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Farnesoid X receptor (FXR) agonists are emerging as important potential therapeutics for the treatment of nonalcoholic steatohepatitis (NASH) patients, as they exert positive effects on multiple aspects of the disease. FXR agonists reduce lipid accumulation in the liver, hepatocellular inflammation, hepatic injury, and fibrosis. While there are currently no approved therapies for NASH, the bile acid-derived FXR agonist obeticholic acid (OCA; 6-ethyl chenodeoxycholic acid) has shown promise in clinical studies. Previously, we described the discovery of tropifexor (LJN452), the most potent non-bile acid FXR agonist currently in clinical investigation. Here, we report the discovery of a novel chemical series of non-bile acid FXR agonists based on a tricyclic dihydrochromenopyrazole core from which emerged nidufexor (LMB763), a compound with partial FXR agonistic activity in vitro and FXR-dependent gene modulation in vivo. Nidufexor has advanced to Phase 2 human clinical trials in patients with NASH and diabetic nephropathy.

Farnesoid X receptor (FXR) agonism is emerging as an important potential therapeutic mechanism of action for multiple chronic liver diseases. The bile acid‐derived FXR agonist obeticholic acid (OCA) has shown promise in a phase 2 study in patients with nonalcoholic steatohepatitis (NASH). Here, we report efficacy of the novel nonbile acid FXR agonist tropifexor (LJN452) in two distinct preclinical models of NASH. The efficacy of tropifexor at

Influenza virus uses a unique mechanism to initiate viral transcription named cap-snatching. The PB2 subunit of the viral heterotrimeric RNA polymerase binds the cap structure of cellular pre-mRNA to promote its cleavage by the PA subunit. The resulting 11–13 capped oligomer is used by the PB1 polymerase subunit to initiate transcription of viral proteins. VX-787 is an inhibitor of the influenza A virus pre-mRNA cap-binding protein PB2. This clinical stage compound was shown to bind the minimal cap-binding domain of PB2 to inhibit the cap-snatching machinery. However, the binding of this molecule in the context of an extended form of the PB2 subunit has remained elusive. Here we generated a collection of PB2 truncations to identify a PB2 protein representative of its structure in the viral heterotrimeric protein. We present the crystal structure of VX-787 bound to a PB2 construct that recapitulates VX-787's biological antiviral activity in vitro. This co-structure reveals more extensive interactions than previously identified and provides insight into the observed resistance profile, affinity, binding kinetics, and conformational rearrangements induced by VX-787.

Imidazopyridine 1 was identified from a phenotypic screen against P. falciparum (Pf) blood stages and subsequently optimized for activity on liver-stage schizonts of the rodent parasite P. yoelii (Py) as well as hypnozoites of the simian parasite P. cynomolgi (Pc). We applied these various assays to the cell-based lead optimization of the imidazopyrazines, exemplified by 3 (KAI407), and show that optimized compounds within the series with improved pharmacokinetic properties achieve causal prophylactic activity in vivo and may have the potential to target the dormant stages of P. vivax malaria.

On the basis of the initial success of optimization of a novel series of imidazolopiperazines, a second generation of compounds involving changes in the core piperazine ring was synthesized to improve antimalarial properties. These changes were carried out to further improve the potency and metabolic stability of the compounds by leveraging the outcome of a set of in vitro metabolic identification studies. The optimized 8,8-dimethyl imidazolopiperazine analogues exhibited improved potency, in vitro metabolic stability profile and, as a result, enhanced oral exposure in vivo in mice. The optimized compounds were found to be more efficacious than the current antimalarials in a malaria mouse model. They exhibit moderate oral exposure in rat pharmacokinetic studies to achieve sufficient multiples of the oral exposure at the efficacious dose in toxicology studies.

Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca(+2). Comparison of the structures with each other and with other members of the trypsin-like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca(+2) cations. This induced fit active site provides a new possible mode of regulation of trypsin-like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.

