Combinatorial Screening Approach Research Articles

Interfacial Resistive Switching of Niobium-Titanium Anodic Memristors with Self-Rectifying Capabilities.

A broad compositional range of Nb-Ti anodic memristors with volatile and self-rectifying behaviour was studied using a combinatorial screening approach. A Nb-Ti thin-film combinatorial library was co-deposited by sputtering, serving as the bottom electrode for the memristive devices. The library, with a compositional spread ranging between 22 and 64 at.% Ti was anodically oxidised, the mixed oxide being the active layer in MIM-type structures completed by Pt discreet top electrode patterning. By studying I-U sweeps, memristors with self-rectifying and volatile behaviour were identified. Moreover, all the analysed memristors demonstrated multilevel properties. The best-performing memristors showed HRS/LRS (high resistive state/low resistive state) ratios between 4 and 6 × 105 and very good retention up to 106 successive readings. The anodic memristors grown along the compositional spread showed very good endurance up to 106 switching cycles, excluding those grown from alloys containing between 31 and 39 at.% Ti, which withstood only 10 switching cycles. Taking into consideration all the parameters studied, the Nb-46 at.% Ti composition was screened as the parent metal alloy composition, leading to the best-performing anodic memristor in this alloy system. The results obtained suggest that memristive behaviour is based on an interfacial non-filamentary type of resistive switching, which is consistent with the performed cross-sectional TEM structural and chemical characterisation.

De novo development of small cyclic peptides that are orally bioavailable

Cyclic peptides can bind challenging disease targets with high affinity and specificity, offering enormous opportunities for addressing unmet medical needs. However, as with biological drugs, most cyclic peptides cannot be applied orally because they are rapidly digested and/or display low absorption in the gastrointestinal tract, hampering their development as therapeutics. In this study, we developed a combinatorial synthesis and screening approach based on sequential cyclization and one-pot peptide acylation and screening, with the possibility of simultaneously interrogating activity and permeability. In a proof of concept, we synthesized a library of 8,448 cyclic peptides and screened them against the disease target thrombin. Our workflow allowed multiple iterative cycles of library synthesis and yielded cyclic peptides with nanomolar affinities, high stabilities and an oral bioavailability (%F) as high as 18% in rats. This method for generating orally available peptides is general and provides a promising push toward unlocking the full potential of peptides as therapeutics.

Solid-Phase Screening and Synthesis of Molecularly Imprinted Nanoparticles for Selective Recognition and Detection of Brain Natriuretic Peptide.

A new recognition method is explored for the rapid detection of B-type natriuretic peptide (BNP) based on the rational design and solid-phase synthesis of molecularly imprinted nanoparticles (nanoMIP) encapsulated with carbon dots. The nanosized magnetic template is first prepared by attaching the epitope of BNP on amino-functionalized magnetic carriers. High-dilution polymerization of monomers in the presence of magnetic template generates lightly crosslinked imprinted nanoparticles. To obtain the optimal MIP formulation, a new combinatorial screening approach is developed by a competitive fluorescence assay using the magnetic template. The resultant nanoMIP exhibits high affinity and selectivity toward BNP with an equilibrium dissociation constant (KD ) of ≈10-11 m. The proposed assay allows fast BNP detection within ≈7min with a linear range of its concentration from 0.25 to 5000 pg mL-1 and a limit of detection of 0.208 pg mL-1 (S/N = 3). To demonstrate its practicability in clinical diagnosis, unknown real serum samples from 160 individuals are analyzed and the relative standard deviation is less than 4.43%. Compared with the routine electrochemiluminescence detection method that is widely used in hospital, the relative error is less than 4.98% and the correlation coefficient is 0.994.

