Daniel Schlatter

Substrate and Inhibitor Profile of BACE (β-Secretase) and Comparison with Other Mammalian Aspartic Proteases

The full-length and ectodomain forms of β-site APP cleavage enzyme (BACE) have been cloned, expressed in Sf9 cells, and purified to homogeneity. This aspartic protease cleaves the amyloid precursor protein at the β-secretase site, a critical step in the Alzheimers disease pathogenesis. Comparison of BACE to other aspartic proteases such as cathepsin D and E, napsin A, pepsin, and renin revealed little similarity with respect to the substrate preference and inhibitor profile. On the other hand, these parameters are all very similar for the homologous enzyme BACE2. Based on a collection of decameric substrates, it was found that BACE has a loose substrate specificity and that the substrate recognition site in BACE extends over several amino acids. In common with the aspartic proteases mentioned above, BACE prefers a leucine residue at position P1. Unlike cathepsin D etc., BACE accepts polar or acidic residues at positions P2′ and P1 but prefers bulky hydrophobic residues at position P3. BACE displays poor kinetic constants toward its known substrates (wild-type substrate, SEVKM↓DAEFR,K m = 7 μm,K cat = 0.002 s−1; Swedish mutant, SEVNL↓DAEFR, K m = 9 μm,K cat = 0.02 s−1). A new substrate (VVEVDA↓AVTP, K m = 1 μm,K cat = 0.004) was identified by serendipity.

Aleglitazar, a new, potent, and balanced dual PPARα/γ agonist for the treatment of type II diabetes

Design, synthesis, and SAR of novel alpha-alkoxy-beta-arylpropionic acids as potent and balanced PPARalphagamma coagonists are described. One representative thereof, Aleglitazar ((S)-2Aa), was chosen for clinical development. Its X-ray structure in complex with both receptors as well as its high efficacy in animal models of T2D and dyslipidemia are also presented.

Tyramine fragment binding to BACE-1

Fragment screening revealed that tyramine binds to the active site of the Alzheimers disease drug target BACE-1. Hit expansion by selection of compounds from the Roche compound library identified tyramine derivatives with improved binding affinities as monitored by surface plasmon resonance. X-ray structures show that the amine of the tyramine fragment hydrogen-bonds to the catalytic water molecule. Structure-guided ligand design led to the synthesis of further low molecular weight compounds that are starting points for chemical leads.

Molecular recognition at the active site of catechol-o-methyltransferase: energetically favorable replacement of a water molecule imported by a bisubstrate inhibitor.

Biologically active catechols, such as l-DOPA and the neurotransmitter dopamine, are inactivated by methylation. This reaction is catalyzed by the enzyme catechol-O-methyltransferase (COMT) in the presence of S-adenosylmethionine (SAM) and Mg ions. Small nitrocatechol-based inhibitors of COMT find application in the treatment of Parkinson disease by blocking unwanted methylation of the administered l-DOPA, thereby enhancing dopamine levels in the brain. 3] Recent studies have pointed towards additional therapeutic applications of COMT inhibition in other disorders of the central nervous system, such as schizophrenia and depression. We have developed a series of potent bisubstrate inhibitors for COMT which are competitive for both the catechol and the SAM binding sites. Based on the X-ray crystal structure of ligand 1 (IC50 = 9 nm) [7a] in a ternary complex with COMT and a Mg ion (PDB code: 1JR4), we started a detailed exploration of the molecular recognition properties of the entire active site of the enzyme. Importantly, we found that potentially hepatotoxic nitro groups, which are mandatory in catechol-based monosubstrate inhibitors, are not required for high-affinity bisubstrate inhibition. We substituted the nitro group in position 5 of 1 with appropriate lipophilic residues, such as the 4-fluorophenyl ring in 2 (IC50 = 31 nm), and found that the high, competitive inhibitory potency was maintained. Computer modeling studies suggested that the newly introduced lipophilic residue occupies a hydrophobic cleft near the surface of the enzyme. This initial proposal is validated here experimentally by X-ray crystallography. The crystal structure of 1 in a ternary complex with COMT and a Mg ion shows that the adenine moiety forms two hydrogen bonds, a moderately strong and a weak one (d(N···O): 3.0 and 3.4 , respectively), to a water molecule

Generation, characterization and structural data of chymase binding proteins based on the human Fyn kinase SH3 domain

The serine protease chymase (EC = 3.4.21.39) is expressed in the secretory granules of mast cells, which are important in allergic reactions. Fynomers, which are binding proteins derived from the Fyn SH3 domain, were generated against human chymase to produce binding partners to facilitate crystallization, structure determination and structure-based drug discovery, and to provide inhibitors of chymase for therapeutic applications. The best Fynomer was found to bind chymase with a KD of 0.9 nM and koff of 6.6x10−4 s−1, and to selectively inhibit chymase activity with an IC50 value of 2 nM. Three different Fynomers were co-crystallized with chymase in 6 different crystal forms overall, with diffraction quality in the range of 2.25 to 1.4 Å resolution, which is suitable for drug design efforts. The X-ray structures show that all Fynomers bind to the active site of chymase. The conserved residues Arg15-Trp16-Thr17 in the RT-loop of the chymase binding Fynomers provide a tight interaction, with Trp16 pointing deep into the S1 pocket of chymase. These results confirm the suitability of Fynomers as research tools to facilitate protein crystallization, as well as for the development of assays to investigate the biological mechanism of targets. Finally, their highly specific inhibitory activity and favorable molecular properties support the use of Fynomers as potential therapeutic agents.

