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Chromatin remodelers use the energy of ATP hydrolysis to regulate chromatin dynamics. Their impact for development and disease requires strict enzymatic control. Here, we address the differential regulability of the ATPase domain of hSNF2H and hCHD3, exhibiting similar substrate affinities and enzymatic activities. Both enzymes are comparably strongly inhibited in their ATP hydrolysis activity by the competitive ATPase inhibitor ADP. However, the nucleosome remodeling activity of SNF2H is more strongly affected than that of CHD3. Beside ADP, also IP6 inhibits the nucleosome translocation of both enzymes to varying degrees, following a competitive inhibition mode at CHD3, but not at SNF2H. Our observations are further substantiated by mutating conserved Q- and K-residues of ATPase domain motifs. The variants still bind both substrates and exhibit a wild-type similar, basal ATP hydrolysis. Apart from three CHD3 variants, none of the variants can translocate nucleosomes, suggesting for the first time that the basal ATPase activity of the CHD3 is sufficient for nucleosome remodeling. Together with the ADP data our results propose a more efficient coupling of ATP hydrolysis and remodeling in CHD3. This aspect correlates with findings that CHD3 nucleosome translocation is visible at much lower ATP concentrations than SNF2H. We propose sequence differences between the ATPase domains of both enzymes as an explanation for the functional differences and suggest that aa interactions, including the conserved Q- and K-residues distinctly regulate ATPase-dependent functions of both proteins. Our data emphasize the benefits of remodeler ATPase domains for selective drugability and/or regulability of chromatin dynamics.

Targeted protein degradation offers new opportunities to inactivate cancer drivers and has successfully entered the clinic. Ways to induce selective protein degradation include proteolysis targeting chimera (PROTAC) technology and immunomodulatory (IMiDs) / next-generation Cereblon (CRBN) E3 ligase modulating drugs (CELMoDs). Here, we aimed to develop a MYC PROTAC based on the MYC-MAX dimerization inhibitor 10058-F4 derivative 28RH and Thalidomide, called MDEG-541. We show that a subgroup of gastrointestinal cancer cell lines and primary patient-derived organoids are MDEG-541 sensitive. Although MYC expression was regulated in a CRBN-, proteasome- and ubiquitin-dependent manner, we provide evidence that MDEG-541 induced the degradation of CRBN neosubstrates, including G1 to S phase transition 1/2 (GSPT1/2) and the Polo-like kinase 1 (PLK1). In sum, we have established a CRBN-dependent degrader of relevant cancer targets with activity in gastrointestinal cancers.

Previous results indicate that the phosphodiesterase PDE3B hydrolyzes cUMP. Also, almost 50 years ago, cUMP-hydrolytic activity was observed in rat adipose tissue. We intended to characterize the enzyme kinetics of PDE3B-mediated cUMP hydrolysis, to determine the PDE3B binding mode of cUMP, and to analyze cUMP hydrolysis in adipocyte preparations. Educts (cNMPs) and products (NMPs) of the PDE reactions as well as intracellular cNMPs were quantitated by HPLC-coupled tandem mass spectrometry. PDE3B expression was determined by qPCR and Western blot. Docking studies were performed with the PDE3B crystal structure PDB ID 1SO2 (complex with a dihydropyridazine inhibitor). PDE3B hydrolyzed cUMP (K

Epigenetic modifiers of the histone deacetylase (HDAC) family contribute to autoimmunity, cancer, HIV infection, inflammation, and neurodegeneration. Hence, histone deacetylase inhibitors (HDACi), which alter protein acetylation, gene expression patterns, and cell fate decisions, represent promising new drugs for the therapy of these diseases. Whereas pan-HDACi inhibit all 11 Zn2+-dependent histone deacetylases (HDACs) and cause a broad spectrum of side effects, specific inhibitors of histone deacetylase 6 (HDAC6i) are supposed to have less side effects. We present the synthesis and biological evaluation of Marbostats, novel HDAC6i that contain the hydroxamic acid moiety linked to tetrahydro-β-carboline derivatives. Our lead compound Marbostat-100 is a more potent and more selective HDAC6i than previously established well-characterized compounds in vitro as well as in cells. Moreover, Marbostat-100 is well tolerated by mice and effective against collagen type II induced arthritis. Thus, Marbostat-100 repr...

Adenylyl cyclases (ACs) generate the second messenger cAMP from ATP. Mammalian cells express nine transmembrane AC (mAC) isoforms (AC1-9) and a soluble AC (sAC, also referred to as AC10). This review will largely focus on mACs. mACs are activated by the G-protein Gαs and regulated by multiple mechanisms. mACs are differentially expressed in tissues and regulate numerous and diverse cell functions. mACs localize in distinct membrane compartments and form signaling complexes. sAC is activated by bicarbonate with physiologic roles first described in testis. Crystal structures of the catalytic core of a hybrid mAC and sAC are available. These structures provide detailed insights into the catalytic mechanism and constitute the basis for the development of isoform-selective activators and inhibitors. Although potent competitive and noncompetitive mAC inhibitors are available, it is challenging to obtain compounds with high isoform selectivity due to the conservation of the catalytic core. Accordingly, caution must be exerted with the interpretation of intact-cell studies. The development of isoform-selective activators, the plant diterpene forskolin being the starting compound, has been equally challenging. There is no known endogenous ligand for the forskolin binding site. Recently, development of selective sAC inhibitors was reported. An emerging field is the association of AC gene polymorphisms with human diseases. For example, mutations in the AC5 gene (ADCY5) cause hyperkinetic extrapyramidal motor disorders. Overall, in contrast to the guanylyl cyclase field, our understanding of the (patho)physiology of AC isoforms and the development of clinically useful drugs targeting ACs is still in its infancy.

Mutants of the FLT3 receptor tyrosine kinase (RTK) with duplications in the juxtamembrane domain (FLT3-ITD) act as drivers of acute myeloid leukemia (AML). Potent tyrosine kinase inhibitors (TKi) of FLT3-ITD entered clinical trials and showed a promising, but transient success due to the occurrence of secondary drug-resistant AML clones. A further caveat of drugs targeting FLT3-ITD is the co-targeting of other RTKs which are required for normal hematopoiesis. This is observed quite frequently. Therefore, novel drugs are necessary to treat AML effectively and safely. Recently bis(1H-indol-2-yl)methanones were found to inhibit FLT3 and PDGFR kinases. In order to optimize these agents we synthesized novel derivatives of these methanones with various substituents. Methanone 16 and its carbamate derivative 17b inhibit FLT3-ITD at least as potently as the TKi AC220 (quizartinib). Models indicate corresponding interactions of 16 and quizartinib with FLT3. The activity of 16 is accompanied by a high selectivity for FLT3-ITD.