Within our bodies, cells and tissues are constantly being pushed and pulled by their surrounding environment. These mechanical forces are then transformed into electrical or chemical signals by cells. This process is crucial for many biological structures, such as blood vessels, to develop correctly, and is also a key part of our senses of touch and hearing. In 2010, researchers discovered a group of ion channels—proteins embedded in the membrane that surrounds a cell—that open up when a force is applied and allow ions such as calcium, potassium, and sodium to flow. This movement of ions generates the electrical response of the cell to the applied force. However, not much is known about how these ‘Piezo’ ion channels work. To investigate this, it is important to be able to precisely control how and when the Piezo channels open. Many other ion channels are studied by using small chemical compounds to activate them, but there were none that were known to act on Piezo proteins. Syeda et al.—including some of the researchers involved in the 2010 work—screened over three million compounds for their ability to cause calcium ions to flow into human cells to try to identify chemicals that activate the Piezo channels. This revealed one promising candidate named Yoda1, which specifically activated Piezo1: a Piezo protein that had previously been linked to a role in blood vessel development in embryos. To investigate how Yoda1 activates Piezo1, Syeda et al. placed Piezo1 in an artificial cell membrane that did not contain any other cellular components. When Yoda1 was added to this set up, the Piezo1 channels opened up. This suggests that Piezo1 and Yoda1 interact in a manner that does not require additional cellular components other than a cell membrane. Separate work by Cahalan, Lukacs et al. uses Yoda1 to reveal that Piezo1 helps to control the volume of red blood cells, showing that in the future, Yoda1 could be valuable in research that investigates the roles of Piezo1.

The synthesis and structure-activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported. Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral bioavailability over early lead compounds. An X-ray structure of compound 37 bound to the active site of cathepsin S is also reported.

A series of N α -2-benzoxazolyl-α-amino acid-(arylaminoethyl)amides were identified as potent, selective, and noncovalent inhibitors of cathepsin S. Structure–activity relationships including strategies for modulating the selectivities among cathepsins S, K, and L, and in vivo pharmacokinetics are discussed. A X-ray structure of compound 3 bound to the active site of cathepsin S is also reported.

A series of Nα-acyl-α-amino acid-(arylaminoethyl)amides were found to be potent and noncovalent cathepsin S inhibitors. Compound 20 possessed high cathepsin S affinity (Ki = 3.3 nM) and showed excellent selectivity over cathepsin K, L, F, and V. Molecular modeling, design, synthesis, and in vitro activity are described.

Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.

Low-polydispersity poly(vinyl oxazolone) has been prepared using nitroxide-mediated living free radical polymerization. Bulk homopolymerization of 2-vinyl-4,4-dimethyl-5-oxazolone (VDMO) in the presence of the α-hydrido alkoxyamine initiator 2 and the corresponding nitroxide 1 proceeds to high conversion with polydispersities of less than 1.10. Accurate molecular weight control and low polydispersities (∼1.04−1.10) were obtained on statistical copolymerization with styrene. Well-defined reactive statistical copolymers with polydispersities ranging from 1.05 to 1.30 were also prepared by copolymerization of oxazolone-functional monomers with acrylates, acrylamides, and N-vinylamides. Reactive block copolymers were prepared by polymerization of VDMO from poly(n-butyl acrylate) starting blocks as well as by polymerization of styrene from poly(VDMO) starting blocks. New polymers were prepared from VDMO-containing polymers and copolymers by nucleophilic ring-opening of the pendant oxazolone rings with amines.

A high throughput screening campaign identified 5-(2-chlorophenyl)indazole compound 4 as an antagonist of the transient receptor potential A1 (TRPA1) ion channel with IC50 = 1.23 μM. Hit to lead medicinal chemistry optimization established the SAR around the indazole ring system, demonstrating that a trifluoromethyl group at the 2-position of the phenyl ring in combination with various substituents at the 6-position of the indazole ring greatly contributed to improvements in vitro activity. Further lead optimization resulted in the identification of compound 31, a potent and selective antagonist of TRPA1 in vitro (IC50 = 0.015 μM), which has moderate oral bioavailability in rodents and demonstrates robust activity in vivo in several rodent models of inflammatory pain.