Design and Biological Evaluation of Novel, Balanced Dual PPARα/γ Agonists

An X‐ray‐guided design approach led to the identification of a novel, balanced class of α‐ethoxy‐phenylpropionic acid‐derived dual PPARα/γ agonists. The series shows a wide range of PPARα/γ ratios within a rather narrow structural space. Advanced compounds possess favorable physicochemical and pharmacokinetic profiles and show a high efficacy in T2D and dyslipidemia animal models.

Crystal Engineering Yields Crystals of Cyclophilin D Diffracting to 1.7 A Resolution

In the pharmaceutical industry, knowledge of the three-dimensional structure of a specific target facilitates the drug-discovery process. Despite possessing favoured analytical properties such as high purity and monodispersion in light scattering, some proteins are not capable of forming crystals suitable for X-ray analysis. Cyclophilin D, an isoform of cyclophilin that is expressed in the mitochondria, was selected as a drug target for the treatment of cardiac disorders. As the wild-type enzyme defied all attempts at crystallization, protein engineering on the enzyme surface was performed. The K133I mutant gave crystals that diffracted to 1.7 A resolution using in-house X-ray facilities and were suitable for soaking experiments. The crystals were very robust and diffraction was maintained after soaking in 25% DMSO solution: excellent conditions for the rapid analysis of complex structures including crystallographic fragment screening.

Molecular Recognition at the Active Site of Catechol-O-methyltransferase (COMT): Adenine Replacements in Bisubstrate Inhibitors

L-Dopa, the standard therapeutic for Parkinsons disease, is inactivated by the enzyme catechol-O-methyltransferase (COMT). COMT catalyzes the transfer of an activated methyl group from S-adenosylmethionine (SAM) to its catechol substrates, such as L-dopa, in the presence of magnesium ions. The molecular recognition properties of the SAM-binding site of COMT have been investigated only sparsely. Here, we explore this site by structural alterations of the adenine moiety of bisubstrate inhibitors. The molecular recognition of adenine is of special interest due to the great abundance and importance of this nucleobase in biological systems. Novel bisubstrate inhibitors with adenine replacements were developed by structure-based design and synthesized using a nucleosidation protocol introduced by Vorbrüggen and co-workers. Key interactions of the adenine moiety with COMT were measured with a radiochemical assay. Several bisubstrate inhibitors, most notably the adenine replacements thiopyridine, purine, N-methyladenine, and 6-methylpurine, displayed nanomolar IC(50) values (median inhibitory concentration) for COMT down to 6 nM. A series of six cocrystal structures of the bisubstrate inhibitors in ternary complexes with COMT and Mg(2+) confirm our predicted binding mode of the adenine replacements. The cocrystal structure of an inhibitor bearing no nucleobase can be regarded as an intermediate along the reaction coordinate of bisubstrate inhibitor binding to COMT. Our studies show that solvation varies with the type of adenine replacement, whereas among the adenine derivatives, the nitrogen atom at position 1 is essential for high affinity, while the exocyclic amino group is most efficiently substituted by a methyl group.

Design of Novel Aminopyrrolidine Factor Xa Inhibitors from a Screening Hit.

Starting from a hit identified by focused screening, 3-aminopyrrolidine factor Xa inhibitors were designed. The binding mode as determined by X-ray structural analysis as well as the pharmacokinetic behaviour of selected compounds is discussed.

Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones

The aspartic protease BACE2 is responsible for the shedding of the transmembrane protein Tmem27 from the surface of pancreatic β-cells, which leads to inactivation of the β-cell proliferating activity of Tmem27. This role of BACE2 in the control of β-cell maintenance suggests BACE2 as a drug target for diabetes. Inhibition of BACE2 has recently been shown to lead to improved control of glucose homeostasis and to increased insulin levels in insulin-resistant mice. BACE2 has 52% sequence identity to the well studied Alzheimers disease target enzyme β-secretase (BACE1). High-resolution BACE2 structures would contribute significantly to the investigation of this enzyme as either a drug target or anti-target. Surface mutagenesis, BACE2-binding antibody Fab fragments, single-domain camelid antibody VHH fragments (Xaperones) and Fyn-kinase-derived SH3 domains (Fynomers) were used as crystallization helpers to obtain the first high-resolution structures of BACE2. Eight crystal structures in six different packing environments define an ensemble of low-energy conformations available to the enzyme. Here, the different strategies used for raising and selecting BACE2 binders for cocrystallization are described and the crystallization success, crystal quality and the time and resources needed to obtain suitable crystals are compared.