Coexistence of memory and threshold resistive switching identified by combinatorial screening in niobium-tantalum system

Memristive devices exhibiting improved performance and memory characteristics are revealed by using a combinatorial screening approach. An Nb-Ta thin film library, with a total compositional spread ranging between 13 and 80 at.% Ta, is co-sputtered as the bottom electrode, while their anodic oxides served as active layers. Such metal–insulator-metal (MIM) specimens under investigation are finalized with patterned Pt top electrodes. Memristors based on mixed Nb2O5-Ta2O5 have been grouped according to their common switching characteristics in four different compositional zones. Reversible switching between threshold and non-volatile mode was observed for devices grown on Nb-39 at.% Ta alloy. The switching mode was dictated by applying different current compliances. Moreover, devices based on Nb-36 at.% Ta switch at minimum seven distinct resistive levels. Apart from the electrical measurements, the structure of samples and components distribution in cross-section geometry is studied via TEM techniques before and after electrical treatment. Electrolyte incorporation and the exact composition of the identified high-performance memristors (resulting from the combinatorial screening) are analyzed by HAXPES. The memristive devices based on the two singled-out Nb-Ta alloys show enhanced memory and electrical characteristics, thus being excellent candidates for neuromorphic applications.

Synthetic gene circuits for cell state detection and protein tuning in human pluripotent stem cells.

During development, cell state transitions are coordinated through changes in the identity of molecular regulators in a cell type- and dose-specific manner. The ability to rationally engineer such transitions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein, we report the generation of synthetic gene circuits that can detect a desired cell state using AND-like logic integration of endogenous miRNAs (classifiers) and, upon detection, produce fine-tuned levels of output proteins using an miRNA-mediated output fine-tuning technology (miSFITs). Specifically, we created an "hPSC ON" circuit using a model-guided miRNA selection and circuit optimization approach. The circuit demonstrates robust PSC-specific detection and graded output protein production. Next, we used an empirical approach to create an "hPSC-Off" circuit. This circuit was applied to regulate the secretion of endogenous BMP4 in a state-specific and fine-tuned manner to control the composition of differentiating hPSCs. Our work provides a platform for customized cell state-specific control of desired physiological factors in hPSC, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond.

Targeting fungal membrane homeostasis with imidazopyrazoindoles impairs azole resistance and biofilm formation

Fungal infections cause more than 1.5 million deaths annually. With an increase in immune-deficient susceptible populations and the emergence of antifungal drug resistance, there is an urgent need for novel strategies to combat these life-threatening infections. Here, we use a combinatorial screening approach to identify an imidazopyrazoindole, NPD827, that synergizes with fluconazole against azole-sensitive and -resistant isolates of Candida albicans. NPD827 interacts with sterols, resulting in profound effects on fungal membrane homeostasis and induction of membrane-associated stress responses. The compound impairs virulence in a Caenorhabditis elegans model of candidiasis, blocks C. albicans filamentation in vitro, and prevents biofilm formation in a rat model of catheter infection by C. albicans. Collectively, this work identifies an imidazopyrazoindole scaffold with a non-protein-targeted mode of action that re-sensitizes the leading human fungal pathogen, C. albicans, to azole antifungals.

Discovery of Thieno[2,3-d]pyrimidine-based KRAS G12D inhibitors as potential anticancer agents via combinatorial virtual screening

A series of novel thieno[2,3-d]pyrimidine analogs were designed and synthesized as KRAS G12D inhibitors via combinatorial virtual screening approach. Most compounds exhibited potent antiproliferative activity on KRAS G12D mutated cancer cell lines (Panc1, SW1990 and CT26) with IC50 values in the low micromolar range. Among them, compound KD-8 showed the highest antiproliferative activity with an average IC50 of 2.1μM against three KRAS G12D-mutated cells (Panc1, SW1990 and CT26). KD-8 decreased the active form of KRAS (KRAS-GTP) in KRAS G12D mutated cancer cell lines (CT26 and SW1990) but not in KRAS G13D mutated cancer cells (HCT116). Moreover, KD-8 down-regulated the phosphorylated Raf and Erk in CT26 and SW1990 cancer cell lines but not in HeLa cells (KRAS WT). The binding affinity of KD-8 was further confirmed by the isothermal titration calorimetry (ITC) assay in which KD-8 exhibited a KD of 33nM for binding to KRAS G12D protein. In addition, KD-8 (40mg/kg or 60mg/kg) exhibited significant antitumor efficacy in a CT26 tumor model with a tumor growth inhibition (TGI) of 42% or 53% without causing apparent toxicity. Taken together the above results suggest that KD-8 is a promising KRAS G12D inhibitor deserving further investigation.

