Joseph Rahuel

Structure-based design of aliskiren, a novel orally effective renin inhibitor

Hypertension is a major risk factor for cardiovascular diseases such as stroke, myocardial infarction, and heart failure, the leading causes of death in the Western world. Inhibitors of the renin-angiotensin system (RAS) have proven to be successful treatments for hypertension. As renin specifically catalyses the rate-limiting step of the RAS, it represents the optimal target for RAS inhibition. Several peptide-like renin inhibitors have been synthesized previously, but poor pharmacokinetic properties meant that these compounds were not clinically useful. We employed a combination of molecular modelling and crystallographic structure analysis to design renin inhibitors lacking the extended peptide-like backbone of earlier inhibitors, for improved pharmacokinetic properties. This led to the discovery of aliskiren, a highly potent and selective inhibitor of human renin in vitro, and in vivo; once-daily oral doses of aliskiren inhibit renin and lower blood pressure in sodium-depleted marmosets and hypertensive human patients. Aliskiren represents the first in a novel class of renin inhibitors with the potential for treatment of hypertension and related cardiovascular diseases.

Structure-based drug design: the discovery of novel nonpeptide orally active inhibitors of human renin

BACKGROUND The aspartic proteinase renin plays an important physiological role in the regulation of blood pressure. It catalyses the first step in the conversion of angiotensinogen to the hormone angiotensin II. In the past, potent peptide inhibitors of renin have been developed, but none of these compounds has made it to the end of clinical trials. Our primary aim was to develop novel nonpeptide inhibitors. Based on the available structural information concerning renin-substrate interactions, we synthesized inhibitors in which the peptide portion was replaced by lipophilic moieties that interact with the large hydrophobic S1/S3-binding pocket in renin. RESULTS Crystal structure analysis of renin-inhibitor complexes combined with computational methods were employed in the medicinal-chemistry optimisation process. Structure analysis revealed that the newly designed inhibitors bind as predicted to the S1/S3 pocket. In addition, however, these compounds interact with a hitherto unrecognised large, distinct, sub-pocket of the enzyme that extends from the S3-binding site towards the hydrophobic core of the enzyme. Binding to this S3(sp) sub-pocket was essential for high binding affinity. This unprecedented binding mode guided the drug-design process in which the mostly hydrophobic interactions within subsite S3(sp) were optimised. CONCLUSIONS Our design approach led to compounds with high in vitro affinity and specificity for renin, favourable bioavailability and excellent oral efficacy in lowering blood pressure in primates. These renin inhibitors are therefore potential therapeutic agents for the treatment of hypertension and related cardiovascular diseases.

Effect of Potent and Selective Inhibitors of the Grb2 SH2 Domain on Cell Motility

Cell motility has been correlated both with oncogenic invasiveness and metastatic potential. The development of selective inhibitors of motility has thus great potential importance. Grb2 is a SH2/SH3 domain-containing adaptor protein that links growth factor receptor tyrosine kinases to the Ras signaling pathway. We have developed specific small molecule inhibitors of the Grb2 SH2 domain as potential leads for drug discovery. Synthesis of the inhibitors and their effects on growth factor-induced growth in cells have been reported previously. In the current study, we establish that these inhibitors inhibit hepatocyte growth factor/scatter factor-induced A431 and Madin-Darby canine kidney cell motility and various cell motility-related events, including epidermal growth factor-induced ruffling of A431 cells and epidermal growth factor-induced translocation of the small GTPase Rac in these cells. We demonstrate for the first time a direct role for Grb2 in cell motility and indicate a new avenue for cancer therapeutics.

Refined crystal structures of subtilisin Novo in complex with wild-type and two mutant eglins: Comparison with other serine proteinase inhibitor complexes

The crystal structures of the complexes formed between subtilisin Novo and three inhibitors, eglin c, Arg45-eglin c and Lys53-eglin c have been determined using molecular replacement and difference Fourier techniques and refined at 2.4 A, 2.1 A, and 2.4 A resolution, respectively. The mutants Arg45-eglin c and Lys53-eglin c were constructed by site-directed mutagenesis in order to investigate the inhibitory specificity and stability of eglin c. Arg45-eglin became a potent trypsin inhibitor, in contrast to native eglin, which is an elastase inhibitor. This specificity change was rationalized by comparing the structures of Arg45-eglin and basic pancreatic trypsin inhibitor and their interactions with trypsin. The residue Arg53, which participates in a complex network of hydrogen bonds formed between the core and the binding loop of eglin c, was replaced with the shorter basic amino acid lysine in the mutant Lys53-eglin. Two hydrogen bonds with Thr44, located in the binding loop, can no longer be formed but are partially restored by a water molecule bound in the vicinity of Lys53. Eglin c in complexes with both subtilisin Novo and subtilisin Carlsberg was crystallized in two different space groups. Comparison of the complexes showed a rigid body rotation for the eglin c core of 11.5 degrees with respect to the enzyme, probably caused by different intermolecular contacts in both crystal forms.

Embryonic stem cell-derived neurons as a cellular system to study gene function: lack of amyloid precursor proteins APP and APLP2 leads to defective synaptic transmission.

