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Biomedical subjects

Peter M Fischer

Publications and source records attributed to Peter M Fischer.

At least 37 records · Page 2Linked to original sources
PubMed2004

2-Anilino-4-(thiazol-5-yl)pyrimidine CDK inhibitors: synthesis, SAR analysis, X-ray crystallography, and biological activity.

Following the identification through virtual screening of 4-(2,4-dimethyl-thiazol-5-yl)pyrimidin-2-ylamines as moderately potent inhibitors of cyclin-dependent kinase-2 (CDK2), a CDK inhibitor analogue program was initiated. The first aims were to optimize potency and to evaluate the cellular mode of action of lead candidate molecules. Here the synthetic chemistry, the structure-guided design approach, and the structure-activity relationships (SARs) that led to the discovery of 2-anilino-4-(thiazol-5-yl)pyrimidine ATP-antagonistic CDK2 inhibitors, many with very low nM K(i)s against CDK2, are reported. Furthermore, X-ray crystal structures of four representative analogues from our chemical series in complex with CDK2 are presented, and these structures are used to rationalize the observed biochemical SARs. Finally results are reported that show, using the most potent CDK2 inhibitor compound from the current series, that the observed antiproliferative and proapoptotic effects are consistent with cellular CDK2 and CDK9 inhibition.

Aniline Compounds↗
PubMed2004

Structural determinants of CDK4 inhibition and design of selective ATP competitive inhibitors.

A number of selective inhibitors of the CDK4/cyclin D1 complex have been reported recently. Due to the absence of an experimental CDK4 structure, the ligand and protein determinants contributing to CDK4 selectivity are poorly understood at present. Here, we report the use of computational methods to elucidate the characteristics of selectivity and to derive the structural basis for specific, high-affinity binding of inhibitors to the CDK4 active site. From these data, the hypothesis emerged that appropriate incorporation of an ionizable function into a CDK2 inhibitor results in more favorable binding to CDK4. This knowledge was applied to the design of compounds in the otherwise CDK2-selective 2-anilino-4-(thiazol-5-yl)pyrimidine pharmacophore that are potent and highly selective ATP antagonists of CDK4/cyclin D1. The findings of this study also have significant implications in the design of CDK4 mimic structures based on CDK2.

Adenosine Triphosphate↗
PubMed2004

Cassette dosing pharmacokinetics of a library of 2,6,9-trisubstituted purine cyclin-dependent kinase 2 inhibitors prepared by parallel synthesis.

Determination of pharmacokinetic properties in the intact animal remains a major bottleneck in drug discovery. Cassette dosing involves administration of a cocktail of drugs to individual animals. Here we describe the cassette dosing properties of a 107-membered library of 2,6,9-trisubstituted purine cyclin-dependent kinase 2 (CDK2) inhibitors. A three-step parallel synthesis approach produced compounds with purity ranging from 63% to 100%. Cassette dosing was validated by comparing the pharmacokinetic parameters obtained following i.v. administration of a mixture of olomoucine, R-roscovitine (CYC202), and bohemine, each at 16.6 mg/kg, with results for administration of single agents at 50 mg/kg. No significant difference was observed between the pharmacokinetic parameters of agents when dosed in combination compared with those of individual compounds. CYC202 showed the highest area under the curve (AUC) and the longest elimination half-life (t(1/2)). Further cassettes evaluated the library of trisubstituted purines with CYC202 and purvalanol A included as pharmacokinetic standards in a validated limited sampling strategy. The ratios of pharmacokinetic parameters to that of CYC202 [AUC, maximum concentration (C(max)), and t(1/2)] remained similar when compounds were tested in two different cassettes or as individual compounds. Following dosing of the same cassette on three different days, there was less than 20% variation in pharmacokinetic parameters between days. The structure-pharmacokinetics relationship showed that the favored purine substituents are benzylamine and veratrylamine at position 6, amino-2 propanol at position 2, and methylpropyl or hydroxyethyl at position 9. Without cassette dosing, this study would have used 3 times as many animals and would have taken 4 times longer, illustrating the power of this method in lead optimization.