Assessing site-specific PEGylation of TEM-1 β-lactamase with cell-free protein synthesis and coarse-grained simulation

PEGylation is a broadly used strategy to enhance the pharmacokinetic properties of therapeutic proteins. It is well established that the location and extent of PEGylation have a significant impact on protein properties. However, conventional PEGylation techniques have limited control over PEGylation sites. Emerging site-specific PEGylation technology provides control of PEG placement by conjugating PEG polymers via click chemistry reaction to genetically encoded non-canonical amino acids. Unfortunately, a method to rapidly determine the optimal PEGylation location has yet to be established. Here we seek to address this challenge. In this work, coarse-grained molecular dynamic simulations are paired with high-throughput experimental screening utilizing cell-free protein synthesis to investigate the effect of site-specific PEGylation on the two-state folder protein TEM-1 β-lactamase. Specifically, the conjugation efficiency, thermal stability, and enzymatic activity are studied for the enzyme PEGylated at several different locations. The results of this analysis confirm that the physical properties of the PEGylated protein vary considerably with PEGylation site and that traditional design recommendations are insufficient to predict favorable PEGylation sites. In this study, the best predictor of the most favorable conjugation site is coarse-grained simulation. Thus, we propose a dual combinatorial screening approach in which coarse-grained molecular simulation informs site selection for high-throughput experimental verification.

Combinatorial screening of ionic liquid extractant for removal of methanol from methylal

A combinatorial screening approach combining quantum chemical (QC) calculation and molecular dynamics (MD) simulation was presented for the selection of ionic liquid (IL) extractant to remove methanol from methylal. The thermodynamic properties of the distributional coefficient and selectivity were calculated by COSMO-RS based on QC calculation to qualitatively narrow down the IL candidates. The separation performance of these IL candidates was further evaluated through MD to determine the best IL extractant. Then, the methanol removal performance of the best IL was further evaluated by the liquid – liquid equilibrium experiments and the process simulation. Finally, the separation mechanism of the IL was investigated through QC calculations, wavefunction and MD analysis. The consistency between simulations and experiments indicates that this approach is a very useful tool to screen ILs for specific separation tasks.

Mixed anodic oxides for forming-free memristors revealed by combinatorial screening of hafnium-tantalum system

A combinatorial screening approach has been designed for identification of Hf-Ta anodic memristors with enhanced properties. Three distinct compositional zones with similar characteristics have been identified, two of which are relevant for memristive applications. In a Hf-poor zone, (< 50 at.% Hf), the memristive behavior is similar to that of anodic Ta2O5. A second compositional zone (Hf content between 50 and 70 at.%) produced anodic memristors which dramatically differ. Devices in this region resemble forming-free unipolar behavior and show resistive states ratios of more than 8 orders of magnitude while retaining and enduring more than one million switching cycles. Photoelectron spectroscopy with soft and hard X-rays has been applied for understanding the chemistry of these special anodic structures and enrichment of Hf species close to the surface of anodic oxides has been evidenced. Unlike bipolar memristors from the first zone, the newly identified unipolar memristors show an in-depth homogeneous local chemical state of Hf and lower electronic polarizabilities for both O and Hf. High-resolution transmission electron microscopy reveals HfO2 crystallites embedded into an amorphous oxide matrix. These entities formed during the anodization process are considered to be responsible for the coexistence of threshold and non-volatile memristive characteristics.

A Peptidomimetic Ligand Targeting the Chromodomain of MPP8 Reveals HRP2's Association with the HUSH Complex.

The interpretation of histone post-translational modifications (PTMs), specifically lysine methylation, by specific classes of "reader" proteins marks an important aspect of epigenetic control of gene expression. Methyl-lysine (Kme) readers often regulate gene expression patterns through the recognition of a specific Kme PTM while participating in or recruiting large protein complexes that contain enzymatic or chromatin remodeling activity. Understanding the composition of these Kme-reader-containing protein complexes can serve to further our understanding of the biological roles of Kme readers, while small molecule chemical tools can be valuable reagents in interrogating novel protein-protein interactions. Here, we describe our efforts to target the chromodomain of M-phase phosphoprotein 8 (MPP8), a member of the human silencing hub (HUSH) complex and a histone 3 lysine 9 trimethyl (H3K9me3) reader that is vital for heterochromatin formation and has specific roles in cancer metastasis. Utilizing a one-bead, one-compound (OBOC) combinatorial screening approach, we identified UNC5246, a peptidomimetic ligand capable of interacting with the MPP8 chromodomain in the context of the HUSH complex. Additionally, a biotinylated derivative of UNC5246 facilitated chemoproteomics studies which revealed hepatoma-derived growth factor-related protein 2 (HRP2) as a novel protein associated with MPP8. HRP2 was further shown to colocalize with MPP8 at the E-cadherin gene locus, suggesting a possible role in cancer cell plasticity.