The in vitro generation of uniform populations of neurons from mouse embryonic stem cells (ESCs) provides a novel opportunity to study gene function in neurons. This is of particular interest when mutations lead to lethal in vivo phenotypes. Although the amyloid precursor protein (APP) and its proteolysis are regarded as key elements of the pathology of Alzheimers disease, the physiological function of APP is not well understood and mice lacking App and the related gene Aplp2 die early postnatally without any obvious histopathological abnormalities. Here we show that glutamatergic neurons differentiated from ESCs lacking both genes reveal a decreased expression of the vesicular glutamate transporter 2 (VGLUT2) both at the mRNA and protein level, as well as a reduced uptake and/or release of glutamate. Blocking γ‐secretase cleavage of APP in wild‐type neurons resulted in a similar decrease of VGLUT2 expression, whereas VGLUT2 levels could be restored in App−/−Aplp2−/− neurons by a construct encompassing the C‐terminal intracellular domain of APP. Electrophysiological recordings of hippocampal organotypic slice cultures prepared from corresponding mutant mice corroborated these observations. Gene expression profiling and pathway analysis of the differentiated App−/−Aplp2−/− neurons identified dysregulation of additional genes involved in synaptic transmission pathways. Our results indicate a significant functional role of APP and amyloid precursor‐like protein 2 (APLP2) in the development of synaptic function by the regulation of glutamatergic neurotransmission. Differentiation of ESCs into homogeneous populations thus represents a new opportunity to explore gene function and to dissect signaling pathways in neurons.

Structure-based design of a non-peptidic antagonist of the SH2 domain of GRB2

The structure-based design and synthesis of a completely non-peptidic, micromolar antagonist of the SH2 domain of Grb2 is presented. The compound mimics the two main pharmacophores of the natural ligand, the phenylphosphate of the phosphotyrosine residue and the beta-carboxamide of the X+2 asparagine, which are linked by a rigid aromatic spacer.

Mapping the X+1 binding site of the Grb2-SH2 domain with α,α-disubstituted cyclic α-amino acids

Abstract A series of phosphopeptides containing α,α-disubstituted cyclic α-amino acids (Ac n c, 3 ≤n≤ 7; n refers to the number of carbons in the ring) at the X +1 position of Ac-Tyr(PO 3 H 2 )-X +1 -Asn-NH 2 has been synthesised and their inhibitory activity as antagonists of the Grb2-SH2 domain has been determined in competitive binding assays. The SAR data obtained have been interpreted by using models constructed from the X-ray structure of the ligand-bound Grb2-SH2 domain. The used of α,α-disubstituted cyclic α-amino acids to map the binding pockets of proteins expands the classical alanine scan concept and takes advantage of the known conformational preferences of these amino acids.

Structure-based design of peptidomimetic ligands of the Grb2-SH2 domain

We have designed and synthesized a (3-aminomethyl-phenyl)-urea scaffold to mimic the X+1-Asn part of the minimal phosphopeptide sequence, Ac-pTyr-X+1-Asn-NH2, recognized by the Grb2-SH2 domain. The resulting compounds show the same degree of affinity as their peptide counterparts for the Grb2-SH2 domain. This is the first example reported to date of ligands of the Grb2-SH2 domain with substantially reduced peptidic character.

X-ray crystal structure of the serine proteinase inhibitor eglin c at 1.95 Å resolution

The crystal structure eglin c. naturally occurring in the leech Hirudo medicinalis, is known from its complexes with various serine proteinases, but the crystallization of free eglin c has not yet been reported. A method is described for growing well‐diffracing crystals of free eglic c from highly concentrated protein solutions (≈2OO mg/ml). The space group of the orthorhombic crystals was determined to be P212121 with unit cell parameters a = 32.6. b = 42.0. c = 44.1 Å. The structure or free eglin c was resolved at 1.95 Å resolution by Patterson search methods. The final model contains all 70 amino acids of eglin c and 125 water molecules. In comparison to the eglin structure known from its complexes with proteinases, only small differences have been observed in free eglin c. However, the reactive site‐binding loop and a few residues on the surface of eglin have been found in different conformations due to crystal contacts. In contrast to the complex structures, the first seven amino acids of the highly flexible amino terminus can be located. Crystallographic refinement comprised molecular dynamics refinement, classical retrained least‐squares refinement and individual isotropic atomic temperature refinement. The final R‐factor is 15.8%.

Scaled-up production of recombinant human renin in CHO cells for enzymatic and X-ray structure analysis.

A process was developed to produce recombinant human renin for X-ray analysis and enzyme inhibition studies. An expression vector containing a human prorenin cDNA and expressing a mouse dihydrofolate reductase selection marker was transfected into dhfr-minus Chinese hamster ovary cells. After selection of cell strains with an increased gene copy number with methotrexate, cultures of the recombinant cells were scaled-up in serum-free media. Major improvements in cellular productivity were achieved by using continuous suspension cultures with cell recycling instead of an adherent culture system or batch-mode suspension cultures. The recombinant zymogen prorenin was purified and preparatively activated with trypsin. Enzymatic properties of the recombinant active renin are described.