Animals↗
PubMed2003

New lupane derived compounds with pro-apoptotic activity in cancer cells: synthesis and structure-activity relationships.

Cellular screening of various synthetic triterpenoid compounds formally derived from lupane has identified a number of analogues as potential anticancer drug candidates. Here we describe the synthesis and structure-activity relationships of betulin and betulinic acid derivatives containing an E-ring modified with different oxygen functions. Thus compounds containing the lup-18-en-21-one, lup-18-ene-21,22-dione, 18,19-secolupane, and the highly oxygenated 18,19-secolupane systems, as well as des-E-lupane derivatives, were prepared from the readily available natural pentacyclic triterpene betulin using oxidative procedures. These compounds were named betulinines. We demonstrate that only selected compounds, particularly those containing a lupane E-ring-derived unsaturated ketone or diketone function, possessed in vitro cytotoxic activity against tumor cell lines, suggesting a structure-activity relationship.

Animals↗
PubMed2003

Diketopiperazines in peptide and combinatorial chemistry.

Diketopiperazines (DKPs), the smallest cyclic peptides, represent an important class of biologically active natural products and their research has been fundamental to many aspects of peptide chemistry. The advent of combinatorial chemistry has revived interest in DKPs for two reasons: firstly, they are simple heterocyclic scaffolds in which diversity can be introduced and stereochemically controlled at up to four positions; secondly, they can be prepared from readily available alpha-amino acids using very robust chemistry. Here synthetic methods, conformation, as well as applications of DKPs are summarized and discussed critically.

Amino Acids↗
PubMed2003

CDK versus GSK-3 inhibition: a purple haze no longer?

The ubiquitous ATP binding site offers a global target for protein kinase inhibitors. The corollary is that molecular selectivity with such agents may be difficult to achieve and ascertain. A relevant example is discussed in terms of design and biomedical rationale.

Adenosine Triphosphate↗
PubMed2003

Insights into cyclin groove recognition: complex crystal structures and inhibitor design through ligand exchange.

Inhibition of CDK2/CA (cyclin-dependent kinase 2/cyclin A complex) activity through blocking of the substrate recognition site in the cyclin A subunit has been demonstrated to be an effective method for inducing apoptosis in tumor cells. We have used the cyclin binding motif (CBM) present in the tumor suppressor proteins p21(WAF1) and p27(KIP1) as a template to optimize the minimal sequence necessary for CDK2/CA inhibition. A series of peptides were prepared, containing nonnatural amino acids, which possess nano- to micromolar CDK2-inhibitory activity. Here we present X-ray structures of the protein complex CDK2/CA, together with the cyclin groove-bound peptides H-Ala-Ala-Abu-Arg-Ser-Leu-Ile-(p-F-Phe)-NH(2) (peptide 1), H-Arg-Arg-Leu-Ile-Phe-NH(2) (peptide 2), Ac-Arg-Arg-Leu-Asn-(m-Cl-Phe)-NH(2) (peptide 3), H-Arg-Arg-Leu-Asn-(p-F-Phe)-NH(2) (peptide 4), and H-Cit-Cit-Leu-Ile-(p-F-Phe)-NH(2) (peptide 5). Some of the peptide complexes presented here were obtained through the novel technique of ligand exchange within protein crystals. This method may find general application for obtaining complex structures of proteins with surface-bound ligands.

Amino Acid Motifs↗
PubMed2003

Discovery of a novel family of CDK inhibitors with the program LIDAEUS: structural basis for ligand-induced disordering of the activation loop.

A family of 4-heteroaryl-2-amino-pyrimidine CDK2 inhibitor lead compounds was discovered with the new database-mining program LIDAEUS through in silico screening. Four compounds with IC(50) values ranging from 17 to 0.9 microM were selected for X-ray crystal analysis. Two distinct binding modes are observed, one of which resembles the hydrogen bonding pattern of bound ATP. In the second binding mode, the ligands trigger a conformational change in the activation T loop by inducing movement of Lys(33) and Asp(145) side chains. The family of molecules discovered provides an excellent starting point for the design and synthesis of tight binding inhibitors, which may lead to a new class of antiproliferative drugs.

