Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels.

Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore.

Unravelling the intricate cooperativity of subunit gating in P2X2 ion channels.

Ionotropic purinergic (P2X) receptors are trimeric channels that are activated by the binding of ATP. They are involved in multiple physiological functions, including synaptic transmission, pain and inflammation. The mechanism of activation is still elusive.

Relating ligand binding to activation gating in P2X2 receptors using a novel fluorescent ATP derivative.

Ionotropic purinergic receptors (P2X receptors) are non-specific cation channels that are activated by the binding of ATP at their extracellular side. P2X receptors contribute to multiple functions, including the generation of pain, inflammation, or synaptic transmission. The channels are trimers and structural information on several of their isoforms is available.

Novel Fluorescent Cyclic Nucleotide Derivatives to Study CNG and HCN Channel Function.

A highly specific molecular interaction of diffusible ligands with their receptors belongs to the key processes in cellular signaling. Because an appropriate method to monitor the unitary binding events is still missing, most of our present knowledge is based on ensemble signals recorded from a big number of receptors, such as ion currents or fluorescence changes of suitably labeled receptors, and reasoning from these data to the ligand binding. To study the binding process itself, appropriately tagged ligands are required that fully activate the receptors and report the binding at the same time.

Hydrophobic alkyl chains substituted to the 8-position of cyclic nucleotides enhance activation of CNG and HCN channels by an intricate enthalpy - entropy compensation.

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are tetrameric non-specific cation channels in the plasma membrane that are activated by either cAMP or cGMP binding to specific binding domains incorporated in each subunit. Typical apparent affinities of these channels for these cyclic nucleotides range from several hundred nanomolar to tens of micromolar. Here we synthesized and characterized novel cAMP and cGMP derivatives by substituting either hydrophobic alkyl chains or similar-sized more hydrophilic heteroalkyl chains to the 8-position of the purine ring with the aim to obtain full agonists of higher potency.

Tick-borne Diseases (Borreliosis, Anaplasmosis, Babesiosis) in German and Austrian Dogs: Status quo and Review of Distribution, Transmission, Clinical Findings, Diagnostics and Prophylaxis.

Tick-borne diseases (TBD) in dogs have gained in significance in German and Austrian veterinary practices. The widespread European tick species Ixodes ricinus represents an important vector for spirochaetes of the Borrelia burgdorferi sensu lato group and Rickettsiales such as Anaplasma phagocytophilum. The meadow or ornate dog tick (Dermacentor reticulatus) is an important vector for Babesia canis, as is the brown dog tick (Rhipicephalus sanguineus) for Babesia vogeli in the Mediterranean region.

Correlating structure and ligand affinity in drug discovery: a cautionary tale involving second shell residues.

A high-resolution crystallographic structure determination of a protein-ligand complex is generally accepted as the 'gold standard' for structure-based drug design, yet the relationship between structure and affinity is neither obvious nor straightforward. Here we analyze the interactions of a series of serine proteinase inhibitors with trypsin variants onto which the ligand-binding site of factor Xa has been grafted. Despite conservative mutations of only two residues not immediately in contact with ligands (second shell residues), significant differences in the affinity profiles of the variants are observed.

Use of IHC and newly designed matriptase inhibitors to elucidate the role of matriptase in pancreatic ductal adenocarcinoma.

Matriptase, also known as MT-SP1, is a type II transmembrane serine protease strongly implicated in both the development and progression of a variety of epithelial cancers. Evidence comes from studies of its expression in human cancers and from mouse models of spontaneous cancer. Matriptase is considered to be a major activator of two key stimulators of invasive growth, namely hepatocyte growth factor/scatter factor and urokinase-type plasminogen activator.

Incorporation of neutral C-terminal residues in 3-amidinophenylalanine-derived matriptase inhibitors.

A novel series of matriptase inhibitors based on previously identified tribasic 3-amidinophenylalanine derivatives was prepared. The C-terminal basic group was replaced by neutral residues to reduce the hydrophilicity of the inhibitors. The most potent compound 22 inhibits matriptase with a K(i) value of 0.

Modification of the N-terminal sulfonyl residue in 3-amidinophenylalanine-based matriptase inhibitors.

Replacement of the N-terminal beta-alanyl-amide moiety in previously identified matriptase inhibitors by non-charged aryl groups caused a slightly decreased potency and partially reduced selectivity, especially towards thrombin. However, some of these analogues are still potent matriptase inhibitors with K(i)-values <10nM. In contrast, improved activity was observed for newly designed tribasic analogues, especially for compound 21, which inhibits matriptase with an K(i)-value of 80pM.

Highly potent and selective substrate analogue factor Xa inhibitors containing D-homophenylalanine analogues as P3 residue: part 2.

A series of highly potent substrate-analogue factor Xa inhibitors containing D-homophenylalanine analogues as the P3 residue has been identified by systematic optimization of a previously described inhibitor structure. An initial lead, benzylsulfonyl-D-hPhe-Gly-4-amidinobenzylamide (3), inhibits fXa with an inhibition constant of 6.0 nM.

