Combinatorial Format Research Articles

Late-Stage Functionalisation of Peptides on the Solid Phase by an Iodination-Substitution Approach.

The functionalisation of peptides at a late synthesis stage holds great potential, for example, for the synthesis of peptide pharmaceuticals, fluorescent biosensors or peptidomimetics. Here we describe an on‐resin iodination‐substitution reaction sequence on homoserine that is also suitable for peptide modification in a combinatorial format. The reaction sequence is accessible to a wide range of sulfur nucleophiles with various functional groups including boronic acids, hydroxy groups or aromatic amines. In this way, methionine‐like thioethers or thioesters and thiosulfonates are accessible. Next to sulfur nucleophiles, selenols, pyridines and carboxylic acids were successfully used as nucleophiles, whereas phenols did not react. The late‐stage iodination‐substitution approach is not only applicable to short peptides but also to the more complex 34‐amino‐acid WW domains. We applied this strategy to introduce 7‐mercapto‐4‐methylcoumarin into a switchable ZnII responsive WW domain to design an iFRET‐based ZnII sensor.

Palbociclib-based high-throughput combination drug screening identifies synergistic therapeutic options in HPV-negative head and neck squamous cell carcinoma

BackgroundDeregulation of cell-cycle pathway is ubiquitously observed in human papillomavirus negative (HPVneg) head and neck squamous cell carcinoma (HNSCC). Despite being an attractive target, CDK4/6 inhibition using palbociclib showed modest or conflicting results as monotherapy or in combination with platinum-based chemotherapy or cetuximab in HPVneg HNSCC. Thus, innovative agents to augment the efficacy of palbociclib in HPVneg HNSCC would be welcomed.MethodsA collection of 162 FDA-approved and investigational agents was screened in combinatorial matrix format, and top combinations were validated in a broader panel of HPVneg HNSCC cell lines. Transcriptional profiling was conducted to explore the molecular mechanisms of drug synergy. Finally, the most potent palbociclib-based drug combination was evaluated and compared with palbociclib plus cetuximab or cisplatin in a panel of genetically diverse HPVneg HNSCC cell lines and patient-derived xenograft models.ResultsPalbociclib displayed limited efficacy in HPVneg HNSCC as monotherapy. The high-throughput combination drug screening provided a comprehensive palbociclib-based drug-drug interaction dataset, whereas significant synergistic effects were observed when palbociclib was combined with multiple agents, including inhibitors of the PI3K, EGFR, and MEK pathways. PI3K pathway inhibitors significantly reduced cell proliferation and induced cell-cycle arrest in HPVneg HNSCC cell lines when combined with palbociclib, and alpelisib (a PI3Kα inhibitor) was demonstrated to show the most potent synergy with particularly higher efficacy in HNSCCs bearing PIK3CA alterations. Notably, when compared with cisplatin and cetuximab, alpelisib exerted stronger synergism in a broader panel of cell lines. Mechanistically, RRM2-dependent epithelial mesenchymal transition (EMT) induced by palbociclib, was attenuated by alpelisib and cetuximab rather than cisplatin. Subsequently, PDX models with distinct genetic background further validated that palbociclib plus alpelisib had significant synergistic effects in models harboring PIK3CA amplification.ConclusionsThis study provides insights into the systematic combinatory effect associated with CDK4/6 inhibition and supports further initiation of clinical trials using the palbociclib plus alpelisib combination in HPVneg HNSCC with PIK3CA alterations.

Isoquinolone-4-Carboxylic Acids by Ammonia-Ugi-4CR and Copper-Catalyzed Domino Reaction.

Highly substituted isoquinolone-4-carboxylic acid is an important bioactive scaffold; however, it is challenging to access it in a general and short way. A Cu-catalyzed cascade reaction was successfully designed involving the Ugi postcyclization strategy by using ammonia and 2-halobenzoic acids as crucial building blocks. Privileged polysubstituted isoquinolin-1(2H)-ones were constructed in a combinatorial format with generally moderate to good yields. The protocol, with a ligand-free catalytic system, shows a broad substrate scope and good functional group tolerance toward excellent molecular diversity. Free 4-carboxy-isoquinolone is now for the first time generally accessible by a convergent multicomponent reaction protocol.