Functional Analysis of Non-Genetic Resistance to Platinum in Epithelial Ovarian Cancer Reveals a Role for the MBD3-NuRD Complex in Resistance Development.

Simple SummaryMost epithelial ovarian cancer (EOC) patients, although initially responsive to standard treatment with platinum-based chemotherapy, develop platinum resistance over the clinical course and succumb due to drug-resistant metastases. It has long been hypothesized that resistance to platinum develops as a result of epigenetic changes within tumor cells evolving over time. In this study, we investigated epigenomic changes in EOC patient samples, as well as in cell lines, and showed that profound changes at enhancers result in a platinum-resistant phenotype. Through correlation of the epigenomic alterations with changes in the transcriptome, we could identify potential novel prognostic biomarkers for early patient stratification. Furthermore, we applied a combinatorial RNAi screening approach to identify suitable targets that prevent the enhancer remodeling process. Our results advance the molecular understanding of epigenetic mechanisms in EOC and therapy resistance, which will be essential for the further exploration of epigenetic drug targets and combinatorial treatment regimes.Epithelial ovarian cancer (EOC) is the most lethal disease of the female reproductive tract, and although most patients respond to the initial treatment with platinum (cPt)-based compounds, relapse is very common. We investigated the role of epigenetic changes in cPt-sensitive and -resistant EOC cell lines and found distinct differences in their enhancer landscape. Clinical data revealed that two genes (JAK1 and FGF10), which gained large enhancer clusters in resistant EOC cell lines, could provide novel biomarkers for early patient stratification with statistical independence for JAK1. To modulate the enhancer remodeling process and prevent the acquisition of cPt resistance in EOC cells, we performed a chromatin-focused RNAi screen in the presence of cPt. We identified subunits of the Nucleosome Remodeling and Deacetylase (NuRD) complex as critical factors sensitizing the EOC cell line A2780 to platinum treatment. Suppression of the Methyl-CpG Binding Domain Protein 3 (MBD3) sensitized cells and prevented the establishment of resistance under prolonged cPt exposure through alterations of H3K27ac at enhancer regions, which are differentially regulated in cPt-resistant cells, leading to a less aggressive phenotype. Our work establishes JAK1 as an independent prognostic marker and the NuRD complex as a potential target for combinational therapy.

Combinatorial Virtual Library Screening Study of Transforming Growth Factor-β2-Chondroitin Sulfate System.

Transforming growth factor-beta (TGF-β), a member of the TGF-β cytokine superfamily, is known to bind to sulfated glycosaminoglycans (GAGs), but the nature of this interaction remains unclear. In a recent study, we found that preterm human milk TGF-β2 is sequestered by chondroitin sulfate (CS) in its proteoglycan form. To understand the molecular basis of the TGF-β2–CS interaction, we utilized the computational combinatorial virtual library screening (CVLS) approach in tandem with molecular dynamics (MD) simulations. All possible CS oligosaccharides were generated in a combinatorial manner to give 24 di- (CS02), 192 tetra- (CS04), and 1536 hexa- (CS06) saccharides. This library of 1752 CS oligosaccharides was first screened against TGF-β2 using the dual filter CVLS algorithm in which the GOLDScore and root-mean-square-difference (RMSD) between the best bound poses were used as surrogate markers for in silico affinity and in silico specificity. CVLS predicted that both the chain length and level of sulfation are critical for the high affinity and high specificity recognition of TGF-β2. Interestingly, CVLS led to identification of two distinct sites of GAG binding on TGF-β2. CVLS also deduced the preferred composition of the high specificity hexasaccharides, which were further assessed in all-atom explicit solvent MD simulations. The MD results confirmed that both sites of binding form stable GAG–protein complexes. More specifically, the highly selective CS chains were found to engage the TGF-β2 monomer with high affinity. Overall, this work present key principles of recognition with regard to the TGF-β2–CS system. In the process, it led to the generation of the in silico library of all possible CS oligosaccharides, which can be used for advanced studies on other protein–CS systems. Finally, the study led to the identification of unique CS sequences that are predicted to selectively recognize TGF-β2 and may out-compete common natural CS biopolymers.