From selective substrate analogue factor Xa inhibitors to dual inhibitors of thrombin and factor Xa. Part 3.

Highly potent and selective substrate analogue factor Xa inhibitors were obtained by incorporation of non-basic or modestly basic P1 residues known from the development of thrombin inhibitors. The modification of the P2 and P3 amino acids strongly influenced the selectivity and provided potent dual factor Xa and thrombin inhibitors without affecting the fibrinolytic enzymes. Several inhibitors demonstrated excellent anticoagulant efficacy in standard clotting assays in human plasma.

New substrate analogue inhibitors of factor Xa containing 4-amidinobenzylamide as P1 residue: part 1.

The trypsin-like serine protease factor Xa (fXa) is located at the convergence point of the intrinsic and extrinsic coagulation cascade, and therefore has emerged as an attractive target for the design of novel anticoagulants. During the development of substrate-analogue urokinase inhibitors we have found that the protection of the P3-dSer side chain leads to a scaffold of potent fXa inhibitors with the general structure R1-SO2-dSer(R2)-Gly-4-amidinobenzylamide. The first lead (3) with an N-terminal benzylsulfonyl group and dSer(tBu) as P3 residue inhibits human fXa with a Ki of 14 nM.

Secondary amides of sulfonylated 3-amidinophenylalanine. New potent and selective inhibitors of matriptase.

Matriptase is an epithelium-derived type II transmembrane serine protease and has been implicated in the activation of substrates such as pro-HGF/SF and pro-uPA, which are likely involved in tumor progression and metastasis. Through screening, we have identified bis-basic secondary amides of sulfonylated 3-amidinophenylalanine as matriptase inhibitors. X-ray analyses of analogues 8 and 31 in complex with matriptase revealed that these inhibitors occupy, in addition to part of the previously described S4-binding site, the cleft formed by the molecular surface and the unique 60 loop of matriptase.

In vitro inhibition of matriptase prevents invasive growth of cell lines of prostate and colon carcinoma.

Matriptase, also known as membrane-type-serine-protease 1 (MT-SP 1), is a type II transmembrane serine protease involved in the activation of the precursor form of hepatocyte growth factor/scatter factor (pro-HGF/SF). Since HGF/SF is a well-known extracellular signal, which plays a key role in the control of invasive growth, we investigated the effects of matriptase inhibition in cell lines derived from colon (DLD-1) or prostate (PC-3) carcinomas. Biochemical analysis showed that matriptase was very efficient in the proteolytic conversion of the inactive HGF/SF precursor into HGF/SF.

Design of novel and selective inhibitors of urokinase-type plasminogen activator with improved pharmacokinetic properties for use as antimetastatic agents.

The serine protease urokinase-type plasminogen activator (uPA) interacts with a specific receptor (uPAR) on the surface of various cell types, including tumor cells, and plays a crucial role in pericellular proteolysis. High levels of uPA and uPAR often correlate with poor prognosis of cancer patients. Therefore, the specific inhibition of uPA with small molecule active-site inhibitors is one strategy to decrease the invasive and metastatic activity of tumor cells.

Increase of anti-metastatic efficacy by selectivity- but not affinity-optimization of synthetic serine protease inhibitors.

Although tumors frequently show elevated protease activities, the concept of anti-proteolytic cancer therapy has lost momentum after failure of clinical trials with broad-spectrum matrix metalloproteinase inhibitors. Thus we need to adapt our design strategies for protease inhibitors. Here, we employed a series of seven structurally fine-modulated and pharmacokinetically closely related synthetic 4-amidinobenzylamine-based inhibitors with distinct selectivity for prototypical serine proteases in a murine T cell lymphoma liver metastasis model.

Crystals of urokinase type plasminogen activator complexes reveal the binding mode of peptidomimetic inhibitors.

Urokinase type plasminogen activator (uPA), a trypsin-like serine proteinase, plays an important role in normal tissue re-modelling, cell adhesion, and cell motility. In addition, studies utilizing normal animals and potent, selective uPA inhibitors or genetically modified mice that lack functional uPA genes have demonstrated that uPA can significantly enhance tumor initiation, growth, progression and metastasis, strongly suggesting that this enzyme may be a promising anti-cancer target. We have investigated the structure-activity relationship (SAR) of peptidomimetic inhibitors of uPA and solved high resolution X-ray structures of key, lead small molecule inhibitors (e.

Structure-activity relationships of new NAPAP-analogs.

Several new analogs of the known thrombin inhibitor NAPAP were synthesized, in which the P2 glycine residue was substituted by natural and unnatural amino acids. The thrombin inhibitory potency was comparable to that of NAPAP. Several of the compounds had inhibition constants lower than 10 nM and a very high selectivity compared to trypsin, factor Xa and plasmin.

4-amidinobenzylamine-based inhibitors of urokinase.

A series of 4-amidinobenzylamine-based peptidomimetic inhibitors of urokinase was synthesized. The most potent one, benzylsulfonyl-D-Ser-Ala-4-amidinobenzylamide 16, inhibits uPA with a K(i) of 7.7 nM but is less selective than 10 with a Gly as P2 residue.