Acyl ureas as human liver glycogen phosphorylase inhibitors for the treatment of type 2 diabetes.

Using a focused screening approach, acyl ureas have been discovered as a new class of inhibitors of human liver glycogen phosphorylase (hlGPa). The X-ray structure of screening hit 1 (IC50 = 2 microM) in a complex with rabbit muscle glycogen phosphorylase b reveals that 1 binds at the AMP site, the main allosteric effector site of the dimeric enzyme. A first cycle of chemical optimization supported by X-ray structural data yielded derivative 21, which inhibited hlGPa with an IC50 of 23 +/- 1 nM, but showed only moderate cellular activity in isolated rat hepatocytes (IC50 = 6.2 microM). Further optimization was guided by (i) a 3D pharmacophore model that was derived from a training set of 24 compounds and revealed the key chemical features for the biological activity and (ii) the 1.9 angstroms crystal structure of 21 in complex with hlGPa. A second set of compounds was synthesized and led to 42 with improved cellular activity (hlGPa IC50 = 53 +/- 1 nM; hepatocyte IC50 = 380 nM). Administration of 42 to anaesthetized Wistar rats caused a significant reduction of the glucagon-induced hyperglycemic peak. These findings are consistent with the inhibition of hepatic glycogenolysis and support the use of acyl ureas for the treatment of type 2 diabetes.

Mode of action of the microbial metabolite GE23077, a novel potent and selective inhibitor of bacterial RNA polymerase.

GE23077, a novel microbial metabolite recently isolated from Actinomadura sp. culture media, is a potent and selective inhibitor of bacterial RNA polymerase (RNAP). It inhibits Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) RNAPs with IC50 values (i.e. the concentration at which the enzyme activity is inhibited by 50%) in the 10(-8) m range, whereas it is not active on E. coli DNA polymerase or on eukaryotic (wheat germ) RNAP II (IC50 values > 10(-4) m in both cases). In spite of its potent activity on purified bacterial RNAPs, GE23077 shows a narrow spectrum of antimicrobial activity on Gram-positive and Gram-negative bacteria. To investigate the molecular basis of this behaviour, the effects of GE23077 on macromolecular biosynthesis were tested in E. coli cells permeabilized under different conditions. The addition of GE23077 to plasmolyzed cells resulted in an immediate and specific inhibition of intracellular RNA biosynthesis, in a dose-response manner, strongly suggesting that cell penetration is the main obstacle for effective antimicrobial activity of the antibiotic. Biochemical studies were also conducted with purified enzymes to obtain further insights into the mode of action of GE23077. Interestingly, the compound displays a behaviour similar to that of rifampicin, an antibiotic structurally unrelated to GE23077: both compounds act at the level of transcription initiation, but not on the sigma subunit and not on the formation of the promoter DNA-RNAP complex. Tests on different rifampicin-resistant E. coli RNAPs did not show any cross-resistance between the two compounds, indicating distinct binding sites on the target enzyme. In conclusion, GE23077 is an interesting new molecule for future mechanistic studies on bacterial RNAP and for its potential in anti-infective drug discovery.

Antibiotics GE23077, novel inhibitors of bacterial RNA polymerase. I. Taxonomy, isolation and characterization.

GE 23077 factors A1, A2, B1 and B2 are novel antibiotics isolated from fermentation broths of an Actinomadura sp. strain. GE23077 antibiotics are cyclic peptides, which inhibit Escherichia coli RNA polymerase at nM concentrations. Both rifampicin-sensitive and rifampicin-resistant polymerases are inhibited, whereas E. coli DNA polymerase and wheat germ RNA polymerase are substantially not affected. In spite of the potent activity on the enzyme, the antibiotics generally show poor activity against whole cell bacteria. The spectrum of activity is restricted to Moraxella catarrhalis, including clinical isolates, with partial activity against Neisseria gonorrhoeae and Mycobacterium smegmatis.

Requirements for specific binding of low affinity inhibitor fragments to the SH2 domain of (pp60)Src are identical to those for high affinity binding of full length inhibitors.