Drug Screening Reveals That Ibrutinib (PCI-32765) Exhibits Synergy With BCL-2 and Proteasome Inhibitors In Models Of Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL)

BackgroundIbrutinib, a Bruton's Tyrosine Kinase inhibitor, is clinically effective in relapsed MCL and CLL, but responses are often partial and resistance eventually develops. To identify agents that synergize with ibrutinib, we performed an in vitro combinatorial drug screen and evaluated the most promising combinations in additional models. MethodsDrug screens were performed in JVM2 and Z138 MCL cell lines, combining ibrutinib with a diverse panel of secondary agents inhibiting targets within the B-cell receptor (BCR) pathway (SC514, PD325901, enzastaurin, R788, temsirolimus, dasatinib, idelalisib) and agents inhibiting targets outside the BCR pathway (ABT-199, bendamustine, carfilzomib, bortezomib, geldanamycin, panobinostat, PD0332991). Three doses (IC10, IC20, IC 30) for each drug were chosen and combined in a 3x3 format. Bliss Model of Additivity was used to quantitate synergistic cytotoxicity. Levels of cleaved PARP or Annexin V/PI staining were assessed for cell lines exposed to ibrutinib and synergistic agents, including bortezomib, carfilzomib, and ABT-199. The Ibrutinib + carfilzomib combination was further tested in MCL cell lines Rec1 and Jeko, in primary MCL and CLL patient (pt) samples, and in a MCL-SCID-hu mouse. Molecular analysis of the mechanistic interactions of the agents has been initiated using forward-phase antibody arrays (R and D Systems). ResultsInhibitors of targets within the BCR pathway were ineffective at potentiating the effects of ibrutinib (magnitude of synergy ranging -12%-9% in Z138, -25%-8% in JVM2). However, substantial synergistic cytotoxicity was seen with some agents that target outside the BCR pathway (magnitude of synergy ranging -5%-37% in Z138, -17%-48% in JVM2). Notably, we observed robust and reproducible synergistic cytotoxicity by combining ibrutinib with proteasome inhibitors carfilzomib and bortezomib as well as BCL-2 inhibitor ABT-199.The degree of synergyof ibrutinib + ABT-199 in MCL cell lines was durable across all 9 dosing combinations, (23-32% in Z-138, 10-26% in JVM-2). In CLL pt samples, ibrutinib + ABT-199 conferred modest synergy (10-20%) and near-complete cytotoxicity (61-70%). Flow cytometry for cleaved PARP demonstrated a high degree of apoptosis when combining ibrutinib with ABT-199 (94.5% in Z-138, 82.1% in Z-138).At optimal dosing, bortezomib conferred 19% synergy with ibrutinib in Z138 and 27% in JVM2. The best synergistic effect of carfilzomib and ibrutinib was 50% in Z138 and 65% in JVM2. The synergistic effect between carfilzomib and ibrutinib was maintained in Rec1 and Jeko MCL cell lines and in primary MCL pt samples. In CLL pt samples, ibrutinib + proteasome inhibition also conferred high degrees of total cytotoxicity (58-71% for carfilzomib, 42-62% for bortezomib). Flow cytometry for cleaved PARP or Annexin V/PI demonstrated increased apoptosis for ibrutinib +carfilzomib (92.3% in Z138, 61.2% in JVM2, 95% in Rec1, 90% in Jeko). The combination treatment increased the percent survival of MCL-SCID-hu mice in three-fold over single agent treatment in a 125-day trial.Preliminary analysis of proteomic changes induced by ibrutinib+ carfilzomib indicates synergistic alterations in cell cycle proteins and in apoptosis-regulating proteins, although the specific alterations differ between Z138 and JVM2 cell lines. DiscussionWhile agents within the BCR pathway did not display synergistic cytotoxicity with ibrutinib in our drug screen, several agents outside the BCR pathway did synergize with ibrutinib, notably ABT-199, bortezomib, and carfilzomib in two MCL cell lines. For all three drug combinations, this effect appeared to be mediated via apoptosis. The synergistic effect of ibrutinib + carfilzomib was further studied in 2 additional MCL cell lines, 3 MCL pt samples, and CLL pt samples. MCL-SCID-hu mice demonstrated three-fold increased survival when treated in combination over animals treated with either agent alone. The identification of unexpected synergistic cytotoxic interactions between inhibitors of apparently unrelated pathways is a benefit of our combinatorial screening approach, although the molecular mechanisms remain to be elucidated. The combination of ibrutinib with either proteasome or BCL-2 inhibition is proposed as promising novel treatments for MCL and CLL. A phase I/II clinical trial has been designed to test ibrutinib + carfilzomib in relapsed or refractory MCL and CLL. Disclosures:Williams:Celgene: Consultancy, Independent Data Safety Committee member Other, Research Funding; Genentech: Consultancy, Research Funding; Janssen: Research Funding; Millennium: Consultancy, Independent Data Safety Committee member, Independent Data Safety Committee member Other, Research Funding; Pharmacyclics: Consultancy, Research Funding; Onyx: Research Funding; Gilead: Research Funding. Wang:Onyx: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Millennium: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Novartis: Research Funding.

