Small Substrates Research Articles

Activated Factor IX As a Candidate for the Initial Protease in the Coagulation Cascade

As currently understood, activated factor VII is the initiating protease of the coagulation process. The identity of the protease(s) that cleaves factor VII to yield its circulating activated form (~1% of the total factor VII protein in normal individuals) remains uncertain. Although, measurement of plasma concentrations of factor VIIa in normal human subjects and those with specific factor deficiencies suggests that factor IX has a factor VIII-independent role in generating circulating factor VIIa (Blood80:25-28, 1992).Previous work using purified plasma proteins yielded evidence that factor IX cleaved only at residue R226 (factor IXaα) was a competent protease, yet did not efficiently bind factor VIIIa (JBC23:14484-14490, 1995). We investigated whether this form of factor IX might activate factor VII, as it seems to have the appropriate characteristics (proteolytic activity that is VIIIa independent). We created and purified recombinant mutant forms of factor IX and factor VII that enabled exploration of the functions of these separate cleavages without the complication of unanticipated proteolysis that is inherent when using plasma derived proteins.Wild-type factor IX and R191H factor IX (the Hemophilia B Chapel Hill variant) were activated to yield factor IXaβ and IXaα, respectively, as demonstrated by SDS-PAGE under reducing and non-reducing conditions, and incorporation of fluoresceinated EGR-chloromethyl ketone into the active sites, confirming that activity correlated with the appropriate molecular mass subunit. As predicted, factor IXaβ and R191H IXaα demonstrated identical catalytic efficiency in the amidolytic cleavage of a small molecular weight substrate, Pefachrome IXa. In contrast, in the presence of factor VIIIa-phospholipid, compared to factor IXaβ, R191H IXaα exhibited greatly reduced ability to cleave the physiologic macromolecular substrate, factor X. Consistent with previous findings, R191H factor IXaα had appreciably lower affinity for factor VIIIa than did factor IXaβ, and this likely explains the difference in factor X activating activity. This difference in activity was also seen in a plasma-based system, where R191H factor IXaα demonstrated only 1.6% of the coagulant activity of factor IXaβ in an aPTT assay in factor IX immunodepleted plasma.There was a distinct difference with a second physiologic macromolecular substrate. Factor R191H factor IXaα and factor IXaβ activated factor VII to VIIa with identical kinetic parameters in a purified system. Intriguingly, the addition of factor VIIIa did not change the kinetic parameters of factor VII activation by either form of factor IXa. This observation is unlikely to be due to factor VIIa contaminating the factor VII preparation, as catalytically incompetent factor VII (S344A) was cleaved at indistinguishable rates by factors R191H IXaα and IXaβ.Experiments with control proteins that also contained mutations at the active site, showed that factor IX R191Q;S411A demonstrated the appropriate sized bands on SDS-PAGE for factor IXaα, but did not incorporate fluoresceinated EGR-chloromethyl ketone, and had no detectable amidolytic, proteolytic, or coagulant activity, consistent with the active site serine mutation. Factor IX R191Q;R226Q was not cleaved by factor VIIa-tissue factor, and factor IX R226Q was cleaved to subunits consistent with factor IXα. Neither of these proteins incorporated fluoresceinated EGR-chloromethyl ketone, and neither demonstrated detectable amidolytic activity.These experiments with recombinant forms of factor IX support the hypothesis that factor IXaα exhibits active site enzymatic competency equal to factor IXaβ, and that cleavage at R191 is required to generate fully competent factor VIIIa binding determinants. Moreover, factor IXaα and factor IXaβ are fully competent to activate factor VII, without influence by factor VIIIa. Given this mechanistic demonstration and the previously noted clinical observations (Blood 80:25-28, 1992), we propose that activated forms of factor IX are responsible for generating circulating plasma factor VIIa, which is available for the initiation phase of coagulation. These data suggest that activated factor IX is as plausible a candidate for the initiating protease of the coagulation cascade as is activated factor VII. DisclosuresNo relevant conflicts of interest to declare.