Renewed global efforts toward malaria eradication have highlighted the need for novel antimalarial agents with activity against multiple stages of the parasite life cycle. We have previously reported the discovery of a novel class of antimalarial compounds in the imidazolopiperazine series that have activity in the prevention and treatment of blood stage infection in a mouse model of malaria. Consistent with the previously reported activity profile of this series, the clinical candidate KAF156 shows blood schizonticidal activity with 50% inhibitory concentrations of 6 to 17.4 nM against P. falciparum drug-sensitive and drug-resistant strains, as well as potent therapeutic activity in a mouse models of malaria with 50, 90, and 99% effective doses of 0.6, 0.9, and 1.4 mg/kg, respectively. When administered prophylactically in a sporozoite challenge mouse model, KAF156 is completely protective as a single oral dose of 10 mg/kg. Finally, KAF156 displays potent Plasmodium transmission blocking activities both in vitro and in vivo . Collectively, our data suggest that KAF156, currently under evaluation in clinical trials, has the potential to treat, prevent, and block the transmission of malaria.

The highly compact and globular shape, as well as the uniform size and plurifunctionality of dendrimers make them ideal molecular building blocks for a wide range of interfacial materials involving self-assembled monolayers, Langmuir films, multilayers, and other surface-confined assemblies. Moreover, the study of the behavior of dendrimers at surfaces and interfaces provides unique insight into their chemical and physical properties. Dendritic macromolecules play an increasingly important role in the materials and surface sciences, where applications utilizing polymer thin films can benefit from their distinctive chemical and physical properties. Recent investigations have highlighted the use of dendrimers as functional surfaces and as interfacial materials for applications in membranes, adhesion, microelectronics or in chemical and biological sensing.

Individual peptides with lysine at the C-terminus as well as protein tryptic digests were reacted with 2-methoxy-4,5-dihydro-1H-imidazole, converting lysine residues to their 4,5-dihydro-1H-imidazol-2-yl derivatives. The mass spectra of derivatized digests exhibit a greater number of more intense features than their underivatized counterparts, thus increasing the information obtained in peptide mapping experiments. Additionally, MS/MS spectra of the derivatized peptides are greatly simplified in comparison to their native species, yielding primarily an easily interpretable series of y-ions. Finally, this novel label also enables differential quantitation studies, as a stable isotopic form containing four deuterium atoms can readily be produced.

Dendrimer monolayers have been designed for use as positive resists for scanning probe lithography (SPL). Several new amphiphilic poly(benzyl ether) dendrimers with carboxylic acids at either the focal point or the “periphery” have been prepared. These can form ionically bound dendrimer monolayers that may serve as either positive or negative tone resists for SPL. The amphiphilic dendrimers self-assemble onto (3-aminopropyl)silanized Si(100) wafer surfaces to afford ultrathin films. The dendrimer monolayers were characterized by AFM, ellipsometry, and contact angle goniometry. Patterning a singly charged dendrimer monolayer results in the formation of positive tone holes ∼35 nm in width. Similarly, patterning a multiply charged dendrimer monolayer in a direct-write manner with the scanning probe microscope results in the formation of negative tone oxide features ∼80 nm in width.

Poly(amidoamine) (PAMAM) dendrimers, generations 1−6, were peripherally modified with cationically substituted naphthalene diimide groups. Several monomeric diimides were also prepared as models. The structure and loading of the dendrimers were determined by vis, IR, and NMR spectroscopies, elemental analysis, and coulometric analysis. In general it was possible to obtain loadings greater than 70% even from generation 6 dendrimer where there are 192 amine groups to be substituted. These polymers and monomers were reduced using sodium dithionite or electrochemically with one electron per imide group converting each imide into its anion radical. Near-infrared (NIR) spectroscopy showed that in D2O or formamide solutions the anion radicals aggregated into π-dimers and π-stacks. Cyclic voltammograms are interpreted in terms of anion radical aggregation and precipitation of the reduced dendrimers. The CV and NIR results for the various dendrimer generations were quite similar. These results allow insight into t...

An electrically conductive polymer is comprised of the reaction product of a dendrimer and a electrophoric compound which exhibits π-stacking upon reduction. The reaction product which is a peripherally modified dendrimer is formed and reduced, either chemically or electrochemically, in a polar solvent. The reduced electrophoric moieties aggregate (form π-stacks) to form electrically conductive pathways. The reduced peripherally modified dendrimers of the invention can be cast into films and other solid forms to provide flexible electrical conductors and semiconductors. The conductive polymers of the invention exhibit electrical conductivities comparable to that of doped polyacetalene, but are oxygen and moisture stable.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria. © 2013 Macmillan Publishers Limited. All rights reserved.