The non-canonical cyclic nucleotide cUMP and the phosphodiesterase PDE9A both occur in neuronal cells. Using HPLC-coupled tandem mass spectrometry, we characterized the kinetics of PDE9A-mediated cUMP hydrolysis. PDE9A is a low-affinity and high-velocity enzyme for cUMP (Vmax = ~ 6 μmol/min/mg; Km = ~ 401 μM). The PDE9 inhibitor BAY 73-6691 inhibited PDE9A-catalyzed cUMP hydrolysis (Ki = 590 nM). Docking studies indicate two H-bonds between the cUMP uridine moiety and Gln453/Asn405 of PDE9A. By contrast, the guanosine moiety of cGMP forms three H-bonds with Gln453. cCMP is not hydrolyzed at a concentration of 3 μM, but inhibits the PDE9A-catalyzed cUMP hydrolysis at concentrations of 100 μM or more. The probable main reason is that the cytosine moiety cannot act as H-bond acceptor for Gln453. A comparison of PDE9A with PDE7A suggests that the preference of the former for cGMP and cUMP and of the latter for cAMP and cCMP is due to stabilized alternative conformations of the side chain amide of Gln453 and Gln413, respectively. This so-called glutamine switch is known to be involved in the regulation of cAMP/cGMP selectivity of some PDEs.

Various diseases are related to epigenetic modifications. Histone deacetylases (HDACs) and histone acetyl transferases (HATs) determine the pattern of histone acetylation, and thus are involved in the regulation of gene expression. First generation histone deacetylase inhibitors (HDACi) are unselective, hinder all different kinds of zinc dependent HDACs and additionally cause several side effects. Subsequently, selective HDACi are gaining more and more interest. Especially, selective histone deacetylase 6 inhibitors (HDAC6i) are supposed to be less toxic. Here we present a successful optimization study of tubastatin A, the synthesis and biological evaluation of new inhibitors based on hydroxamic acids linked to various tetrahydro-β-carboline derivatives. The potency of our selective HDAC6 inhibitors, exhibiting IC50 values in a range of 1–10 nM towards HDAC6, was evaluated with the help of a recombinant human HDAC6 enzyme assay. Selectivity was proofed in cellular assays by the hyperacetylation of surrogate parameter α-tubulin in the absence of acetylated histone H3 analyzed by Western Blot. We show that all synthesized compounds, with varies modifications of the rigid cap group, were selective and potent HDAC6 inhibitors.

In order to apply structure-based drug design techniques to GPCR complexes, it is essential to model their 3D structure. For this purpose, a multi-component protocol was derived based on protein-protein docking which generates populations of dimers compatible with membrane integration, considering all reasonable interfaces. At the next stage, we applied a scoring procedure based on up to eleven different parameters including shape or electrostatics complementarity. Two methods of consensus scoring were performed: (i) average scores of 100 best scored dimers with respect to each interface, and (ii) frequencies of interfaces among 100 best scored dimers. In general, our multi-component protocol gives correct indications for dimer interfaces that have been observed in X-ray crystal structures of GPCR dimers (opsin dimer, chemokine CXCR4 and CCR5 dimers, κ opioid receptor dimer, β1 adrenergic receptor dimer and smoothened receptor dimer) but also suggests alternative dimerization interfaces. Interestingly, at times these alternative interfaces are scored higher than the experimentally observed ones suggesting them to be also relevant in the life cycle of studied GPCR dimers. Further results indicate that GPCR dimer and higher-order oligomer formation may involve transmembrane helices (TMs) TM1-TM2-TM7, TM3-TM4-TM5 or TM4-TM5-TM6 but not TM1-TM2-TM3 or TM2-TM3-TM4 which is in general agreement with available experimental and computational data.

Membranous adenylyl cyclase 1 (AC1) is associated with memory and learning. AC1 is activated by the eukaryotic Ca(2+)-sensor calmodulin (CaM), which contains nine methionine residues (Met) important for CaM-target interactions. During ageing, Met residues are oxidized to (S)- and (R)-methionine sulfoxide (MetSO) by reactive oxygen species arising from an age-related oxidative stress. We examined how oxidation by H2O2 of Met in CaM regulates CaM activation of AC1. We employed a series of thirteen mutant CaM proteins never assessed before in a single study, where leucine is substituted for Met, in order to analyze the effects of oxidation of specific Met. CaM activation of AC1 is regulated by oxidation of all of the C-terminal Met in CaM, and by two N-terminal Met, M36 and M51. CaM with all Met oxidized is unable to activate AC1. Activity is fully restored by the combined catalytic activities of methionine sulfoxide reductases A and B (MsrA and B), which catalyze reduction of the (S)- and (R)-MetSO stereoisomers. A small change in secondary structure is observed in wild-type CaM upon oxidation of all nine Met, but no significant secondary structure changes occur in the mutant proteins when Met residues are oxidized by H2O2, suggesting that localized polarity, flexibility and structural changes promote the functional changes accompanying oxidation. The results signify that AC1 catalytic activity can be delicately adjusted by mediating CaM activation of AC1 by reversible Met oxidation in CaM. The results are important for memory, learning and possible therapeutic routes for regulating AC1.

There is increasing evidence for a role of cytidine 3',5'-cyclic monophosphate (cCMP) as second messenger. In a recent study, we showed that cCMP activates both purified guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase Iα (PKG Iα) and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) isoenzymes with the regulatory subunits RIα and RIIα. Moreover, the membrane-permeant cCMP analog dibutyryl (DB)-cCMP induces effective vasodilation and inhibition of platelet aggregation via PKG Iα, but not via PKA. These data prompted us to conduct a systematic analysis of the effects of cyclic nucleotide (cNMP) analogs on purified PKG Iα and PKA RIα and RIIα We also studied the effect of DB-cCMP on PKA-dependent phosphorylation of the transcription factor cAMP response-binding protein (CREB) in S49 wild-type lymphoma cells and S49 kin(-) cells, devoid of the catalytic subunit of PKA. The major cellular metabolite of the prodrug DB-cCMP, N(4)-monobutyryl (4-MB)-cCMP, was a partial and low-potency activator of purified PKG Iα and a full and moderate-potency activator of PKA RIα and RIIα. Sp-cCMPS and Sp-cAMPS activated PKA RIα and RIIα with much higher potency and efficacy than PKG Iα. Molecular modeling suggested that the cytidine ring interacts with PKG Iα mainly via hydrophobic interactions, while the butyryl group projects away from the kinase. In contrast to DB-cAMP, DB-cCMP did not induce PKA-dependent phosphorylation in intact cells. Taken together, our data show that N(4)-monobutyryl-cCMP (4-MB-cCMP) activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo. cNMP phosphorothioates constitute a starting point for the development of PKA activators with high selectivity relative to PKG.

Background and purpose Some histamine H4 receptor ligands act as inverse agonists at the human H4 receptor (hH4 R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H4 receptor (mH4 R) and rat H4 receptor (rH4 R). To study molecular determinants of constitutive activity, H4 receptor reciprocal mutants were constructed: single mutants: hH4 R-F169V, mH4 R-V171F, hH4 R-S179A, hH4 R-S179M; double mutants: hH4 R-F169V+S179A, hH4 R-F169V+S179M and mH4 R-V171F+M181S. Experimental approach Site-directed mutagenesis with pVL1392 plasmids containing hH4 or mH4 receptors were performed. Wild-type or mutant receptors were co-expressed with Gαi2 and Gβ1 γ2 in Sf9 cells. Membranes were studied in saturation and competition binding assays ([(3) H]-histamine), and in functional [(35) S]-GTPγS assays with inverse, partial and full agonists of the hH4 receptor. Key results Constitutive activity decreased from the hH4 receptor via the hH4 R-F169V mutant to the hH4 R-F169V+S179A and hH4 R-F169V+S179M double mutants. F169 alone or in concert with S179 plays a major role in stabilizing a ligand-free active state of the hH4 receptor. Partial inverse hH4 receptor agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologues with lower or no constitutive activity. Some partial and full hH4 receptor agonists showed decreased maximal effects and potencies at hH4 R-F169V and double mutants. However, the mutation of S179 in the hH4 receptor to M as in mH4 receptor or A as in rH4 receptor did not significantly reduce constitutive activity. Conclusions and implications F169 and S179 are key amino acids for the high constitutive activity of hH4 receptors and may also be of relevance for other constitutively active GPCRs. Linked articles This article is part of a themed issue on Histamine Pharmacology Update published in volume 170 issue 1. To view the other articles in this issue visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2013.170.issue-1/issuetoc.