Discovery of Selective Inhibitor Leads by Targeting an Allosteric Site in Insulin-Regulated Aminopeptidase.

Insulin-Regulated aminopeptidase (IRAP) is a zinc-dependent aminopeptidase with several important biological functions and is an emerging pharmaceutical target for cognitive enhancement and immune system regulation. Aiming to discover lead-like IRAP inhibitors with enhanced selectivity versus homologous enzymes, we targeted an allosteric site at the C-terminal domain pocket of IRAP. We compiled a library of 2.5 million commercially available compounds from the ZINC database, and performed molecular docking at the target pocket of IRAP and the corresponding pocket of the homologous endoplasmic reticulum aminopeptidase 1 (ERAP1). Of the top compounds that showed high selectivity, 305 were further analyzed by molecular dynamics simulations and free energy calculations, leading to the selection of 33 compounds for in vitro evaluation. Two orthogonal functional assays were employed: one using a small fluorogenic substrate and one following the degradation of oxytocin, a natural peptidic substrate of IRAP. In vitro evaluation suggested that several of the compounds tested can inhibit IRAP, but the inhibition profile was dependent on substrate size, consistent with the allosteric nature of the targeted site. Overall, our results describe several novel leads as IRAP inhibitors and suggest that the C-terminal domain pocket of IRAP is a promising target for developing highly selective IRAP inhibitors.

Therapy Prospects for Mitochondrial DNA Maintenance Disorders.

Mitochondrial DNA depletion and multiple deletions syndromes (MDDS) constitute a group of mitochondrial diseases defined by dysfunctional mitochondrial DNA (mtDNA) replication and maintenance. As is the case for many other mitochondrial diseases, the options for the treatment of these disorders are rather limited today. Some aggressive treatments such as liver transplantation or allogeneic stem cell transplantation are among the few available options for patients with some forms of MDDS. However, in recent years, significant advances in our knowledge of the biochemical pathomechanisms accounting for dysfunctional mtDNA replication have been achieved, which has opened new prospects for the treatment of these often fatal diseases. Current strategies under investigation to treat MDDS range from small molecule substrate enhancement approaches to more complex treatments, such as lentiviral or adenoassociated vector-mediated gene therapy. Some of these experimental therapies have already reached the clinical phase with very promising results, however, they are hampered by the fact that these are all rare disorders and so the patient recruitment potential for clinical trials is very limited.

Extracellular proteolytic activation of Pseudomonas aeruginosa aminopeptidase (PaAP) and insight into the role of its non-catalytic N-terminal domain.

Pseudomonas aeruginosa secretes several endopeptidases, including elastase, alkaline proteinase (Apr), a lysine-specific endopeptidase (LysC), and an aminopeptidase (PaAP), all of which are important virulence factors. Activation of the endopeptidases requires removal of an inhibitory N-terminal propeptide. Activation of pro-PaAP, in contrast, requires C-terminal processing. The activating proteases of pro-PaAP and their cleavage site(s) have not yet been defined. Studying pro-PaAP processing in a wild type P. aeruginosa strain and strains lacking either elastase or both elastase and Apr, we detected three processing variants, each ~56 kDa in size (AP56). Activity assays and N- and C-terminal sequence analyses of these variants pointed at LysC as the principal activating protease, cleaving a Lys512-Ala513 peptide bond at the C-terminal end of pro-PaAP. Elastase and/or Apr are required for activation of LysC, suggesting both are indirectly involved in activation of PaAP. To shed light on the function(s) of the N-terminal domain of AP56, we purified recombinant AP56 and generated from it the 28 kDa catalytic domain (AP28). The kinetic constants (Km and Kcat) for hydrolysis of Leu-, Lys-, Arg- and Met-p-nitroanilide (pNA) derivatives by AP56 and AP28 were then determined. The catalytic coefficients (Kcat/Km) for hydrolysis of all four substrates by AP28 and AP56 were comparable, indicating that the non-catalytic domain is not involved in hydrolysis of small substrates. It may, however, regulate hydrolysis of natural peptides/proteins. Lys-pNA was hydrolyzed 2 to 3-fold more rapidly than Leu-pNA and ~8-fold faster than Arg- or Met-pNA, indicating that Lys-pNA was the preferred substrate.

