Discovery of a 4-aryloxy-1H-pyrrolo[3,2-c]pyridine and a 1-aryloxyisoquinoline series of TRPA1 antagonists

A series of TRPA1 antagonists is described having a 4-aryloxy-1H-pyrrolo[3,2-c]pyridine or a 1-aryloxyisoquinoline scaffold. These compounds have high ligand efficiency and favorable physical properties and may thus serve as scaffolds for further optimization.     

Cyclopentane-based human NK1 antagonists. Part 2: development of potent, orally active, water-soluble derivatives

The synthesis and optimization of a cyclopentane-based hNK1 antagonist scaffold 3, having four chiral centers, will be discussed in the context of its enhanced water solubility properties relative to the marketed anti-emetic hNK1 antagonist EMEND (Aprepitant). Sub-nanomolar hNK1 binding was achieved and oral activity comparable to Aprepitant in two in vivo models will be described.     

Synthesis of some aromatic prostaglandin analogs

Some aromatic prostaglandin analogs, having a benzene (2a, 2b) and a dimethoxybenzene (1) ring in place of the cyclopentane moiety, have been synthesized. The key intermediates in the syntheses were lactols 9 and 5, which were elaborated to the final products via two olefination reactions. Compound 2b was twice as potent as phenylbutazone and nine times as potent as aspirin in inhibiting prostaglandin synthetase activity.     

Synthesis of some aromatic prostaglandin analogs

Some aromatic prostaglandin analogs, having a benzene (2a, 2b) and a dimethoxybenzene (1) ring in place of the cyclopentane moiety, have been synthesized. The key intermediates in the syntheses were lactols 9 and 5, which were elaborated to the final products via two olefination reactions. Compound 2b was twice as potent as phenylbutazone and nine times as potent as aspirin in inhibiting prostaglandin synthetase activity.     

The conformationally constrained N-methanocarba-dT analogue adopts an unexpected C4'-exo sugar pucker in the structure of a DNA hairpin

Incorporation of a bicyclo[3.1.0]hexane scaffold into the nucleoside sugar was devised to lock the embedded cyclopentane ring in conformations that mimic the furanose North and South sugar puckers. To analyze the effects of North-methanocarba-2'-deoxythymidine (N-MCdT) on the B-form DNA, we crystallized d(CGCGAA[mcTmcT]CGCG) with two N-MCdTs. Instead of a duplex, the 12mer forms a tetraloop hairpin, whereby loop N-MCdTs adopt the C4'-exo pucker (NE; P = 50°). Thus, the bicyclic framework does not limit the pucker to the anticipated C2'-exo range (NNW; P = -18°).     

Synthesis and in vitro biological evaluation of ring B abeo-sterols as novel inhibitors of Mycobacterium tuberculosis

A series of 3beta-hydroxy steroid analogues possessing a contracted cyclopentane B-ring were prepared based on the initial activity screening of a recently reported naturally occurring marine 5(6-->7)abeo-sterol against Mycobacterium tuberculosis. All of the novel ring B abeo-sterols synthesized showed good inhibitory activity, whereas none of the starting steroids based on the common 3beta-hydroxy-Delta(5)-cholestane nucleus, proved to be active. Therefore, the 5(6-->7)abeo-sterol nucleus present in compounds 3, 5, 7, 9, and 11 represents a novel scaffold for the development of new antitubercular agents.     

Discovery of a series of aryl-N-(3-(alkylamino)-5-(trifluoromethyl)phenyl)benzamides as TRPA1 antagonists

We describe the discovery and advancement of a novel series of TRPA1 antagonist having an aryl-N-(3-(alkylamino)-5-(trifluoromethyl)phenyl)benzamide scaffold. The physical and in vitro DMPK profiles are discussed.     

Mass spectrometric investigation of minor products in the radiolysis of liquid cyclopentane

The following substances have been identified as minor products in the gamma radiolysis of liquid cyclopentane at 3 X 10(22) eV total dose: 1,4-pentadiene; 2-pentene; cyclopentadiene; 1,3-pentadiene; ethyl cyclopentane; vinyl cyclopentane; n-heptane; n-octane; propyl cyclopentane; propenyl cyclopentane or propyl cyclopentene; n-decane; pentenyl cyclopentane or n-pentyl cyclopentene; n-pentyl cyclopentane; and cyclopentyl cyclopentene. The determination of these products was by parent mass number, gas chromatographic retention times, and/or boiling points of the compounds. Ultraviolet spectroscopic studies of the radiolysis of solutions of cyclopentadiene in cyclopentane are also described.     