Soluble guanylyl cyclase (sGC) plays an important role in cardiovascular function and catalyzes formation of cGMP. sGC is activated by nitric oxide and allosteric stimulators and activators. However, despite its therapeutic relevance, the regulatory mechanisms of sGC are still incompletely understood. A major reason for this situation is that no crystal structures of active sGC have been resolved so far. An important step toward this goal is the identification of high-affinity ligands that stabilize an sGC conformation resembling the active, "fully closed" state. Therefore, we examined inhibition of rat sGCα1β1 by 38 purine- and pyrimidine-nucleotides with 2,4,6,-trinitrophenyl and (N-methyl)anthraniloyl substitutions at the ribosyl moiety and compared the data with that for the structurally related membranous adenylyl cyclases (mACs) 1, 2, 5 and the purified mAC catalytic subunits VC1:IIC2. TNP-GTP [2',3'-O-(2,4,6-trinitrophenyl)-GTP] was the most potent sGCα1β1 inhibitor (Ki, 10.7 nM), followed by 2'-MANT-3'-dATP [2'-O-(N-methylanthraniloyl)-3'-deoxy-ATP] (Ki, 16.7 nM). Docking studies on an sGCαcat/sGCβcat model derived from the inactive heterodimeric crystal structure of the catalytic domains point to similar interactions of (M)ANT- and TNP-nucleotides with sGCα1β1 and mAC VC1:IIC2. Reasonable binding modes of 2'-MANT-3'-dATP and bis-(M)ANT-nucleotides at sGC α1β1 require a 3'-endo ribosyl conformation (versus 3'-exo in 3'-MANT-2'-dATP). Overall, inhibitory potencies of nucleotides at sGCα1β1 versus mACs 1, 2, 5 correlated poorly. Collectively, we identified highly potent sGCα1β1 inhibitors that may be useful for future crystallographic and fluorescence spectroscopy studies. Moreover, it may become possible to develop mAC inhibitors with selectivity relative to sGC.

Mammalian membranous and soluble adenylyl cyclases (mAC, sAC) and soluble guanylyl cyclases (sGC) generate cAMP and cGMP from ATP and GTP, respectively, as substrates. mACs (nine human isoenzymes), sAC, and sGC differ in their overall structures owing to specific membrane-spanning and regulatory domains but consist of two similarly folded catalytic domains C1 and C2 with high structure-based homology between the cyclase species. Comparison of available crystal structures - VC1:IIC2 (a construct of domains C1a from dog mAC5 and C2a from rat mAC2), human sAC and sGC, mostly in complex with substrates, substrate analogs, inhibitors, metal ions, and/or modulators - reveals that especially the nucleotide binding sites are closely related. An evolutionarily well-conserved catalytic mechanism is based on common binding modes, interactions, and structural transformations, including the participation of two metal ions in catalysis. Nucleobase selectivity relies on only few mutations. Since in all cases the nucleoside moiety is embedded in a relatively spacious cavity, mACs, sAC, and sGC are rather promiscuous and bind nearly all purine and pyrimidine nucleotides, including CTP and UTP, and many of their derivatives as inhibitors with often high affinity. By contrast, substrate specificity of mammalian adenylyl and guanylyl cyclases is high due to selective dynamic rearrangements during turnover.

The human histamine receptors hH(1)R and hH(2)R constitute important drug targets, and hH(3)R and hH(4)R have substantial potential in this area. Considering the species-specificity of pharmacology of H(x)R orthologs, it is important to analyze hH(x)Rs. Here, we summarize current knowledge of hH(x)Rs endogenously expressed in human cells and hH(x)Rs recombinantly expressed in mammalian and insect cells. We present the advantages and disadvantages of the various systems. We also discuss problems associated with the use of hH(x)R antibodies, an issue of general relevance for G-protein-coupled receptors (GPCRs). There is much greater overlap in activity of 'selective' ligands for other hH(x)Rs than the cognate receptor subtype than generally appreciated. Studies with native and recombinant systems support the concept of ligand-specific receptor conformations, encompassing agonists and antagonists. It is emerging that for characterization of hH(x)R ligands, one cannot rely on a single test system and a single parameter. Rather, multiple systems and parameters have to be studied. Although such studies are time-consuming and expensive, ultimately, they will increase drug safety and efficacy.

Bivalent histamine H(2) receptor (H(2)R) agonists were synthesized by connecting pharmacophoric 3-(2-amino-4-methylthiazol-5-yl)-, 3-(2-aminothiazol-5-yl)-, 3-(imidazol-4-yl)-, or 3-(1,2,4-triazol-5-yl)propylguanidine moieties by N(G)-acylation with alkanedioic acids of various chain lengths. The compounds were investigated for H(2)R agonism in GTPase and [(35)S]GTPγS binding assays at guinea pig (gp) and human (h) H(2)R-Gsα(S) fusion proteins including various H(2)R mutants, at the isolated gp right atrium, and in GTPase assays for activity on recombinant H(1), H(3), and H(4) receptors. The bivalent ligands are H(2)R partial or full agonists, up to 2 orders of magnitude more potent than monovalent acylguanidines and, with octanedioyl or decanedioyl spacers, up to 4000 times more potent than histamine at the gpH(2)R. In contrast to their imidazole analogues, the aminothiazoles are highly selective for H(2)R vs other HR subtypes. Compounds with (theoretically) sufficient spacer length (20 CH(2) groups) to simultaneously occupy two orthosteric binding sites in H(2)R dimers are nearly inactive, whereas the highest potency resides in compounds with considerably shorter spacers. Thus, there is no evidence for interaction with H(2)R dimers. The high agonistic potency may result from interaction with an accessory binding site at the same receptor protomer.