A Diverse Array of C-C Bonds Formed at a Tantalum Metal Center.

We demonstrate the formation of a diverse array of organic and organometallic products containing newly formed C-C bonds via successive methyl transfers from di-, tri-, and tetramethyl Ta(V) precursors to unsaturated small molecule substrates under mild conditions. The reactions of Ta(V) methyl complexes 1-X [H2B(MesIm)2]TaMe3X (X = Me, Cl; Im = imidazole, Mes = 2,4,6-trimethylphenyl) with CO led to oxo enolate Ta(V) products, in which the enolate ligands were constructed from Ta-Me groups and two equivalents of CO. Similarly, the reaction of 1-Me with CNXyl yielded an imido enamine Ta(V) product. Surprisingly, 1-Cl reacted with CNXyl (1 equiv) at the borate backbone of the [H2B(MesIm)2] ligand with concomitant methyl transfer from the metal center to form a new, dianionic scorpionate ligand that supported a Ta(V) dimethyl chloro complex (6). Treatment of 1-Cl with further CNXyl led to an azaallyl scorpionate complex, and an imido isocyanide scorpionate complex, along with propene and xylyl ketenimine. Complex 6 reacted with CO to yield a pinacol scorpionate complex 10-a new reaction pathway in early transition metal chemistry. Mechanistic studies revealed that this proceeded via migratory insertion of CO into a Ta-Me group, followed by methyl transfer to form an η2-acetone intermediate. Elimination of acetone furnished a CO-stabilized Ta(III) intermediate capable of rebinding and subsequently coupling two equivalents of CO-derived acetone to form the pinacol ligand in 10.

Effect of substrate size and depth on macroalgal communities in unstable marine rocky bottoms

Subtidal unstable bottoms are sensitive to the environmental disturbances that determine the structure of macroalgal communities. Here, three variables (substrate size, depth and site) are tested together to explore their effect on the community structure on cobble and gravel bottoms. Diversity metrics (richness per sample, richness per area, diversity (H '), total cover), cover of life forms (hypnophyceae, ephemerophyceae, phanerophyceae, hemiphanerophyceae, chamaephyceae) and cover of the predominant species (Harveylithon samoënse, Jania pedunculata var. adhaerens, Lobophora spp., Canistrocarpus cervicornis, Halopteris scoparia) are used as descriptors of communities. Descriptors response was modeled using multiple regression analyses, in which each descriptor was considered separately as the dependent variable, while substrate size, depth and site was used as independent variables. Results show the substrate size was the most important variable with depth only being important for some perennial species although there was often site-site variability in these patterns. Richness per sample of macroalgal communities increased with substrate size; however, the highest values of richness per area and total cover increased towards the smallest substrates. Substrate size also positively influenced the cover of the perennial life forms hemiphanerophyceae and phanerophyceae, but do not on the cover of chamaephyceae. The perennial crustose Harveylithon samoënse showed a significant relationship with all variables increasing its cover towards small substrates in shallow, while the perennial erect Lobophora spp. are more abundant on large substrates at greater depths. Together these results showed that the substrate size was consistently the most important factor for algal diversity in unstable-bottom habitats.

Dimensional Reduction of Lewis Acidic Metal-Organic Frameworks for Multicomponent Reactions.