Novel chitosan/collagen scaffold containing transforming growth factor-beta1 DNA for periodontal tissue engineering

The current rapid progression in tissue engineering and local gene delivery system has enhanced our applications to periodontal tissue engineering. In this study, porous chitosan/collagen scaffolds were prepared through a freeze-drying process, and loaded with plasmid and adenoviral vector encoding human transforming growth factor-beta1 (TGF-beta1). These scaffolds were evaluated in vitro by analysis of microscopic structure, porosity, and cytocompatibility. Human periodontal ligament cells (HPLCs) were seeded in this scaffold, and gene transfection could be traced by green fluorescent protein (GFP). The expression of type I and type III collagen was detected with RT-PCR, and then these scaffolds were implanted subcutaneously into athymic mice. Results indicated that the pore diameter of the gene-combined scaffolds was lower than that of pure chitosan/collagen scaffold. The scaffold containing Ad-TGF-beta1 exhibited the highest proliferation rate, and the expression of type I and type III collagen up-regulated in Ad-TGF-beta1 scaffold. After implanted in vivo, EGFP-transfected HPLCs not only proliferated but also recruited surrounding tissue to grow in the scaffold. This study demonstrated the potential of chitosan/collagen scaffold combined Ad-TGF-beta1 as a good substrate candidate in periodontal tissue engineering.     

A scaffold protein JIP-1b enhances amyloid precursor protein phosphorylation by JNK and its association with kinesin light chain 1

Amyloid precursor protein (APP) is the precursor molecule of beta-amyloid peptides, the major components of amyloid plaque in patients with Alzheimer's disease. In this study, we isolated JIP-1b, a JNK signaling scaffold protein, as a binding protein of APP, and analyzed the roles of JIP-1b in APP phosphorylation by JNK and the association of kinesin light chain 1 with APP. APP phosphorylation at threonine 668 by JNK was enhanced on the JIP-1b scaffold in vitro and in cultured cells exogenously expressing APP. APP phosphorylation in nerve growth factor-differentiated PC12 cells was mediated by activation of JNK signaling. JIP-1b also enhanced the association of kinesin light chain 1 with APP. Our results suggest that JIP-1b may function as a protein linking the kinesin-I motor protein to the cargo receptor, APP, and that the JNK signaling pathway may regulate the phosphorylation of this cargo protein through the JIP-1b scaffold.     

Membrane and Actin Tethering Transitions Help IQGAP1 Coordinate GTPase and Lipid Messenger Signaling

The coordination of lipid messenger signaling with cytoskeletal regulation is central to many organelle-specific regulatory processes. This coupling often depends on the function of multidomain scaffolds that orchestrate transient interactions among multiple signaling intermediates and regulatory proteins on organelles. The number of possible scaffold interaction partners and the ability for these interactions to occur at different timescales makes investigations of scaffold functions challenging. This work employs live cell imaging to probe how the multidomain scaffold IQ motif containing GTPase activating protein 1 (IQGAP1) coordinates the activities of proteins affecting local actin polymerization, membrane processing, and phosphoinositide signaling. Using endosomes that are confined by a local actin network as a model system, we demonstrate that IQGAP1 can transition between different actin and endosomal membrane tethered states. Fast scaffold binding/disassociation transitions are shown to be driven by interactions between C-terminal scaffold domains and Rho GTPases at the membrane. Fluctuations in these binding modes are linked to negative regulation of actin polymerization. Although this control governs core elements of IQGAP1 dynamics, actin binding by the N-terminal calponin homology domain of the scaffold is shown to help the scaffold track the temporal development of endosome membrane markers, implying actin associations bolster membrane and actin coordination. Importantly, these effects are not easily distilled purely through standard (static) co-localization analyses or traditional pathway perturbations methods and were resolved by performing dynamic correlation and multiple regression analyses of IQGAP1 scaffold mutants. Using these capabilities with pharmacological inhibition, we provide evidence that membrane tethering is dependent on the activities of the lipid kinase phosphoinositide 3-kinase in addition to the Rho GTPases Rac1 and Cdc42. Overall, these methods and results point to a scaffold tethering mechanism that allows IQGAP1 to help control the amplitude of phosphoinositide lipid messenger signaling by coordinating signaling intermediate activities with the development and disassembly of local actin cytoskeletal networks.     