[Image: see text] Starting from a hit series from a GNF compound library collection and based on a cell-based proliferation assay of Plasmodium falciparum, a novel imidazolopiperazine scaffold was optimized. SAR for this series of compounds is discussed, focusing on optimization of cellular potency against wild-type and drug resistant parasites and improvement of physiochemical and pharmacokinetic properties. The lead compounds in this series showed good potencies in vitro and decent oral exposure levels in vivo. In a Plasmodium berghei mouse infection model, one lead compound lowered the parasitemia level by 99.4% after administration of 100 mg/kg single oral dose and prolonged mice survival by an average of 17.0 days. The lead compounds were also well-tolerated in the preliminary in vitro toxicity studies and represents an interesting lead for drug development.

The highly compact and globular shape, as well as the uniform size and plurifunctionality of dendrimers make them ideal molecular building blocks for a wide range of interfacial materials involving self-assembled monolayers, Langmuir films, multilayers, and other surface-confined assemblies. Moreover, the study of the behavior of dendrimers at surfaces and interfaces provides unique insight into their chemical and physical properties. Dendritic macromolecules play an increasingly important role in the materials and surface sciences, where applications utilizing polymer thin films can benefit from their distinctive chemical and physical properties. Recent investigations have highlighted the use of dendrimers as functional surfaces and as interfacial materials for applications in membranes, adhesion, microelectronics or in chemical and biological sensing.

A novel family of 1H-imidazol-2-yl-pyrimidine-4,6-diamines has been identified with potent activity against the erythrocyte-stage of Plasmodium falciparum (Pf), the most common causative agent of malaria. A systematic SAR study resulted in the identification of compound 40 which exhibits good potency against both wild-type and drug resistant parasites and exhibits good in vivo pharmacokinetic properties.

Graphical abstract, Screening our in-house compound collection using a cell based Plasmodium falciparum proliferation assay we discovered a known pan-kinase inhibitor scaffold as a hit. Further optimization of this series led us to a novel benzamide scaffold which was devoid of human kinase activity while retaining its antiplasmodial activity. The evolution of this compound series leading to optimized candidates with good cellular potency against multiple strains as well as decent in vivo profile is described in this Letter.

Peptidic, non-covalent inhibitors of lysosomal cysteine protease cathepsin S (1 and 2) were investigated due to low oral bioavailability, leading to an improved series of peptidomimetic inhibitors. Utilizing phenyl succinamides as the P2 residue increased the oral exposure of this lead series of compounds, while retaining selective inhibition of the cathepsin S isoform. Concurrent investigation of the P1 and P2 subsites resulted in the discovery of several potent and selective inhibitors of cathepsin S with good pharmacokinetic properties due to the elimination of saturated aliphatic P2 residues.

A systematic study of anilines led to the discovery of a metabolically robust fluoroindoline replacement for the alkoxy aniline toxicophore in 1. Investigations of the P1 pocket resulted in the discovery of a wide tolerance of functionality leading to the discovery of 11 as a potent and selective inhibitor of cathepsin S.

Human prostasin was recently identified as a potential regulator of epithelial sodium channel (ENaC) function. Through the use of positional scanning combinatorial substrate libraries, prostasin was shown to have a preference for poly-basic substrates: in position P4 preference was for arginine or lysine; in P3 preference was for histidine, lysine or arginine; in P2 preference was for basic or large hydrophobic amino acids; and in P1 preference was for arginine and lysine. P1', P2', and P3' displayed broad selectivity with the exception of a lack of activity for isoleucine, and P4' had a preference for small, unbranched, amino acids such as alanine and serine. A prostasin-preferred poly-basic cleavage site was found in the extracellular domains of the ENaC alpha- and beta-subunits, and may present a mechanism for prostasin activation. The absence of activity seen with substrates containing isoleucine in position P1' explains the inability of prostasin to autoactivate and suggests that prostasin proteolytic activity is regulated by an upstream protease. Prostasin activity was highly influenced by mono- and divalent metal ions which were potent inhibitors and substrate specific modulators of enzymatic activity. In the presence of sub-inhibitory concentrations of zinc, the activity of prostasin increased several-fold and its substrate specificity was significantly altered in favor of a strong preference for histidine in positions P3 or P4 of the substrate.