Results from a novel approach which uses protein crystallography for the screening of a low affinity inhibitor fragment library are analyzed by comparing the X-ray structures with bound fragments to the structures with the corresponding full length inhibitors. The screen for new phospho-tyrosine mimics binding to the SH2 domain of (pp60)src was initiated because of the limited cell penetration of phosphates. Fragments in our library typically had between 6 and 30 atoms and included compounds which had either millimolar activity in a Biacore assay or were suggested by the ab initio design program LUDI but had no measurable affinity. All identified fragments were located in the phospho-tyrosine pocket. The most promising fragments were successfully used to replace the phospho-tyrosine and resulted in novel nonpeptidic high affinity inhibitors. The significant diversity of successful fragments is reflected in the high flexibility of the phospho-tyrosine pocket. Comparison of the X-ray structures shows that the presence of the H-bond acceptors and not their relative position within the pharmacophore are essential for fragment binding and/or high affinity binding of full length inhibitors. The X-ray data show that the fragments are recognized by forming a complex H-bond network within the phospho-tyrosine pocket of SH2. No fragment structure was found in which this H-bond network was incomplete, and any uncompensated H-bond within the H-bond network leads to a significant decrease in the affinity of full length inhibitors. No correlation between affinity and fragment binding was found for these polar fragments and hence affinity-based screening would have overlooked some interesting starting points for inhibitor design. In contrast, we were unable to identify electron density for hydrophobic fragments, confirming that hydrophobic interactions are important for inhibitor affinity but of minor importance for ligand recognition. Our results suggest that a screening approach using protein crystallography is particularly useful to identify universal fragments for the conserved hydrophilic recognition sites found in target families such as SH2 domains, phosphatases, kinases, proteases, and esterases.

Evaluation of phenylboronate agarose for industrial-scale purification of erythropoietin from mammalian cell cultures.

The search for novel, cost-effective ways to produce erythropoietin (Epo), the world top-selling biopharmaceutical, is a major challenge for today's biotechnology industry. However, Epo's high glycosylation content (almost 40% of total mass) and the requirement for sialic acid for optimal in vivo activity still make mammalian cells the expression system of choice. In contrast to the abundance of reports on Epo production, robust, cost-effective methods for large-scale Epo purification can hardly be found in literature. To fill this gap, we describe here a process specifically studied for industrial-scale purification of the protein. Our method is based on the ability of phenylboronate agarose (PBA) to form reversible complexes with 1,2-cis-diol-containing molecules, like sugars in glycoproteins. Finding that additional factors (i.e., ionic and hydrophobic interactions) contribute to the Epo-PBA binding reaction, chromatography conditions have been optimized in scale-down experiments to improve selectivity and yield. As a result, the high performance of affinity chromatography has been achieved using a support possessing the robustness, chemical stability and low cost of a small synthetic ligand. By adding an anion exchange chromatography step and gel filtration for polishing, a pure and active product can easily be obtained by an integrated, start-to-end process optimized for industrial-scale operations.

High-affinity Src-SH2 ligands which do not activate Tyr(527)-phosphorylated Src in an experimental in vivo system.

The Src-SH2 domain has been determined to play a key role in many signaling pathways, especially in osteoclast-mediated bone resorption. Therefore, non-peptidic small molecules, mimicking the natural pYEEI peptide ligand, have been designed, to inhibit SH2-mediated protein-protein interactions and provide therapeutic treatment of certain diseases such as osteoporosis. However it has been shown in vitro that phosphopeptidic ligands of the SH2 domain are able to increase Src kinase activity by disrupting the intramolecular interactions between the Tyr(521)-phosphorylated C-terminal tail and the SH2 domain, thereby inducing a change from a "closed" inactive to an "open" active conformation of Src. Thus it was not clear whether non-peptidic ligands would limit their action to the inhibition of the signaling cascade by interfering with the intermolecular SH2 binding, or would activate the enzyme as do phosphopeptides. To address this question we have investigated the effects of a series of both peptidic and non-peptidic ligands of the SH2 domain on Src kinase activation, both in vitro in an ELISA based assay and in vivo using csk and src double transformed Schizosaccharomyces pombe. We found that, in the peptide series, the extent of c-Src activation is directly correlated to the respective binding affinity for Src-SH2. By contrast such correlation is not valid for non-peptidic ligands, some high-affinity SH2 binders showing no detectable Src activation in vivo. These results have significant implications for the design of SH2 binders, as they allow a way to inhibit Src-SH2-mediated signal transduction in target cells, without activating Src in non-target cells, thereby reducing the possibility of side effects.