Synthesis and conformational analysis of a locked analogue of carbovir built on a bicyclo[3.1.0]hex-2-enyl template

The synthesis and biological evaluation of a carbovir analogue (5) built on a bicyclo[3.1.0]hex-2-enyl template is described. A conformational analysis using density functional theory at the B3LYP/6-31G* level has been carried out on the rigid pseudosugar template of 5, the cyclopentene moiety of carbovir and the bicyclo[3.1.0]hex-2-yl pseudosugars of two isomeric carbonucleosides (12 and 13) containing exo- and endo-fused cyclopropane rings. The results show that while the planar configuration of the fused cyclopentane ring of compound 5 helps retain weak anti-HIV activity, the ability of the cyclopentene ring of carbovir to easily adopt a planar or puckered conformation with little energy penalty may prove to be a crucial advantage. The bicyclo[3.1.0]hex-2-yl nucleosides 12 and 13 that were inactive against HIV exhibited stiffer resistance to having a planar, fused cyclopentane moiety.     

Phosphoramidate-based peptidomimetic inhibitors of membrane type-1 matrix metalloproteinase

Membrane-type I matrix metalloproteinases (MT1-MMP) is an enzyme critical to the remodeling and homeostasis of extracellular matrix, and when over expressed it contributes to metastasis and cancer cell progression. Because of its role and implication as a biomarker that is upregulated in various cancers, MT1-MMP has become an attractive target for drug discovery. A small pilot library of peptidomimetics containing a phosphoramidate core as a zinc-binding group was synthesized and tested for inhibitory potency against MT1-MMP. From this library, a novel two residue peptidomimetic scaffold was identified that confers potency against MT1-MMP at submicromolar concentrations. The results of this study confirm that for this scaffold, valine is favored as a P1 residue and leucine in the P1′ position. Furthermore, steric tolerance was observed for the N-terminus, thus implicating that a second-generation library could be constructed to extend the scaffold to P2 without concomitant loss of affinity within the MT1-MMP catalytic domain.     

Discovery of pyrrolo[1,2-b]pyridazine-3-carboxamides as Janus kinase (JAK) inhibitors

A new class of Janus kinase (JAK) inhibitors was discovered using a rationally designed pyrrolo[1,2-b]pyridazine-3-carboxamide scaffold. Preliminary studies identified (R)-(2,2-dimethylcyclopentyl)amine as a preferred C4 substituent on the pyrrolopyridazine core (3b). Incorporation of amino group to 3-position of the cyclopentane ring resulted in a series of JAK3 inhibitors (4g–4j) that potently inhibited IFNγ production in an IL2-induced whole blood assay and displayed high functional selectivity for JAK3–JAK1 pathway relative to JAK2. Further modifications led to the discovery of an orally bioavailable (2-fluoro-2-methylcyclopentyl)amino analogue 5g which is a nanomolar inhibitor of both JAK3 and TYK2, functionally selective for the JAK3–JAK1 pathway versus JAK2, and active in a human whole blood assay.     

Development of a second generation of inhibitors of microsomal prostaglandin E synthase 1 expression bearing the gamma-hydroxybutenolide scaffold

Petrosaspongiolide M (PM), a marine sesterterpene metabolite bearing the gamma-hydroxybutenolide scaffold and displaying a potent inhibitory activity toward PLA(2) enzyme, was selected by us as an attractive target in order to explore its mechanism of action at molecular level. In the course of our investigations we decided to synthetically modify the parent compound to clarify the structural determinants responsible for the activity; in fact, very recently, our research group reported the synthesis and the pharmacological properties of a first collection of PM analogues generated by Ludi approach. The synthesized compounds showed a poor or moderate activity toward PLA(2) enzymes, nevertheless we discovered a potent and selective modulator of the expression of microsomal prostaglandin E synthase 1 (mPGES-1), an enzyme highly involved in the inflammatory response, which represents an interesting target for the development of a new class of anti-inflammatory agents. In this paper we report the synthesis of a further collection of nine analogues, having the same scaffold of PM, the gamma-hydroxybutenolide, and bearing, as side chain, more complex aromatic portions, in substitution of the sesterterpene moiety. Their pharmacological behavior against PLA(2) enzymes as well as to modulate the expression of inducible cyclooxygenase 2 (COX-2) and mPGES-1 enzymes is also described.     

Potent inhibitors of the HIV-1 protease incorporating cyclic urea P1-P2 scaffold

We have developed synthetic approaches to novel analogues of 2-imidazolidinone scaffold 2, which was found to be an effective P1-P2 mimetic in HIV-1 protease inhibitor 4. This enabled a rapid synthesis of analogues of 4 and subsequently allowed us to evaluate and rationalize the SAR. Accordingly, trans relationship of P1 and P2 substituents in the P1-P2 mimetic, as found in a related 2-pyrrolidone-based scaffold 1, was found necessary for high potency against HIV-1 protease. Results of this study provided further rationale towards subsequent optimization of 2-pyrrolidone-based lead 3, which led us to potent and drug-like HIV-1 protease inhibitors described in a follow-on report (Bioorg. Med. Chem. Lett. 2004, 14, in press. ).     