Several new poly(benzyl ether) and poly(benzyl ester) dendrimers that incorporate acid- and thermally- labile peripheral groups have been synthesized. Tertiary butyl ester terminated poly(benzyl ether) dendrimers were synthesized using (alpha) -bromo-t-butyl acetate in the preliminary protection step to afford the first generation alcohol. A standard bromination of the focal point benzylic alcohol was used for the activation step, while standard Williamson ether conditions were used for the coupling steps to afford higher generation poly(benzyl ether) dendrons. Tertiary butyl ester terminated dendrons were then coupled to a difunctional core to produce the [G-3] dendrimer. Tertiary butyl carbonate (t-Boc) terminated poly(benzyl ester) dendrimers were also synthesized. This class of dendrimers was synthesized by first protecting monomeric building block 3,5-dihydroxybenzaldehyde with di-t-butyl dicarbonate. A reductive activation step afforded the [G-1] alcohol. The growth steps were accomplished by either Mitsunobu etherification with 3,5- dihydroxybenzaldehyde or by esterification with 5- hydroxymethylisophthalic acid. Finally, coupling of the benzyl alcohol dendrons to a polyfunctional core afforded second and third generation dendrimers. Chemically amplified resists formulated from both t-butyl ester and t-Boc terminated dendrimers show high sensitivity to DUV and e-beam irradiation. Feature sizes well below 100 nm have been routinely patterned using e-beam lithography.

The application of a hyperbranched polymer with its globular architecture as a chemically amplified resist system isdemonstrated. These hyperbranched poly(esters) based on 3,5-dihydroxybenzoic acid and 4,4-bis(4-hydroxyphenyl)valericacid and obtained by a polycondensation process at high temperatures. Once obtained, the hyperbranched polymers arefunctionalized with acid and thermally labile t-BOC groups by reaction oftheir phenolic groups with di-t-butyl dicarbonate inthe presence of a catalytic amount of potassium t-butoxide. These globular materials have number average molecularweights (Ma) in the range of 5,000 - 20,000 with polydispersities of 1 .5 — 2. Exposure of the hyperbranched resist materialformulated with a photoacid generator was carried out using a direct-write electron-beam (e-beam) tool operating at 50 keV with doses of 1 5 — 40 tC/cm2. Development of these resist materials can be accomplished in either aqueous base developeror organic solvent, thereby allowing access to both the positive and negative tone images. Feature sizes of 100 nm arereadily obtained from these unoptimized materials.Keywords : hyperbranched polymer, photoresist, electron-beam, chemical amplification

Scanning probe lithography (SPL) has recently been shown to be a versatile technique for patterning semiconductor surfaces. The intense electric field emanating from the scanning probe tip can be used for the patterning of carbon- based self-assembled monolayers and thin films, as well as the anodization or field-enhanced oxidation of passivated metal surfaces. We have been investigating the use of monolayers and ultra-thin films made from dendritic polymers as resists for SPL. Dendrimer films have ben prepare by both covalent and ionic binding to the wafer surface. Silicon wafers were treated with dendritic chlorosilanes to afford self-assembled monolayer from the functionalized dendrons. Ionically bound dendrimer films were prepared by treating an aminopropylsilane-modified wafer surface with a dendritic carboxylic acid. These ultra-thin dendrimer films were characterized by atomic force microscopy, contact angle goniometry, and optical ellipsometry. The dendrimer films were shown to be effective resist for SPL, and we have patterned negative tone oxide images onto dendrimer modified wafer surfaces. Pattern transfer can be achieved by a selective wet etch resulting in the formation of positive tone images.

Our current understanding of the role and regulation of protease activity in normal and pathogenic processes is limited by our ability to measure and deconvolute their enzymatic activity. To address this limitation, an approach was developed that utilizes rhodamine-based fluorogenic substrates encoded with PNA tags. The PNA tags address each of the substrates to a predefined location on an oligonucleotide microarray through hybridization, thus allowing the deconvolution of multiple signals from a solution. A library of 192 protease substrates was prepared by split and mix combinatorial synthesis. The methodology and validation of this approach for profiling proteolytic activity from single proteases and from those in crude cell lysates as well as clinical blood samples is described.