Principles governing the binding of a class of non-peptidic inhibitors to the SH2 domain of src studied by X-ray analysis.

A total of 11 structures of the (pp60)src SH2 domain with non-peptidic inhibitors based on the same two closely related inhibitor scaffolds were determined using X-ray crystallography. Surprisingly, the inhibitors that have an IC(50) value between 4 and 2700 nM bind in three different binding modes. Structure comparisons show that the inhibitors aim to maximize the interaction between the hydrophobic substituent and the hydrophobic pY+3 pocket. This is achieved either by an alternative binding mode of the phenyl phosphate or by including water molecules that mediate the interaction between the inhibitor scaffold and a rigid surface of the SH2 domain. The combination of the rigid pY+3 pocket and the rigid protein surface to which the scaffolds bind results in severe distance and angular restraints for putative scaffolds and their substituents. The X-ray data suggest that these restraints seem to be compensated in our system by including water molecules, thereby increasing the flexibility of the system.

SAR and X-ray. A new approach combining fragment-based screening and rational drug design: application to the discovery of nanomolar inhibitors of Src SH2.

(pp60)Src is a protein involved in signal transduction and is mainly expressed in neurones, platelets, and osteoclasts. Its precise biological role was recently discovered with the KO experiments by Soriano that gave rise to no other apparent phenotype than osteopetrosis, a disease resulting in excedent bone formation. The SH2 domain of the Src family specifically recognizes a sequence of tetrapeptide featuring a phosphotyrosine and a lipophilic aminoacid at the +1 and +3 positions. Recently we engaged in the search for SH2 ligands via modular peptidomimicry of this tetrapetide. This gave rise to several families of nanomolar inhibitors; the best one incorporated a caprolactam scaffold, a biphenyl moiety, and a phosphotyrosine. However, these inhibitors still incorporated the phosphate group that confers good binding affinity to the protein. Phosphates have undesirable features for drug candidates, namely, high rate of hydrolysis of the phosphate group by phosphatases and high charge content precluding cell penetration. Therefore, while searching for optimal non-peptide ligands for Src SH2, we looked for phosphate replacements. For this, we have designed an SAR by fragment crystallography approach. The start of this work resulted from two experimental observations. First, the fact that phenyl phosphate itself displayed detectable binding affinity for Src SH2 permitted us to perform a screening of small aromatic compounds as phenyl phosphate surrogates. Second, the obtention of large Src SH2 crystals displaying a channel large enough for soaking purposes allowed structure determination of over 40 of these small aromatic compounds bound in the phosphotyrosine binding pocket. This search and the way it gave rise to low nanomolar range Src SH2 inhibitors devoid of phosphate groups will be the subject of the present paper.

Natural kirromycin resistance of elongation factor Tu from the kirrothricin producer Streptomyces cinnamoneus.

The antibiotic kirromycin (Kr) inhibits bacterial protein synthesis by binding to elongation factor Tu (EF-Tu). Streptomyces cinnamoneus and Nocardia lactamdurans, producers of antibiotics of the Kr class, are known to possess an EF-Tu resistant to Kr. Both micro-organisms appear to possess a single tuf gene and we have characterized the one from S. cinnamoneus, which belongs to the tuf1 family. To assess the molecular determinants of Kr resistance, the S. cinnamoneus tuf gene was expressed in Escherichia coli as a translational fusion to malE, which enabled the recovery by affinity chromatography of the recombinant protein uncontaminated by the host factor. The recombinant EF-Tu was able to catalyse polyU-directed polyPhe synthesis in two heterologous cell-free systems, even as an uncleaved fusion. When tested for antibiotic sensitivity it behaved like the natural S. cinnamoneus protein, showing equivalent resistance to Kr but sensitivity to the antibiotic GE2270, indicating that all the determinants for Kr resistance are intrinsic to the EF-Tu sequence. Multiple sequence analysis of EF-Tu proteins, together with knowledge of mutations conferring Kr resistance, allowed the identification of key residues as likely candidates for the natural Kr resistance of the S. cinnamoneus EF-Tu. One of these, Thr378, was mutated to the consensus Ala and the resulting mutant protein was sensitive to Kr. Interestingly, it retained some activity (30% of the control) even at high Kr concentrations.