Interaction between Epsin/Yap180 adaptors and the scaffolds Ede1/Pan1 is required for endocytosis

The spatial and temporal regulation of the interactions among the approximately 60 proteins required for endocytosis is under active investigation in many laboratories. We have identified the interaction between monomeric clathrin adaptors and endocytic scaffold proteins as a critical prerequisite for the recruitment and/or spatiotemporal dynamics of endocytic proteins at early and late stages of internalization. Quadruple deletion yeast cells (DeltaDeltaDeltaDelta) lacking four putative adaptors, Ent1/2 and Yap1801/2 (homologues of epsin and AP180/CALM proteins), with a plasmid encoding Ent1 or Yap1802 mutants, have defects in endocytosis and growth at 37 degrees C. Live-cell imaging revealed that the dynamics of the early- and late-acting scaffold proteins Ede1 and Pan1, respectively, depend upon adaptor interactions mediated by adaptor asparagine-proline-phenylalanine motifs binding to scaffold Eps15 homology domains. These results suggest that adaptor/scaffold interactions regulate transitions from early to late events and that clathrin adaptor/scaffold protein interaction is essential for clathrin-mediated endocytosis.     

A novel unstructured scaffold based on 4EBP1 enables the functional display of a wide range of bioactive peptides

Target validation using protein aptamers enables the characterization of a specific function of a target protein in an environment that resembles native conditions as closely as possible. A major obstacle to the use of this technology has been the generation of bioactive aptamers, which is dependent on the choice of scaffold. Constraining binding peptides within a particular scaffold does not necessarily result in binding aptamers, as suboptimal presentation of peptides can occur. It is therefore understandable that different peptides might require different scaffolds for optimal presentation. In this article, we describe a novel scaffold protein that bypasses the conventional requirement for scaffolds to have known rigid structures and yet successfully presents several peptides that need to adopt a wide range of conformations for binding to their target protein. Using an unstructured protein, 4EBP1, as scaffold, we successfully construct binding aptamers to three different target proteins: Mdm2, proliferating cell nuclear antigen, and cyclin A. The Mdm2-binding aptamer constructed using 4EBP1 as scaffold demonstrates better stability and bioactivity compared to that constructed using thioredoxin as scaffold. This new scaffold protein, which makes it relatively easy to create bioactive aptamers based on known interaction sequences, will greatly facilitate the aptamer approach to target validation.     

Disassembly of the JIP1/JNK molecular scaffold by caspase-3-mediated cleavage of JIP1 during apoptosis

We report here the cleavage of the c-Jun N-terminal Kinase (JNK) pathway scaffold protein, JNK Interacting Protein-1 (JIP1), by caspases during both Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and staurosporine-induced apoptosis in HeLa cells. During the initiation of apoptosis, maximal JNK activation is observed when JIP1 is intact, whereas cleavage of JIP1 correlates with JNK inactivation and progression of apoptosis. JIP1 is cleaved by caspase-3 at two sites, leading to disassembly of the JIP1/JNK complex. Inhibition of JIP1 cleavage by the caspase-3 inhibitor DEVD.fmk inhibits this disassembly, and is accompanied by sustained JNK activation. These data suggest that TRAIL and staurosporine induce JNK activation in a caspase-3-independent manner and that caspase-3-mediated JIP1 cleavage plays a role in JNK inactivation via scaffold disassembly during the execution phase of apoptosis. Caspase-mediated cleavage of JIP scaffold proteins may therefore represent an important mechanism for modulation of JNK signalling during apoptotic cell death.     

Synthesis of versatile thiol-reactive polymer scaffolds via RAFT polymerization

Well-defined polymer scaffolds convertible to (multi)functional polymer structures via selective and efficient modifications potentially provide an easy, versatile, and useful approach for a wide variety of applications. Considering this, a homopolymer scaffold, poly(pyridyldisulfide ethylmethacrylate) (poly(PDSM)), having pendant groups selectively reactive with thiols, was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Soluble polymers with controlled molecular weights and narrow PDIs were generated efficiently. The versatility of the scaffold to generate random co- and ter-polymers combining multiple functionalities with controlled-composition was shown by separate and simultaneous conjugation of different mercapto-compounds, including a tripeptide in one-step. Conversion of water-insoluble scaffold to peptide-containing water-soluble copolymers was observed to yield nanometer-size particles with narrow polydispersity. The overall results suggest that the well-defined PDSM homopolymer scaffold generated via RAFT polymerization can be a versatile building block for generation of new structures having potential for drug delivery applications via a straightforward synthetic approach.