Owing to hindered diffusions, the application of porous catalytic materials has been limited to relatively simple organic transformations with small substrates. Herein we report a dimensional reduction strategy to construct a two-dimensional metal-organic framework (MOF), Zr6OTf-BTB, with 96% accessible Lewis acidic sites as probed by the bulky Lewis base pivalonitrile. With nearly free substrate accessibility, Zr6OTf-BTB outperformed two three-dimensional MOF counterparts of similar Lewis acidity (Zr6OTf-BPDC and Zr6OTf-BTC) in catalyzing sterically hindered multicomponent reactions (MCRs) for the construction of tetrahydroquinoline and aziridine carboxylate derivatives with high turnover numbers (TONs). Zr6OTf-BTB was also superior to the homogeneous benchmark Sc(OTf)3 with nearly 14 times higher TON and 9 times longer catalyst lifetime. Furthermore, the topology-activity relationships in these Zr-based Lewis acidic MOFs were rationalized by comparing their Lewis acidity, numbers of Lewis acidic sites, and sterically accessible Lewis acidic sites. Zr6OTf-BTB was successfully used to construct several bioactive molecules via MCRs with excellent efficiency. This dimensional reduction strategy should allow the development of other MOF catalysts for synthetically useful and complicated organic transformations.

Lamellar Enzyme-Metal–Organic Framework Composites Enable Catalysis on Large Substrates

Lamellar Enzyme-Metal–Organic Framework Composites Enable Catalysis on Large Substrates

Determination of the Calcium‐Dependent Activation Mechanism for Metacaspase 1 from Schizophyllum commune

Metacaspases are cysteine proteases implicated in initiating programmed cell death in plants and fungi, and have recently been shown to play a key role in the plant immune response. These proteins are distantly related to caspases, the enzymes that induce apoptosis in animals. Notably, metacaspases require calcium binding for activity, whereas caspases are instead activated by upstream signalling pathways that utilize calcium as a signal transducer. Metacaspase activity is greatest in the presence of millimolar levels of calcium. However, the exact mechanism by which calcium activates the metacaspases is still unclear. Our lab has performed an extensive characterization of metacaspase1 from the fungus Schizophyllum commune (ScMCA-Ia) to determine the likely conformational changes needed for metacaspase activation. The available metacaspase crystal structures from yeast and T. brucei demonstrate calcium-binding to a conserved site of four aspartic acid residues outside of the active site. While calcium itself binds to this allosteric site, the calcium-binding Asps are found on flexible loops and are only a few residues away from the active site. Sequence alignments demonstrate that these Asp residues are conserved in ScMCA-Ia and mutagenesis studies that exchange Asp for Ala reveal that this site is crucial for activity on small peptide substrates. We have used differential scanning fluorimetry to determine the calcium-binding constants of ScMC-Ia and associated mutants. Mutating calcium-binding Asp residues to Glu renders the enzymes inactive, however differential scanning fluorimetry indicates that the mutants still bind to calcium with Kd values similar to wildtype, in the µM range. This suggests that calcium-binding to this high-affinity site changes the position of calcium binding residues, altering the positions of the loops bearing these residues, and ultimately changing the orientation of the active site. Even slight changes to the calcium binding site (Asp to Glu) cannot be tolerated. In addition to the micromolar binding site, a second site that binds calcium in the mM range has been inferred from the low millimolar calcium levels needed for optimal metacaspase activity. Kinetics experiments suggest that the low-affinity calcium binding site is important for substrate binding. Together, these findings comprise a complete model for metacaspase activation in which high-affinity calcium binding reorients the active site for catalysis and low-affinity binding alters the substrate binding pocket to enhance substrate binding and catalysis. This mechanistic understanding of metacaspase activation and conformational changes is needed to design metacaspase-specific inhibitors to treat fungal diseases.

Application of SNAP-Tag in Expansion Super-Resolution Microscopy Using DNA Oligostrands.

Expansion super-resolution technology is a new technology developed in recent years. It anchors the dye on the hydrogel and the dye expands with the expansion of the hydrogel so that a super-resolution map can be obtained under an ordinary microscope. However, by labeling the target protein with a first antibody and secondary antibody, the distance between the fluorescent group and the actual target protein is greatly increased. Although fluorescent proteins can also be used for expansion super-resolution to reduce this effect, the fluorescent protein is often destroyed during sample preparation. To solve this problem, we developed a novel label system for expansion microscopy, based on a DNA oligostrand linked with a fluorescent dye, acrylamide group (linker), and benzoylguanine (BG, a small substrate molecule for SNAP-tag). This protocol greatly reduced the error between the position of fluorescent group and the actual target protein, and also reduced loss of the fluorescent group during sample preparation.