Adenosine Triphosphate↗
PubMed2003

CNIO cancer conference: targeted search for anticancer drugs.

The topics discussed at the conference covered many aspects of cancer research, from the genetic search for new targets, target validation and drug discovery, all the way to preclinical and clinical development of oncology drugs. Here the presentations on new metabolic, angiogenic, cell cycle and other molecular targets, as well as recent developments with experimental drugs with action on some of these targets, are summarised. Particular emphasis is placed on the emerging realisation that changes in the metabolic phenotype lie at the heart of cellular transformation. New insights into the biological links between cancer cell metabolism and the balance between survival and death signalling are likely to lead to the identification of a new category of anticancer targets.

Animals↗
PubMed2003

CDK inhibitors in clinical development for the treatment of cancer.

Cyclin-dependent protein kinases (CDKs) are key regulators of the cell division cycle, whose various checkpoints proliferating cells must traverse. Since CDK deregulation, either through direct or indirect means, is found in most cancer cells, pharmacological CDK inhibition has become an attractive strategy towards mechanism-based and non-genotoxic therapies in oncology. Over the last decade, discovery and lead optimisation efforts have provided a wealth of potential drug candidate molecules capable of inhibiting CDKs, blocking cell-cycle progression, modulating transcription and inducing apoptosis selectively in cancer cells. However, only few such agents have as yet reached clinical evaluation. Here, the preclinical and clinical results obtained so far with flavopiridol (L868275, HMR1275; Aventis), 7-hydroxystaurosporine (UCN-01, KW-2401; Kyowa Hakko Kogyo) and roscovitine (R-roscovitine, CYC202; Cyclacel) are summarised. Furthermore, the potential for monotherapy and applications in combination with existing drugs are discussed.

Animals↗
PubMed2003

The p53-Mdm2 pathway: targets for the development of new anticancer therapeutics.

The tumour suppressor p53 is at the centre of a network of regulatory pathways that guard over the continued integrity of the living cell and its progeny after exposure to different forms of stress, particularly those capable of inducing DNA damage. Tumour cells very frequently circumvent this control by disabling the function of p53, or other proteins in the p53 network, through mutation. Here we review the different therapeutic strategies that have been adopted to exploit common neoplastic aberrations in the p53 pathways. We emphasise in particular those approaches where modulation with pharmaceutical agents has already shown some promise, including pharmacological rescue of mutant p53, modulation of the protein-protein interaction between p53 and one of its negative regulators, Mdm2, as well as interference with downstream targets.

Antineoplastic Agents↗
PubMed2003

Peptidomimetic design of CDK inhibitors targeting the recruitment site of the cyclin subunit.

The recognition of cyclin-dependent kinase (CDK)/cyclin complexes by various cell-cycle regulatory proteins, including certain tumour suppressors and transcription factors, occurs at least in part through a protein-protein interaction with a binding groove on the cyclin subunit. Since CDK function is generally deregulated in tumour cells, blocking of this recruitment site prevents recognition and subsequent phosphorylation of CDK substrates and offers a therapeutic approach towards restoration of checkpoint control in transformed cells. Here we discuss the finding that peptides derived from such cyclin-interacting proteins, and rendered permeable through conjugation to cellular delivery vectors, can apparently induce tumour cells to undergo apoptosis selectively. We review the current status of 3D-structural information available on cyclin-peptide interactions and we summarise our extensive peptide structure-activity relationship studies in light of this information. We also show how a combination of molecular modelling and introduction into synthetic peptides of peptidomimetic elements, such as non-natural amino acid residues and conformational constraints, is being used hopefully to arrive at drug candidates capable of modulating CDK function in a selective mechanism-based approach rather than through ATP antagonism.