Antidepressants and antipsychotics affect multiple molecular targets. Consequently, these drugs exhibit not only unique profiles of therapeutic effects but also several undesired effects. Histamine receptors (H₁R, H₂R, H₃R, and H₄R) belong to the large family of G protein-coupled receptors and are very important drug targets. All four H(x)R subtypes are expressed in the CNS. Interactions of lipophilic, blood-brain barrier-penetrating drugs with H(x)Rs could contribute to therapeutic and unwanted effects. Therefore, we investigated potencies H(x)R as well as potencies and (inverse) agonistic efficacies of 34 antidepressants and antipsychotics at HxRs in functional assays. We expressed human H(x)Rs in Sf9 insect cells and conducted radioligand competition binding experiments and functional steady-state GTPase assays. Ligand affinities and potencies were compared with literature data and related to therapeutic reference ranges. Almost all antidepressants and antipsychotics displayed high binding affinities to H₁R and behaved as antagonists. The atypical antidepressant trimipramine behaved as a high-affinity/high-potency H₂R antagonist (pK(i), 7.39; pK(B), 7.36; pA₂, 7.55). Docking to an H₂R model suggested a probable binding mode. The affinity of antidepressants and antipsychotics for H₃R was low. The atypical antipsychotic clozapine, known to induce agranulocytosis, exhibited partial H₄R agonism for which docking experiments provided a molecular basis. Clozapine also exhibited H₂R antagonism. We observed dissociations between pK(i) and pK (B) values as well as between pK(i) and pIC₅₀ values for H(x)Rs. Antidepressants and antipsychotics interact differentially with H(x)Rs. The concept of functional selectivity (also referred to as ligand-specific receptor conformations or biased signaling) explains dissociations between pK(i) and pK(B) values as well as differences between pK(i) and pIC₅₀ values. The H₁R antagonism of numerous antidepressants and antipsychotics is very pronounced. The H₂R antagonism of trimipramine and partial H₄R agonism of clozapine may be clinically relevant. We also discuss the possible role of the H₂R antagonism of clozapine for neutropenia/agranulocytosis induced by this compound. Finally, we discuss the methodological, conceptual, and clinical limitations of our study.

The histamine H4 receptor (H4R) is expressed on cells of the immune system including eosinophils, dendritic cells, and T cells and plays an important role in the pathogenesis of bronchial asthma, atopic dermatitis, and pruritus. Analysis of the H4R in these diseases depends on the use of animal models. However, there are substantial pharmacological differences between various H4R species orthologs. The purpose of this study was to analyze the pharmacological properties of canine, rat, and murine H4R in comparison to human H4R expressed in Sf9 insect cells. Only hH4R and cH4R exhibited a sufficiently high [3H]histamine affinity for radioligand binding studies. Generally, cH4R exhibited lower ligand-affinities than hH4R. Similarly, in high-affinity GTPase studies, ligands were more potent at hH4R than at other H4R species orthologs. Unlike the other H4R species orthologs, hH4R exhibited high agonist-independent (constitutive) activity. Most strikingly, the prototypical H4R antagonist (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) (JNJ7777120) exhibited partial agonistic activity at cH4R, rH4R, and mH4R, whereas at hH4R, JNJ7777120 was a partial inverse agonist. H4R agonists from the class of NG-acylated imidazolylpropylguanidines and cyanoguanidines exhibited substantial differences in terms of affinity, potency, and efficacy among H4R species orthologs, too. The species-dependent pharmacological profiles are not due to the highly variable amino acid sequence position 341. Finally, H4R species orthologs differ from each other in terms of regulation by NaCl. Collectively, there are profound pharmacological differences between H4R species orthologs. Most importantly, caution must be exerted when interpreting pharmacological effects of “the prototypical H4R antagonist” JNJ7777120 as H4R antagonism.

Since its discovery 10 years ago the histamine H(4) receptor (H(4)R) has attracted attention as a potential drug target, for instance, for the treatment of inflammatory and allergic diseases. Potent and selective ligands including agonists are required as pharmacological tools to study the role of the H(4)R in vitro and in vivo. Many H(4)R agonists, which were identified among already known histamine receptor ligands, show only low or insufficient H(4)R selectivity. In addition, the investigation of numerous H(4)R agonists in animal models is hampered by species-dependent discrepancies regarding potencies and histamine receptor selectivities of the available compounds, especially when comparing human and rodent receptors. This article gives an overview about structures, potencies, and selectivities of various compounds showing H(4)R agonistic activity and summarizes the structure-activity relationships of selected compound classes.

A series of 51 5-HT(2A) partial agonistic arylethylamines (primary or benzylamines) from different structural classes (indoles, methoxybenzenes, quinazolinediones) was investigated by fragment regression analysis (FRA), docking and 3D-QSAR approaches. The data, pEC(50) values and intrinsic activities (E(max)) on rat arteries, show high variability of pEC(50) from 4 to 10 and of E(max) from 15 to 70%. FRA indicates which substructures affect potency or intrinsic activity. The high contribution of halogens in para position of phenethylamines to pEC(50) points to a specific hydrophobic pocket. Other results suggest the significance of hydrogen bonds of the aryl moiety for activation and the contrary effect of benzyl groups on affinity (increasing) and intrinsic activity (decreasing). Results from fragment regression and data on all available mutants were considered to derive a common binding site at the rat 5-HT(2A) receptor. After generation and MD simulations of a receptor model based on the β(2)-adrenoceptor structure, typical derivatives were docked, leading to the suggestion of common interactions, e.g., with serines in TM3 and TM5 and with a cluster of aromatic amino acids in TM5 and TM6. The whole series was aligned by docking and minimization of the complexes. The pEC(50) values correlate well with Sybyl docking energies and hydrophobicity of the aryl moieties. With this alignment, CoMFA and CoMSIA approaches based on a training set of 36 and a test set of 15 compounds were performed. The correlation of pEC(50) with steric, electrostatic, hydrophobic and H-bond acceptor fields resulted in sufficient fit (q (2): 0.75-0.8, r (2): 0.92-0.95) and predictive power (r (pred) (2) : 0.85-0.88). The important interaction regions largely reflect the patterns provided by the putative binding site. In particular, the fit of the aryl moieties and benzyl substituents to two hydrophobic pockets is evident.

Whooping cough is caused by Bordetella pertussis and still constitutes one of the top five causes of death in young children, particularly in developing countries. The calmodulin-activated adenylyl cyclase (AC) toxin CyaA substantially contributes to disease development. Thus, potent and selective CyaA inhibitors would be valuable drugs for the treatment of whooping cough. However, it has been difficult to obtain potent CyaA inhibitors with selectivity relative to mammalian ACs. Selectivity is important to reduce potential toxic effects. In a previous study we serendipitously found that bis-methylanthraniloyl (bis-MANT)-IMP is a more potent CyaA inhibitor than MANT-IMP (Gottle et al., Mol Pharmacol 72:526-535 (2007)). These data prompted us to study the effects of a series of 32 bulky mono- and bis-anthraniloyl (ANT)-substituted nucleotides on CyaA and mammalian ACs. The novel nucleotides differentially inhibited CyaA and ACs 1, 2 and 5. Bis-ANT-nucleotides inhibited CyaA competitively. Most strikingly, bis-Cl-ANT-ATP inhibited CyaA with a potency ≥ 100-fold higher than ACs 1, 2 and 5. In contrast to MANT-ATP, bis-MANT-ATP exhibited low intrinsic fluorescence, thereby substantially enhancing the signal-to noise ratio for the analysis of nucleotide binding to CyaA. The high sensitivity of the fluorescence assay revealed that bis-MANT-ATP binds to CyaA already in the absence of calmodulin. Molecular modeling showed that the catalytic site of CyaA is sufficiently spacious to accommodate both MANT substituents. Collectively, we have identified the first potent CyaA inhibitor with high selectivity relative to mammalian ACs. The fluorescence properties of bis-ANT-nucleotides facilitate development of a high-throughput screening assay.

Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) are second messengers for numerous mammalian cell functions. The natural occurrence and synthesis of a third cyclic nucleotide (cNMP), cyclic cytidine 3',5'-monophosphate (cCMP), is a matter of controversy, and almost nothing is known about cyclic uridine 3',5'-monophosphate (cUMP). Bacillus anthracis and Bordetella pertussis secrete the adenylyl cyclase (AC) toxins edema factor (EF) and CyaA, respectively, weakening immune responses and facilitating bacterial proliferation. A cell-permeable cCMP analogue inhibits human neutrophil superoxide production. Here, we report that EF and CyaA also possess cytidylyl cyclase (CC) and uridylyl cyclase (UC) activity. CC and UC activity was determined by a radiometric assay, using [alpha-(32)P]CTP and [alpha-(32)P]UTP as substrates, respectively, and by a high-performance liquid chromatography method. The identity of cNMPs was confirmed by mass spectrometry. On the basis of available crystal structures, we developed a model illustrating conversion of CTP to cCMP by bacterial toxins. In conclusion, we have shown both EF and CyaA have a rather broad substrate specificity and exhibit cytidylyl and uridylyl cyclase activity. Both cCMP and cUMP may contribute to toxin actions.

A set of chiral imidazolylpropylguanidines and 2-aminothiazolylpropylguanidines bearing N(G)-3-phenyl- or N(G)-3-cyclohexylbutanoyl residues was synthesized and investigated for histamine H(2) receptor (H(2)R) agonism (guinea pig (gp) right atrium, GTPase assay on recombinant gp and human (h)H(2)R) and for hH(2)R selectivity compared to hH(1)R, hH(3)R and hH(4)R. In contrast to previous studies on arpromidine derivatives, the present investigation of acylguanidine-type compounds revealed only very low eudismic ratios (1.1-3.2), indicating the stereochemistry of the acyl moiety to play only a minor role in this series of H(2)R agonists.

It is assumed that many G protein-coupled receptors (GPCRs) are restrained in an inactive state by the "ionic lock," an interaction between an arginine in transmembrane domain (TM) 3 (R3.50) and a negatively charged residue in TM6 (D/E6.30). In the human histamine H4 receptor (hH4R), alanine is present in position 6.30. To elucidate whether this mutation causes the high constitutive activity of hH4R, we aimed to reconstitute the ionic lock by constructing the A6.30E mutant. The role of R3.50 was investigated by generating hH4R-R3.50A. Both mutants were expressed alone or together with Galpha(i2) and Gbeta1gamma2 in Sf9 cells and characterized in GTPase, 35S-labeled guanosine 5'-[gamma-thio]triphosphate binding, and high-affinity agonist binding assays. Unexpectedly, compared with hH4R, hH4R-A6.30E showed only nonsignificant reduction of constitutive activity and G protein-coupling efficiency. The KD of [3H]histamine was unaltered. By contrast, hH4R-R3.50A did not stimulate G proteins. Thioperamide affinity at hH(4)R-R3.50A was increased by 300 to 400%, whereas histamine affinity was reduced by approximately 50%. A model of the active hH4R state in complex with the Galpha(i2) C terminus was compared with the crystal structures of turkey beta1 and human beta2 adrenoceptors. We conclude that 1) constitutive activity of hH4R is facilitated by the salt bridge D5.69-R6.31 rather than by the missing ionic lock, 2) Y3.60 may form alternative locks in active and inactive GPCR states, 3) R3.50 is crucial for hH4R-G protein coupling, and 4) hH4R-R3.50A represents an inactive state with increased inverse agonist and reduced agonist affinity. Thus, the ionic lock, although stabilizing the inactive rhodopsin state, is not generally important for all class A GPCRs.

Inhibitors of histone deacetylases are a new class of cancer therapeutics with possibly broad applicability. Combinations of HDAC inhibitors with the kinase inhibitor 1 (Imatinib) in recent studies showed additive and synergistic effects. Here we present a new concept by combining inhibition of protein kinases and HDACs, two independent pharmacological activities, in one synthetic small molecule. In general, the HDAC inhibition profile, the potencies, and the probable binding modes to HDAC1 and HDAC6 were similar as for 6 (SAHA). Inhibition of Abl kinase in biochemical assays was maintained for most compounds, but in general the kinase selectivity profile differed from that of 1 with nearly equipotent inhibition of the wild-type and the Imatinib resistant Abl T(315)I mutant. A potent cellular inhibition of PDGFR and cytotoxicity toward EOL-1 cells, a model for idiopathic hypereosinophilic syndrome (HES), are restored or enhanced for selected analogues (12b, 14b, and 18b). Cytotoxicity was evaluated by using a broad panel of tumor cell lines, with selected analogues displaying mean IC(50) values between 3.6 and 7.1 muM.

The bioisosteric replacement of the guanidino group in arpromidine-like histamine H(2) receptor (H(2)R) agonists by an acylguanidine moiety is a useful approach to obtain potent H(2)R agonists with improved oral bioavailability and blood-brain barrier penetration. Unfortunately, the selectivity of such N(G)-acylated imidazolylpropylguanidines for the H(2)R is poor, in particular versus histamine H(3) (H(3)R) and H(4) receptors (H(4)R). This drawback appears to depend on the "privileged" imidazolylpropylguanidine structure. The 2-amino-4-methylthiazol-5-yl moiety is a bioisostere of the imidazole ring in the moderately potent H(2)R-selective histamine analogue amthamine. This approach was successfully applied to acylguanidine-type H(2)R agonists. The aminothiazoles are nearly equipotent to the corresponding imidazoles as H(2)R agonists. Compared with histamine, the potency is increased up to 40-fold on the guinea pig right atrium, and up to 125- and 280-fold in GTPase assays with human and guinea pig H(2)R-G(salphaS) fusion proteins expressed in Sf9 insect cells, respectively. Docking studies on H(2)R models support the hypothesis that 2-aminothiazolyl and imidazolyl derivatives interact with H(2)Rs as bioisosteres. In contrast to the imidazoles, the aminothiazoles are devoid of agonistic or relevant antagonistic effects on H(1), H(3), and H(4) receptors. Moreover, unlike amthamine, the 4-methyl group does not significantly contribute to the H(2)R agonism of N(G)-acylated 2-amino-4-methylthiazol-5-ylpropylguanidines.

Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins: lethal factor, protective antigen, and edema factor (EF), a highly active calmodulin-dependent adenylyl cyclase (AC). However, conventional antibiotic treatment is ineffective against either toxemia or antibiotic-resistant strains. Thus, more effective drugs for anthrax treatment are needed. Previous studies from our laboratory showed that mammalian membranous AC (mAC) exhibits broad specificity for purine and pyrimidine nucleotides (Mol Pharmacol 70 878-886,200616766715). Here, we investigated structural requirements for EF inhibition by natural purine and pyrimidine nucleotides and nucleotides modified with N-methylanthraniloyl (MANT)- or anthraniloyl groups at the 2′(3′)-O-ribosyl position. MANT-CTP was the most potent EF inhibitor (Ki, 100 nM) among 16 compounds studied. MANT-nucleotides inhibited EF competitively. Activation of EF by calmodulin resulted in effective fluorescence resonance energy transfer (FRET) from tryptophan and tyrosine residues located in the vicinity of the catalytic site to MANT-ATP, but FRET to MANT-CTP was only small. Mutagenesis studies revealed that Phe586 is crucial for FRET to MANT-ATP and MANT-CTP and that the mutations N583Q, K353A, and K353R differentially alter the inhibitory potencies of MANT-ATP and MANT-CTP. Docking approaches relying on crystal structures of EF indicate similar binding modes of the MANT nucleotides with subtle differences in the region of the nucleobases. In conclusion, like mAC, EF accommodates both purine and pyrimidine nucleotides. The unique preference of EF for the base cytosine offers an excellent starting point for the development of potent and selective EF inhibitors.

