1, indicating a common mechanistic end point. Compounds of the class typified by 16 may form the basis for the development of novel therapeutic agents within the area of immunoregulation.

Amino Acid Sequence↗
PubMed1997

Unexpected lability of cysteine acetamidomethyl thiol protecting group. Tyrosine ring alkylation and disulfide bond formation upon acidolysis.

Cleavage and deprotection of the peptidyl resin H-Asn-Gly-Gly-Cys (Acm)-Glu(OBu(t))-Gln-Tyr(Bu(t))-Cys(Acm)-Ser(Bu(t))-Asp( OBU(t))-[(p-alkoxy)benzyloxy polystyrene resin] using standard conditions with various trifluoroacetic acid-containing mixtures were found to result in partial removal of ordinarily acid-stable S-Acm groups. Thus, apart from the desired peptide H-Asn-Gly-Gly-Cys (Acm)-Glu-Gln-Tyr-Cys(Acm)-Ser-Asp-OH, a disulfide-cyclic peptide derivative was also isolated. Furthermore, it was found that in another major by-product of the peptide resin cleavage the tyrosine side chain had been alkylated with an Acm group in a position ortho to the phenolic function. The formation of both by-products could be suppressed by carrying out the cleavage/deprotection reaction at higher dilution and by inclusion of scavengers such as phenol. An authentic sample of the disulfide-cyclic peptide was obtained by oxidation of H-Asn-Gly-Gly-Cys-Glu-Gln-Tyr-Cys-Ser-Asp-OH using Ellman's reagent.

Alkylation↗
PubMed1997

Peptides corresponding to the second epidermal growth factor-like domain of human blood coagulation factor VII: synthesis, folding and biological activity.

Factor VIIa (FVIIa) is the enzymatically active constituent of the FVIIa/tissue factor (TF) complex, the initiator of the extrinsic pathway of blood coagulation. The zymogen FVII and FVIIa are composed of discrete domains, two of which are homologous to the epidermal growth factor (EGF). This investigation examined the significance of the FVII EGF-2 domain in the processes leading to activation of factor X (FX). Peptides 47 residues in length and corresponding to the amino acid sequence of the EGF-2 domain of human FVII were prepared by solid-phase synthesis methods. Peptide variants with all six Cys residues replaced by L-2-aminobutyryl residues (1), or containing one (2a-c), two (3a,b) or three (4) disulfide bonds, were obtained by application of various S-protecting groups and oxidation methods. Peptide 4, containing the cystine bridge arrangement corresponding to that found in the native protein, was prepared by a two-step regioselective disulfide bond formation method. An evaluation of the anti-coagulant properties of peptides 1-4 revealed that all peptides, with the exception of the two-cystine isomer containing non-native disulfide pairings (3b), were potent inhibitors of TF/FVIIa-mediated activation of FX. The fully constrained peptide 4 was found to be twice as active as its completely non-constrained counterpart 1, the two peptides showing IC50 values of 1.6 +/- 0.5 microM (1) and 0.8 +/- 0.2 microM (4) with respect to TF/FVIIa-dependent FX activation. The results of this study demonstrate the functional importance of the EGF-2 domain of FVII in the induction of coagulation by the extrinsic pathway.

Amino Acid Sequence↗