Animals↗
PubMed2003

Cyclin-dependent kinase inhibitors.

Cyclin-dependent kinases are involved in diverse cellular processes that include cell cycle control, apoptosis, neuronal physiology, differentiation, and transcription. Intensive screening and drug design based on CDK/inhibitor co-crystal structures and on SAR studies have led to the identification and characterization of a large variety of chemical inhibitors of CDKs. Although they all act by competing with ATP for binding at the catalytic site of the kinase, their kinase selectivity varies greatly and remains to be studied in most cases. The requirement for CDKs in many physiological processes justifies their evaluation as potential therapeutic targets against a much larger scope of diseases than initially anticipated.

Adenosine Triphosphate↗
PubMed2002

In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-roscovitine).

CDK2 inhibitors have been proposed as effective anti-cancer therapeutics. We show here that CYC202 (R-roscovitine) is a potent inhibitor of recombinant CDK2/cyclin E kinase activity (IC(50) = 0.10 microM) with an average cytotoxic IC(50) of 15.2 microM in a panel of 19 human tumour cell lines, and we also demonstrate selectivity for rapidly proliferating cells over non-proliferating cells. A study of the cell cycle effects of CYC202 in Lovo colorectal carcinoma cells showed that the major effect was not the predicted arrest in one part of the cycle, but rather an induction of cell death from all compartments of the cell cycle. The maximum tolerated dose given intravenously to mice was in excess of 20 mg/kg. Doses up to 2,000 mg/kg were tolerated when administered orally in mice. Following repeated intraperitoneal administration (3 times daily for 5 days) of 100 mg/kg to nude mice bearing the Lovo human colorectal tumour, CYC202 induced a significant antitumour effect with a 45% reduction in tumour growth compared to controls. A second experiment using the human uterine xenograft MESSA-DX5 treated with orally administered CYC202 (500 mg/kg 3 times daily for 4 days) also exhibited a significant reduction in the rate of growth of the tumour (62%). These data, showing enzyme and cellular potency together with antitumour activity, confirm the potential of CDK2 inhibitors such as CYC202 as anticancer drugs.

Animals↗
PubMed2002

Peptide inhibitors of CDK2-cyclin A that target the cyclin recruitment-site: structural variants of the C-terminal Phe.

A focused series of octapeptides based on the lead compound H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH(2) 1, in which the C-terminal phenylalanine residue was replaced by alpha and/or beta-modified variants, was synthesized using solid-phase chemistry. Both the L-threo-beta-hydroxy-phenylalanine (beta-phenylserine, Pse) and (2S)-phenylalaninol derivatives, as competitive binders at the cyclin-recruitment site, displayed potent inhibitory activity towards the CDK2-cyclin A complex. Unexpectedly, the D-threo-Pse derivatives also showed inhibitory activity.

Amino Acid Sequence↗
PubMed2002

Liquid-phase peptide synthesis on polyethylene glycol (PEG) supports using strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group: application of PEGylated peptides in biochemical assays.

Stepwise synthetic assembly of polypeptide chains reversibly linked to polyethylene glycol represents a hybrid between traditional solution and solid-phase chemistries and combines the inherent advantages of both approaches. The technical simplicity and scalability of the liquid-phase peptide synthesis method renders it particularly attractive for multiple parallel syntheses, combinatorial approaches and the large-scale preparation of peptides. The versatile protection strategy based on the N alpha-fluorenylmethoxycarbonyl group commonly used in solid-phase peptide synthesis was adapted to the liquid-phase approach. Fluoride ions were used rather than the conventional organic base piperidine for the repetitive amino-deprotection step. Using a range of acid- and base-labile linkers between the polymer and the peptide, it was shown that free and fully side-chain protected peptides can be obtained using our version of the liquid-phase peptide synthesis method. Protocols for simultaneous multiple syntheses requiring a minimum of equipment are presented and the use of polyethylene glycol-bound peptides in biochemical binding and functional assay systems is demonstrated.

Amino Acids↗