N1-Aryl(heteroaryl)alkyl-N2-[3-(1H-imidazol-4-yl)propyl]guanidines are potent histamine H2-receptor (H2R) agonists, but their applicability is compromised by the lack of oral bioavailability and CNS penetration. To improve pharmacokinetics, we introduced carbonyl instead of methylene adjacent to the guanidine moiety, decreasing the basicity of the novel H2R agonists by 4-5 orders of magnitude. Some acylguanidines with one phenyl ring were even more potent than their diaryl analogues. As demonstrated by HPLC-MS, the acylguanidines (bioisosteres of the alkylguanidines) were absorbed from the gut of mice and detected in brain. In GTPase assays using recombinant receptors, acylguanidines were more potent at the guinea pig than at the human H2R. At the hH1R and hH3R, the compounds were weak to moderate antagonists or partial agonists. Moreover, potent partial hH4R agonists were identified. Receptor subtype selectivity depends on the imidazolylpropylguanidine moiety (privileged structure), opening an avenue to distinct pharmacological tools including potent H4R agonists.

Residues in the second extracellular loop (e2) play a role in ligand binding in certain aminergic G protein coupled receptors (GPCRs). N-[3-(1H-Imidazol-4-yl)propyl)]guanidines and N (G)-acylated derivatives are more efficacious and potent agonists at fusion proteins of the guinea pig histamine H(2) receptor and the short splice variant of G(salpha), G(salphaS) (gpH(2)R-G(salphaS)) than at the human isoform (hH(2)R-G(salphaS)). To elucidate the structural basis for this species-selectivity, we generated a mutant hH(2)R-G(salphaS) fusion protein with the four e2 residues differing in both species isoforms mutated into the gpH(2)R sequence, and a reverse mutant of the gpH(2)R-G(salphaS) with the corresponding mutations into the human species. In a steady-state GTPase activity assay, efficacies and potencies of guanidine-type agonists were similar at mutant and wild-type receptors indicating that e2 does not contribute to the species-selectivity. In several class 1 GPCRs, amino acids in the vicinity of a highly conserved cysteine in e2 participate in ligand binding. A three-dimensional homology model of the hH(2)R predicted Lys-173 and Lys-175, adjacent to Cys-174 in e2, to be in close proximity to the binding pocket of guanidine-type agonists. To elucidate the putative role of both residues for interactions with the agonists, two hH(2)R-G(salphaS) fusion proteins, with single-point mutations of Lys-173--Ala-173 and Lys-175--Ala-175 respectively, were generated. With these mutants, the efficacies and potencies of small and bulky H(2)R agonists did not significantly change. However, increases in GTPase activity upon agonist stimulation were reduced, suggesting an impact of both residues on the efficiency of receptor coupling to G(salphaS). In conclusion, none of the point mutations generated within this study substantially altered the efficacies and potencies of guanidine-type agonists relative to the wild-type receptors, suggesting that these residues do not directly face the H(2)R guanidine-binding pocket. Thus, agonist binding to residues in e2 is relevant for some but not all aminergic GPCRs.

Previous studies revealed pharmacological differences between human and guinea pig histamine H(2) receptors (H(2)Rs) with respect to the interaction with guanidine-type agonists. Because H(2)R species variants are structurally very similar, comparative studies are suited to relate different properties of H(2)R species isoforms to few molecular determinants. Therefore, we systematically compared H(2)Rs of human (h), guinea pig (gp), rat (r), and canine (c). Fusion proteins of hH(2)R, gpH(2)R, rH(2)R, and cH(2)R, respectively, and the short splice variant of G(salpha), G(salphaS), were expressed in Sf9 insect cells. In the membrane steady-state GTPase activity assay, cH(2)R-G(salphaS) but neither gpH(2)R-G(salphaS) nor rH(2)R-G(salphaS) showed the hallmarks of increased constitutive activity compared with hH(2)R-G(salphaS), i.e., increased efficacies of partial agonists, increased potencies of agonists with the extent of potency increase being correlated with the corresponding efficacies at hH(2)R-G(salphaS), increased inverse agonist efficacies, and decreased potencies of antagonists. Furthermore, in membranes expressing nonfused H(2)Rs without or together with mammalian G(salphaS) or H(2)R-G(salpha) fusion proteins, the highest basal and GTP-dependent increases in adenylyl cyclase activity were observed for cH(2)R. An example of ligand selectivity is given by metiamide, acting as an inverse agonist at hH(2)R-G(salphaS), gpH(2)R-G(salphaS), and rH(2)R-G(salphaS) in the GTPase assay in contrast to being a weak partial agonist with decreased potency at cH(2)R-G(salphaS). In conclusion, the cH(2)R exhibits increased constitutive activity compared with hH(2)R, gpH(2)R, and rH(2)R, and there is evidence for ligand-specific conformations in H(2)R species isoforms.

Mammalian membranous and soluble adenylyl cyclases (mAC, sAC) and soluble guanylyl cyclases (sGC) generate cAMP and cGMP from ATP and GTP, respectively, as substrates. mACs (nine human isoenzymes), sAC, and sGC differ in their overall structures owing to specific membrane-spanning and regulatory domains but consist of two similarly folded catalytic domains C1 and C2 with high structure-based homology between the cyclase species. Comparison of available crystal structures – VC1:IIC2 (a construct of domains C1a from dog mAC5 and C2a from rat mAC2), human sAC and sGC, mostly in complex with substrates, substrate analogs, inhibitors, metal ions, and/or modulators – reveals that especially the nucleotide binding sites are closely related. An evolutionarily well-conserved catalytic mechanism is based on common binding modes, interactions, and structural transformations, including the participation of two metal ions in catalysis. Nucleobase selectivity relies on only few mutations. Since in all cases the nucleoside moiety is embedded in a relatively spacious cavity, mACs, sAC, and sGC are rather promiscuous and bind nearly all purine and pyrimidine nucleotides, including CTP and UTP, and many of their derivatives as inhibitors with often high affinity. By contrast, substrate specificity of mammalian adenylyl and guanylyl cyclases is high due to selective dynamic rearrangements during turnover.

Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these residues was investigated by site-directed mutagenesis and a functional luciferase assay reflecting NF-κB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL-1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the αC' helix (Q469-E473 in mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.

Functional selectivity is well established as an underlying concept of ligand-specific signaling via G protein-coupled receptors (GPCRs). Functionally, selective drugs could show greater therapeutic efficacy and fewer adverse effects. Dual coupling of the β2-adrenoceptor (β2AR) triggers a signal transduction via Gsα and Giα proteins. Here, we examined 12 fenoterol stereoisomers in six molecular and cellular assays. Using β2AR-Gsα and β2AR-Giα fusion proteins, (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol were identified as biased ligands with preference for Gs. G protein-independent signaling via β-arrestin-2 was disfavored by these ligands. Isolated human neutrophils constituted an ex vivo model of β2AR signaling and demonstrated functional selectivity through the dissociation of cAMP accumulation and the inhibition of formyl peptide-stimulated production of reactive oxygen species. Ligand bias was calculated using an operational model of agonism and revealed that the fenoterol scaffold constitutes a promising lead structure for the development of Gs-biased β2AR agonists.

Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.

Previously, we identified ascorbic acid 6-O-hexadecanoate as an up to 1500 times more potent inhibitor of bacterial and bovine hyaluronidases than the parent compound, vitamin C, and determined a crystal structure of hyaluronidase from Streptococcus pneumoniae in complex with the inhibitor. As the alkanoyl chain interacts with a hydrophobic patch of the enzyme we synthesized other 6-O-acylated vitamin C derivatives bearing various lipophilic residues and investigated the inhibition of Streptococcus agalactiae strain 4755 hyaluronidase (SagHyal(4755)) and of bovine testicular hyaluronidases (BTH) in a turbidimetric assay. All compounds showed selectivity for the bacterial enzyme. Whereas vitamin C 6-O-hexanoate only weakly inhibited SagHyal(4755), the inhibition of both enzymes increased with the length of the aliphatic chain. In the case of the 6-O-octadecanoate, IC(50) values of 0.9 and 39microM for SagHyal(4755) and BTH, respectively, were determined. Partial replacement of the aliphatic chain with a phenyl, p-phenylene or p-biphenylyl group resulted in inhibitors with activity in the lower micromolar range, too. The title compounds are among the most potent inhibitors of both enzymes known to date.

FLT3 receptor tyrosine kinase is aberrantly active in many cases of acute myeloid leukemia (AML). Recently, bis(1H-indol-2-yl)methanones were found to inhibit FLT3 and PDGFR kinases. To optimize FLT3 activity and selectivity, 35 novel derivatives were synthesized and tested for inhibition of FLT3 and PDGFR autophosphorylation. The most potent FLT3 inhibitors 98 and 102 show IC50 values of 0.06 and 0.04 microM, respectively, and 1 order of magnitude lower PDGFR inhibiting activity. The derivatives 76 and 82 are 20- to 40-fold PDGFR selective. Docking at the recent FLT3 structure suggests a bidentate binding mode with the backbone of Cys-694. Activity and selectivity can be related to interactions of one indole moiety with a hydrophobic pocket including Phe-691, the only different binding site residue (PDGFR Thr-681). Compound 102 inhibited the proliferation of 32D cells expressing wildtype FLT3 or FLT3-ITD similarly as FLT3 autophosphorylation, and induced apoptosis in primary AML patient blasts.

The bacterial hyaluronan lyases (Hyals) that degrade hyaluronan, an important component of the extracellular matrix, are involved in microbial spread. Inhibitors of these enzymes are essential in investigation of the role of hyaluronan and Hyal in bacterial infections and constitute a new class of antibiotics against Hyal-producing bacteria. Recently, we identified 1,3-diacetylbenzimidazole-2-thione and related molecules as inhibitors of streptococcal Hyal. One of such compounds, 1-decyl-2-(4-sulfamoyloxyphenyl)-1-indol-6-yl sulfamate, was co-crystallized in a complex with Streptococcus pneumoniae Hyal and its structure elucidated. The resultant X-ray structure demonstrates that this inhibitor fits in the enzymatic active site via interactions resembling the binding mode of the natural hyaluronan substrate. X-ray structural analysis also indicates binding interactions with the catalytic residues and those of a catalytically essential hydrophobic patch. An IC50 value of 11 microM for Hyal from Streptococcus agalactiae (strain 4755) qualifies this phenylindole compound as one of the most potent Hyal inhibitors known to date. The structural data suggested a similar binding mode for N-(3-phenylpropionyl)-benzoxazole-2-thione. This new compound's inhibitory properties were confirmed resulting in discovery of yet another Hyal inhibitor (IC50 of 15 microM). These benzoxazole-2-thiones constitute a new class of inhibitors of bacterial Hyals and are well suited for further optimization of their selectivity, potency, and pharmacokinetic properties.

Hyaluronidases depolymerize hyaluronic acid, an important component of the extracellular matrix. Bacterial hyaluronan lyases (EC 4.2.2.1) contribute to the spreading of microorganisms in tissues. Potent and selective inhibitors are not known up to now, but would be interesting pharmacological tools and potential drugs for the treatment of bacterial infections. Starting from two crystal structures of streptococcal hyaluronan lyases, a homology model of the S. agalactiae strain 4755 enzyme hylB4755 was generated and applied to structure-based inhibitor design with the de novo design program LUDI. The databases LeadQuest® Vol. 1&2, Accelrys and ChemACX containing 228000 compounds were screened resulting in 1275 hits. Based on high LUDI scores, synthetic feasibility and commercial availability, 19 of these hits were investigated for inhibition of hylB4755. 13 compounds were active in the milli- and submillimolar range. 1,3-diacetylbenzimidazole-2-thione with IC50 values of 5 M and 160 M at physiological and optimum pH, respectively, is one of the two most potent inhibitors of hyaluronan lyases known to date. Depending on the LUDI sphere radius, it was docked in two different poses with similar scores. To analyze whether the hits represent (physico)chemical properties typical for drugability and optimal pharmacokinetics, the distributions of six descriptors within all hits from LeadQuest® and ChemACX and within the original databases were compared. Generally, the hit distributions represent the type of the original ones, but are narrower with slightly different medians. More than 80% of the hits have molecular weights between 150 and 350, XLOGP values between 1 and 4, one H-donor, 1-3 H-acceptors, 0-3 rotatable bonds and 1-3 rings. In conclusion, the application of LUDI to both databases has led to the accumulation of relatively rigid, drug-like molecules.

Recent research on histamine H2 receptor agonists was focused on quantitative structure-activity relationships and receptor models explaining the activity of imidazolylpropylguanidines. Their selectivity for guinea pig vs. human isoforms was investigated using H2 receptor-Gsalpha fusion proteins and attributed to amino acid differences in transmembrane domains 1 and 7. New antagonists result from approaches to improve pharmacokinetic properties and to design hybrid drugs which additionally have gastroprotective or anti H. pylori activity.

Hyaluronidases are enzymes that degrade hyaluronan, an important component of the extracellular matrix. The mammalian hyaluronidases are considered to be involved in many (patho)physiological processes like fertilization, tumor growth, and metastasis. Bacterial hyaluronidases, also termed hyaluronate lyases, contribute to the spreading of microorganisms in tissues. Such roles for hyaluronidases suggest that inhibitors could be useful pharmacological tools. Potent and selective inhibitors are not known to date, although L-ascorbic acid has been reported to be a weak inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL). The x-ray structure of SpnHL complexed with L-ascorbic acid has been elucidated suggesting that additional hydrophobic interactions might increase inhibitory activity. Here we show that L-ascorbic acid 6-hexadecanoate (Vcpal) is a potent inhibitor of both streptococcal and bovine testicular hyaluronidase (BTH). Vcpal showed strong inhibition of Streptococcus agalactiae hyaluronate lyase with an IC(50) of 4 microM and weaker inhibition of SpnHL and BTH with IC(50) values of 100 and 56 microM, respectively. To date, Vcpal has proved to be one of the most potent inhibitors of hyaluronidase. We also determined the x-ray structure of the SpnHL-Vcpal complex and confirmed the hypothesis that additional hydrophobic interactions with Phe-343, His-399, and Thr-400 in the active site led to increased inhibition. A homology structural model of BTH was also generated to suggest binding modes of Vcpal to this hyaluronidase. The long alkyl chain seemed to interact with an extended, hydrophobic channel formed by mostly conserved amino acids Ala-84, Leu-91, Tyr-93, Tyr-220, and Leu-344 in BTH.