A multi-omic screening approach for the discovery of thermoactive glycoside hydrolases.

Next-generation sequencing and computational biology have facilitated the implementation of new combinatorial screening approaches to discover novel enzymes of biotechnological interest. In this study, we describe the successful establishment of a multi-omic approach for the identification of thermostable hydrolase-encoding genes by determination of gene expression levels. We applied this combinatorial approach using an anaerobic enrichment culture from an Azorean hot spring sample grown on green coffee beans as recalcitrant substrate. An in-depth analysis of the microbial community resulted in microorganisms capable of metabolizing the selected substrate, such as the genera Caloramator,Dictyoglomus and Thermoanaerobacter as active and abundant microorganisms. To discover glycoside hydrolases, 90,342 annotated genes were screened for specific reaction types. A total number of 106 genes encoding cellulases (EC 3.2.1.4), beta-glucosidases (EC 3.2.1.21) and endo-1,4-beta-mannosidases (EC 3.2.1.78) were selected. Mapping of RNA-Seq reads to the related metagenome led to expression levels for each gene. Amongst those, 14 genes, encoding glycoside hydrolases, showed highest expression values, and were used for further cloning. Four proteins were biochemically characterized and were identified as thermoactive glycoside hydrolases with a broad substrate range. This work demonstrated that a combinatory omic approach is a suitable strategy identifying unique thermoactive enzymes from environmental samples.

An oxindole efflux inhibitor potentiates azoles and impairs virulence in the fungal pathogen Candida auris

Candida auris is an emerging fungal pathogen that exhibits resistance to multiple drugs, including the most commonly prescribed antifungal, fluconazole. Here, we use a combinatorial screening approach to identify a bis-benzodioxolylindolinone (azoffluxin) that synergizes with fluconazole against C. auris. Azoffluxin enhances fluconazole activity through the inhibition of efflux pump Cdr1, thus increasing intracellular fluconazole levels. This activity is conserved across most C. auris clades, with the exception of clade III. Azoffluxin also inhibits efflux in highly azole-resistant strains of Candida albicans, another human fungal pathogen, increasing their susceptibility to fluconazole. Furthermore, azoffluxin enhances fluconazole activity in mice infected with C. auris, reducing fungal burden. Our findings suggest that pharmacologically targeting Cdr1 in combination with azoles may be an effective strategy to control infection caused by azole-resistant isolates of C. auris.

Structure-guided screening strategy combining surface plasmon resonance with nuclear magnetic resonance for identification of small-molecule Argonaute 2 inhibitors.