Polymer Coatings on Virus-like Particle Nanoreactors at Low Ionic Strength-Charge Reversal and Substrate Access.

Virus-like particles (VLPs) are a class of biomaterials which serve as platforms for achieving the desired functionality through interior and exterior modifications. Through ionic strength-mediated electrostatic interactions, VLPs have been assembled into hierarchically ordered materials. This work builds on predictive models to prepare polymer-coated VLP clusters at very low ionic strength. Zeta potential measurements showed that the clusters carried a strongly positive charge, a complete charge reversal from the VLP building block. SAXS analysis confirmed polymer adsorption onto the VLP exterior. We then studied the activity of an encapsulated enzyme toward small molecular and macromolecular substrates to determine the effect of each component of the hierarchically assembled material. We found that while encapsulation and polymer coating did not have a large effect on access to the enzyme by its native, small molecular substrate, substrate modification with a macromolecule caused the polymer coating and encapsulation to affect the access to the enzyme.

Molecular Dynamics-Guided Design of a Functional Protein-ATRP Conjugate That Eliminates Protein-Protein Interactions.

Even the most advanced protein-polymer conjugate therapeutics do not eliminate antibody-protein and receptor-protein recognition. Next-generation bioconjugate drugs will need to replace stochastic selection with rational design to select desirable levels of protein-protein interaction while retaining function. The "Holy Grail" for rational design would be to generate functional enzymes that are fully catalytic with small molecule substrates while eliminating interaction between the protein surface and larger molecules. Using chymotrypsin, an important enzyme that is used to treat pancreatic insufficiency, we have designed a series of molecular chimeras with varied grafting densities and shapes. Guided by molecular dynamic simulations and next-generation molecular chimera characterization with asymmetric flow field-flow fractionation chromatography, we grew linear, branched, and comb-shaped architectures from the surface of the protein by atom-transfer radical polymerization. Comb-shaped polymers, grafted from the surface of chymotrypsin, completely prevented enzyme inhibition with protein inhibitors without sacrificing the ability of the enzyme to catalyze the hydrolysis of a peptide substrate. Asymmetric flow field-flow fractionation coupled with multiangle laser light scattering including dynamic light scattering showed that nanoarmor designed with comb-shaped polymers was particularly compact and spherical. The polymer structure significantly increased protein stability and reduced protein-protein interactions. Atomistic molecular dynamic simulations predicted that a dense nanoarmor with long-armed comb-shaped polymer would act as an almost perfect molecular sieve to filter large ligands from substrates. Surprisingly, a conjugate that was composed of 99% polymer was needed before the elimination of protein-protein interactions.

Self-reconstructing Bessel beam created by two-photon-polymerized micro-axicon for light-sheet fluorescence microscopy

Observing micro-organisms with depth-resolving capability is important in optical microscopy for biomedical sciences and industries. We demonstrate the fabrication and use of a two-photon polymerized micro-axicon lens that generates a self-reconstructing pencil-like Bessel beam for light-sheet fluorescence microscopy (LSFM), providing 3D internal structures of micro-organisms. The fabricated SU-8 micro-axicon of 100 µm diameter transforms the input Gaussian beam from a single-mode fiber into a non-diffractive Bessel-Gauss beam. The focused spot size of the Bessel-Gauss beam is 2.3 ± 0.25 µm with a long propagation distance over 160 µm, which is well-suited for LSFM. The self-reconstruction capability of the generated Bessel-Gauss beam is investigated thoroughly through both simulations and experiments. Since this micro-axicon can be directly 3D-printed on single-mode fibers’ end facets or small mobile substrates, this can replace the bulky objective lens from conventional light-sheet microscopes. This will facilitate the wide-spread use of 3D tomographic imaging of micro-organisms, especially in compact micro-fluidic devices and lab-on-a-chip architectures.