The Toll/interleukin 1 (IL-1) receptor family plays an important role in both innate and adaptive immunity. These receptors are characterized by a C-terminal homology motif called the Toll/IL-1 receptor (TIR) domain. A principal function of the TIR domain is mediating homotypic protein-protein interactions in the signal transduction pathway. To suggest interaction sites of TIR domains in the IL-1 receptor complex, we modeled the putative three-dimensional structure of the TIR domain within the co-receptor chain, IL-1 receptor accessory protein. The model was based on homology with the crystal structures of human TLR1 and TLR2. The final structure of the IL-1 receptor accessory protein TIR domain suggests the conserved regions box 1 and 2, including Pro-446, as well as box 3 within the C-terminal α-helix as possible protein-protein interaction sites due to their exposure and their electrostatic potential. Pro-446, corresponding to the Pro/His mutation in dominant negative TLR4, is located in the third loop at the outmost edge of the TIR domain and does not play any structural role. Inhibition of IL-1 responsiveness seen after substitution of Pro-446 by charged amino acids is due to the loss of an interaction site for other TIR domains. Amino acids 527–534 as part of the loop close to the conserved box 3 are critical for recruitment of myeloid differentiation factor 88 and to a lesser extent for IL-1 responsiveness. Modeling suggests that native folding of the TIR domain may be approached by the responsive deletion mutants Δ528–534 and Δ527–533, whereas the C-terminal β-strand and/or α-helix is displaced in the nonresponsive mutant Δ527–534.

It is unknown why the potencies and efficacies of long-chained guanidine-type histamine H2-receptor (H2R) agonists are lower at the H2R of human neutrophils than at the H2R of the guinea pig atrium. To elucidate these differences, we analyzed fusion proteins of the human H2R (hH2R) and guinea pig H2R (gpH2R), respectively, and the short splice variant of Gsalpha (GsalphaS) expressed in Sf9 cells. The potencies and efficacies of small H2R agonists in the GTPase assay and the potencies of antagonists at inhibiting histamine-stimulated GTP hydrolysis by hH2R-GsalphaS and gpH2R-GsalphaS were similar. In contrast, the potencies and efficacies of guanidines were lower at hH2R-GsalphaS than at gpH2R-G(salphaS). Guanidines bound to hH2R-GsalphaS with lower affinity than to gpH2R-GsalphaS, and high-affinity binding of guanidines at gpH2R-GsalphaS was more resistant to disruption by GTPgammaS than binding at hH2R-GsalphaS. Molecular modeling suggested that the nonconserved Asp-271 in transmembrane domain 7 of gpH2R (Ala-271 in hH2R) confers high potency to guanidines. This hypothesis was confirmed by Ala-271-->Asp-271 mutation in hH2R-GsalphaS. Intriguingly, the efficacies of guanidines at the Ala-271-->Asp-271 mutant and at hH2R/gpH2R chimeras were lower than at gpH2R. Our model suggests that a Tyr-17/Asp-271 H-bond, present only in gpH2R-GsalphaS but not the other constructs studied, stabilizes the active guanidine-H2R state. Collectively, our data show 1) distinct interaction of H2R species isoforms with guanidines, 2) that a single amino acid in transmembrane domain 7 critically determines guanidine potency, and 3) that an interaction between transmembrane domains 1 and 7 is important for guanidine efficacy.

Analogues of BIBP 3226, (R)-N(alpha)-diphenylacetyl-N-(4-hydroxybenzyl)argininamide, were synthesized and investigated for Y1 antagonism (Ca2+-assay, HEL cells) and binding on Y1, Y2 and Y5 receptors. Replacing the benzylamino by a tetrahydrobenzazepinyl group preserves most of the Y1 activity. Combination with a N(G)-phenylpropyl arginine and a N(alpha)-p-biphenylylacetyl moiety shifted the NPY receptor selectivity towards Y5.

The design of non-peptide, Y1-selective antagonists of neuropeptide Y (NPY) as pharmacological tools is in progress and is increasingly important as therapeutic applications are expected. Starting from the potent histamine H2agonist and weak NPY Y1antagonist arpromidine, 16 imidazolylpropylguanidine derivatives were synthesized and tested for Y1antagonistic activity (inhibition of NPY-stimulated Ca2+increase in human erythroleukemic cells), where the pheniramine-like moiety of arpromidine was replaced with 2-pyridylaminoalkyl, benzyl-(2-pyridyl)aminoalkyl, and phenyl-(2-pyridyl)alkylaminoalkyl partial structures derived from mepyramine. The pA2values of the most active compounds are in the range of 6.2-6.5. Quantitative structure-activity relationships (QSAR) were investigated by fragment regression analysis. Results indicate that a tetramethylene spacer between the guanidino group and the amino nitrogen is optimal. For an at least moderate degree of Y1antagonistic activity, a second benzyl or phenyl group must be present in addition to the 2-pyridyl ring. At this second group, hydrophobic substituents such as 3,4-di-Cl and 4-Br further enhance Y1antagonism. The most active derivative additionally bears a 5-Br substituent at the 2-pyridyl moiety. Structure-activity relationships suggest that the compounds might be able to partially imitate the role of NPY when interacting with Y1receptors and thus behave as moderate non-peptide NPY Y1antagonists.Key words : neuropeptide Y Y1antagonists, imidazolylpropylguanidines, quantitative structure-activity relationships.

On s'interesse de plus en plus a concevoir des antagonistes non peptidiques selectifs du recepteur Y 1 du neuropeptide Y (NPY), les travaux en cours visant a les utiliser comme outils pharmacologiques dans de futures applications therapeutiques. Nous avons employe le puissant antagoniste H 2 a l'histamine et faible antagoniste du recepteur Y 1 du NPY, apromidine, pour synthetiser des derives de la 16-imidazolylpropylguanidine et tester leur activite antagoniste envers le recepteur Y 1 (inhibition de l'augmentation de Ca 2+ stimulee par le NPY dans les cellules HEL); dans ce cas, le fragment de type pheniramine de l'apromidine a ete remplace par les structures partielles 2-pyridylaminoalkyl, benzyl-(2-pyridyl)aminoalkyl et phenyl-(2-pyridyl)alkylaminoalkyl derivees de la mepyramine. Les valeurs de pA 2 des composes les plus actifs se situent entre 6,2 et 6,5. Les relations structure-activite quantitatives (RSAQ) ont ete examinees par l'analyse de regression des fragments. Les resultats indiquent la presence d'un espaceur tetramethylene optimal entre le groupe guanidino et l'azote amine. A tout le moins a un niveau modere, l'activite antagoniste envers Y 1 requiert la presence d'un deuxieme groupe benzyl ou phenyl en plus du noyau 2-pyridyl. Pour ce qui est de ce deuxieme groupe, des substituants hydrophobes comme le 3,4-di-Cl et le 4-Br ajoutent a l'antagonisme. De plus, le derive le plus actif porte un substituant 5-Br au niveau du fragment 2-pyridyl. Les relations structure-activite suggerent que les composes pourraient imiter partiellement le NPY lorsqu'ils interagissent avec les recepteurs Y 1 et ainsi se comporter comme des antagonistes non peptidiques moderes a leur egard.