Argonaute (AGO) proteins are the key component of the RNA interference machinery that suppresses gene expression by forming an RNA-induced silencing complex (RISC) with microRNAs (miRNAs). Each miRNA is involved in various cellular processes, such as development, differentiation, tumorigenesis, and viral infection. Thus, molecules that regulate miRNA function are expected to have therapeutic potential. In addition, the biogenesis of miRNA is a multistep process involving various proteins, although the complete pathway remains to be elucidated. Therefore, identification of molecules that can specifically modulate each step will help understand the mechanism of gene suppression. To date, several AGO2 inhibitors have been identified. However, these molecules were identified through a single screening method, and no studies have specifically evaluated a combinatorial strategy. Here, we demonstrated a combinatorial screening (SCR) approach comprising an in silico molecular docking study, surface plasmon resonance (SPR) analysis, and nuclear magnetic resonance (NMR) analysis, focusing on the strong binding between the 5'-terminal phosphate of RNA and the AGO2 middle (MID) domain. By combining SPR and NMR, we identified binding modes of amino acid residues binding to AGO2. First, using a large chemical library (over 6,000,000 compounds), 171 compounds with acidic functional groups were screened using in silico SCR. Next, we constructed an SPR inhibition system that could analyze only the 5'-terminal binding site of RNA, and nine molecules that strongly bound to the AGO2 MID domain were selected. Finally, using NMR, three molecules that bound to the desired site were identified. The RISC inhibitory ability of the “hit” compounds was analyzed in human cell lysate, and all three hit compounds strongly inhibited the binding between double-stranded RNA and AGO2.

High‐Throughput Characterization of Perovskite Solar Cells for Rapid Combinatorial Screening

To discover the ideal perovskite material for solar cell application, a large parameter space (composition, surrounding condition, fabrication technique, etc.) must first be explored. Therefore, screening this parameter space using a rapid combinatorial screening approach can drastically speed up the rate of discovery. During the last decade, these discoveries and optimization processes of perovskite materials have been achieved using simple lab‐scale deposition techniques and characterization methods, resulting in a substantial time‐consuming process, slowing the rate of progress in the field of photovoltaics. Thus, the benefits of developing fully automated, high‐throughput characterization techniques become apparent. Herein, a high‐throughput solar cell testing system that enables parallel, real‐time, and comprehensive measurements is detailed, allowing for 16 solar cells to be characterized simultaneously. The importance of measurement reproducibility, condition verification, and structured data postprocessing is shown.

Simultaneous Control of Endogenous and User-Defined Genetic Pathways Using Unique ecDHFR Pharmacological Chaperones

Destabilizing domains (DDs), such as a mutated formof Escherichia coli dihydrofolate reductase (ecDHFR), confer instability and promote protein degradation. However, when combined with small-molecule stabilizers (e.g., the antibiotic trimethoprim), DDs allow positive regulation of fusion proteinabundance. Using a combinatorial screening approach, we identified and validated 17unique 2,4-diaminopyrimidine/triazine-based ecDHFR DD stabilizers, at least 15 of which were ineffective antibiotics against E. coli and S. aureus. Identified stabilizers functioned invivo to control an ecDHFR DD-firefly luciferase in the mouse eye and/or the liver. Next, stabilizers were leveraged to perform synergistic dual functions invitro (HeLa cell death sensitization) and invivo (repression of ocular inflammation) by stabilizing a user-defined ecDHFR DD while also controlling endogenous signaling pathways. Thus, these newly identified pharmacological chaperones allow for simultaneous control of compound-specific endogenous and user-defined genetic pathways, the combination of which may provide synergistic effects in complex biological scenarios.

A Combinatorial Virtual Screening Approach Driving the Synthesis of 2,4-Thiazolidinedione-Based Molecules as New Dual mPGES-1/5-LO Inhibitors.

Dual inhibition of microsomal prostaglandin E2 synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO), two key enzymes involved in pro-inflammatory eicosanoid biosynthesis, represents a new strategy for treating inflammatory disorders. Herein we report the discovery of 2,4-thiazolidinedione-based mPGES-1/5-LO dual inhibitors following a multidisciplinary protocol, involving virtual combinatorial screening, chemical synthesis, and validation of the biological activities for the selected compounds. Following the multicomponent-based chemical route for the decoration of the 2,4-thiazolidinedione core, a large library of virtual compounds was built (∼2.0×104 items) and submitted to virtual screening. Nine selected molecules were synthesized and biologically evaluated, disclosing among them four compounds able to reduce the activity of both enzymes in the mid- and low- micromolar range of activities. These results are of interest for further expanding the chemical diversity around the 2,4-thiazolidinedione central core, facilitating the identification of novel anti-inflammatory agents endowed with a promising and safer pharmacological profile.