Insulinase-like Protease 1 Contributes to Macrogamont Formation in Cryptosporidium parvum.

The apicomplexan parasite Cryptosporidium parvum contains an expanded family of 22 insulinase-like proteases (INS), a feature that contrasts with their otherwise streamlined genome. Here, we examined the function of INS1, which is most similar to the human insulinase protease that cleaves a variety of small peptide substrates. INS1 is an M16A clan member and contains a signal peptide, an N-terminal domain with the HXXEH active site, followed by three inactive domains. Unlike previously studied C. parvum INS proteins that are expressed in sporozoites and during merogony, INS1 was expressed exclusively in macrogamonts, where it was localized in small cytoplasmic vesicles. Although INS1 did not colocalize with the oocyst wall protein recognized by the antibody OW50, immune-electron microscopy indicated that INS1 resides in small vesicles in the secretory system. Notably, these small INS1-positive vesicles were often in close proximity to large OW50-positive vacuoles resembling wall-forming bodies, which contain precursors for oocyst wall formation. Genetic deletion of INS1, or replacement with an active-site mutant, resulted in lower formation of macrogamonts in vitro and reduced oocyst shedding in vivo Our findings reveal that INS1 functions in the formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice.IMPORTANCE Cryptosporidiosis is a debilitating diarrheal disease in young children in developing countries. The absence of effective treatments or vaccines makes this infection very difficult to manage in susceptible populations. Although the oral dose of oocysts needed to cause infection is low, infected individuals shed very high numbers of oocysts, readily contaminating the environment. Our studies demonstrate that the protease INS1 is important for formation of female sexual stages and that in its absence, parasites produce fewer oocysts and are attenuated in immunocompromised mice. These findings suggest that mutants lacking INS1, or related proteases, are useful for further characterizing the pathway that leads to macrogamont maturation and oocyst wall formation.

Kinesin-1 activity recorded in living cells with a precipitating dye

Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. The ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate (QPD-OTf) for kinesin-1 that yields a precipitating dye along its walking path on microtubules (MTs). QPD-OTf enables to monitor native kinesin-1 transport activity in cellulo without external modifications. In vitro assays show that kinesin-1 and MTs are sufficient to yield fluorescent crystals; in cells, kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time. These findings are further supported by docking studies, which suggest the binding of the activity-based substrate in the nucleotide binding site of kinesin-1.

Not all methods are created equal: assessment of sampling methods for crayfishes and fishes in southern Appalachian streams

We compared the effectiveness of three sampling methods (kick seining, electrofishing, and nest trapping) to collect crayfishes and fishes, simultaneously, in southern Appalachian Mountain streams (Alabama, USA). For crayfishes, kick seining collected the highest species richness and most individuals. However, by combining kick seining and electrofishing collections, we decreased the number of sites needed to accurately assess crayfish richness relative to using one method. For 9 of the 13 species collected, no differences in crayfish sizes or sex ratios were detected between electrofishing and kick seining. In the remaining four species, electrofishing collected larger crayfishes and more females than kick seining. For fishes, electrofishing was most effective at assessing fish species richness. Sampled fish richness was higher when electrofishing in streams with higher water temperatures and width-to-depth ratios, as well as lower conductivities and smaller substrates. Electrofishing was the most effective sampling method for collecting Centrarchids, whereas kick seining was most effective at collecting Cyprinids. Nest traps were the least effective sampling method. We conclude that using a combination of kick seining and electrofishing is best for assessing stream fish and crayfish assemblages, simultaneously, which can improve management, biomonitoring, and understanding of the complex relationships between these faunal groups.

Giant Coacervate Vesicles As an Integrated Approach to Cytomimetic Modeling.

Although giant unilamellar vesicles (GUVs) have been extensively studied as synthetic cell-like microcompartments, their applicability as cytomimetic models is severely compromised by low levels of membrane permeability, low encapsulation efficiencies, and high physicochemical instability. Here, we develop an integrated cytomimetic model comprising a macromolecularly crowded interior with high sequestration efficiency and enclosed within a phospholipid membrane that is permeable to molecules below a molecular weight cutoff of ca. 4 kDa. The protocells are readily prepared by spontaneous assembly of a phospholipid membrane on the surface of preformed polynucleotide/polysaccharide coacervate microdroplets and are designated as giant coacervate vesicles (GCVs). Partial anchoring of the GCV membrane to the underlying coacervate phase results in increased robustness, lower membrane fluidity, and increased permeability compared with GUV counterparts. As a consequence, enzyme and ribozyme catalysis can be triggered in the molecularly crowded interior of the GCV but not inside the GUVs when small molecule substrates or inducers are present in the external environment. By integrating processes of membrane-mediated compartmentalization and liquid-liquid microphase separation, GCVs could offer substantial advantages as cytomimetic models, synthetic protocells, and artificial biomolecular microreactors.

Photoinduced Decarboxylative Radical Addition Reactions for Late Stage Functionalization of Peptide Substrates

AbstractPhotoredox chemistry has greatly stimulated the application of radical based transformations in medicinal chemistry and early drug discovery in recent years. Carboxylate groups have been identified as traceless leaving groups that can be converted into radical intermediates capable of undergoing 1,4‐conjugate addition reactions to Michael acceptors. Herein, we show the successful C‐terminal derivatization of small peptide substrates by using this methodology in a parallel synthesis setting. Finally, we outline a general strategy for the γ‐homologation of several drugs derived from α‐amino acids in a late stage functionalization (LSF) approach.

Nanoparticles deposition patterns in evaporating nanofluid droplets on smooth heated hydrophilic substrates: A 2D immersed boundary-lattice Boltzmann simulation

Nanoparticles deposition dynamics and deposition patterns in evaporating nanofluid droplets on a smooth hydrophilic heated substrate in a vapor environment (0.9Tc) under zero gravity are investigated numerically by a 2D immersed boundary-lattice Boltzmann method in combination of the non-isothermal Gong-Cheng liquid-vapor phase change model. The Marangoni flow along droplet interface is found to be the dominated transportation mechanism of nanoparticles inside an evaporating nanofluid droplet when droplet contact line is freely moving. It is demonstrated that the pinning of the triple phase contact line on the substrate having a small contact angle (θ=51°) is a required condition for a ring-like deposition pattern after a nanofluid droplet evaporation. On the other hand, on a substrate with a larger contact angle (θ=74°) where the triple contact line is not pinned, the upward Marangoni flow along droplet interface has a greater strength to resist nanoparticles moving toward the edge of the heated substrate, which result in bump-like deposition patterns after a nanofluid droplet evaporation. A regime map, showing effects of substrate contact angle and substrate temperature on nanoparticle deposition patterns after droplet evaporation, is presented. It is shown that the ring-like deposition pattern occurs when the triple line is strongly pinned (at small contact angle) and the Marangoni flow is weak (small contact angle and low substrate temperature). The bump deposition pattern occurs when the droplet contact line is not pinned (at high contact angle) and the Marangoni flow is strong (large contact angle and high temperature).

Modulators of hERAP2 discovered by high-throughput screening.

Endoplasmic reticulum aminopeptidase 2, ERAP2, is an emerging pharmacological target in cancer immunotherapy and control of autoinflammatory diseases, as it is involved in antigen processing. It has been linked to the risk of development of spondyloarthritis, and it associates with the immune infiltration of tumours and strongly predicts the overall survival for patients receiving check-point inhibitor therapy. While some selective inhibitors of its homolog ERAP1 are available, no selective modulator of ERAP2 has been disclosed so far. In order to identify such compounds, we screened an in-house focused library of 1920 compounds designed to target metalloenzymes. Structure-Activity Relationships and docking around two hits led to the discovery of selective inhibitors of ERAP2. Amid those, some bind to yet untapped amino-acids in the S1 pocket. Importantly, we disclose also the first activator of small substrates hydrolysis by ERAP2. Inhibitors and activators identified in this study could serve as useful starting points for optimization.