The beauty of Dutch: Ex-post split-award auctions in procurement markets with diseconomies of scale

Abstract Ex-post split-award auctions are frequently organized as combinatorial procurement auctions in which the demand for some quantity to be procured is split into multiple shares and suppliers can bid on individual shares and the package. With two shares the auction selects either a single- or dual-source allocation. Markets with diseconomies of scale make the environment strategically challenging, because suppliers need to coordinate on a split outcome in the efficient equilibrium. We provide an equilibrium analysis of three combinatorial formats, namely, the first-price sealed-bid, the Dutch and the Dutch-FPSB split-award auction. We show that, unlike in single-object auctions, first-price sealed-bid and Dutch combinatorial auction formats are not strategically equivalent. While the FPSB auction exhibits a coordination problem for bidders, the Dutch formats have only efficient equilibria. The price information revealed during the Dutch auction formats helps bidders coordinate. In markets with only two bidders equilibria involving high pooling prices emerge in all three formats. However, no such strategy exists anymore when the number of bidders exceeds the number of shares. As a result, qualifying one more supplier has a significant impact as the expected procurement costs are reduced substantially.

Matrix Screen Identifies Synergistic Combination of PARP Inhibitors and Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors in Ewing Sarcoma.

Purpose: Although many cancers are showing remarkable responses to targeted therapies, pediatric sarcomas, including Ewing sarcoma, remain recalcitrant. To broaden the therapeutic landscape, we explored the in vitro response of Ewing sarcoma cell lines against a large collection of investigational and approved drugs to identify candidate combinations.Experimental Design: Drugs displaying activity as single agents were evaluated in combinatorial (matrix) format to identify highly active, synergistic drug combinations, and combinations were subsequently validated in multiple cell lines using various agents from each class. Comprehensive metabolomic and proteomic profiling was performed to better understand the mechanism underlying the synergy. Xenograft experiments were performed to determine efficacy and in vivo mechanism.Results: Several promising candidates emerged, including the combination of small-molecule PARP and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, a rational combination as NAMPTis block the rate-limiting enzyme in the production of nicotinamide adenine dinucleotide (NAD+), a necessary substrate of PARP. Mechanistic drivers of the synergistic cell killing phenotype of these combined drugs included depletion of NMN and NAD+, diminished PAR activity, increased DNA damage, and apoptosis. Combination PARPis and NAMPTis in vivo resulted in tumor regression, delayed disease progression, and increased survival.Conclusions: These studies highlight the potential of these drugs as a possible therapeutic option in treating patients with Ewing sarcoma. Clin Cancer Res; 23(23); 7301-11. ©2017 AACR.

Interferon-γ is a master checkpoint regulator of cytokine-induced differentiation

Cytokines are protein mediators that are known to be involved in many biological processes, including cell growth, survival, inflammation, and development. To study their regulation, we generated a library of 209 different cytokines. This was used in a combinatorial format to study the effects of cytokines on each other, with particular reference to the control of differentiation. This study showed that IFN-γ is a master checkpoint regulator for many cytokines. It operates via an autocrine mechanism to elevate STAT1 and induce internalization of gp130, a common component of many heterodimeric cytokine receptors. This targeting of a receptor subunit that is common to all members of an otherwise diverse family solves the problem of how a master regulator can control so many diverse receptors. When one adds an autocrine mechanism, fine control at the level of individual cells is achieved.

Speech Image in the System of Social Relations: About Certain Stages of Linguistic Image-making (as exemplified by Margaret Thatcher’s Oral Activity)

Objectives: Our task is to describe speech component of image structure on example of an established, conceptually concise image (Margaret Thatcher). Methods: Systematization and theoretical understanding of material. A descriptive method for creating biographical context of understanding Margaret Thatcher's career. Method of diagnostic context, denotative, emotive and structural analysis, semantic differential. Corpus-based and cross-lingual methods. Findings: The research of corpus of Margaret Thatcher's texts suggests that Thatcher's speech image is a combinatorial formation based both on process of personal potential exteriorization (the image of the Iron Lady), and verbal behavior program setup implementing communicative ideal of a mother. Applications/Improvements: Application of results includes linguo-pragmatics, political linguistics, sociolinguistics, discourse theory and practice.

Reprint of: Pendant allyl crosslinking as a tunable shape memory actuator for vascular applications

Thermo-responsive shape memory polymers (SMPs) can be programmed to fit into small-bore incisions and recover their functional shape upon deployment in the body. This property is of significant interest for developing the next generation of minimally-invasive medical devices. To be used in such applications, SMPs should exhibit adequate mechanical strengths that minimize adverse compliance mismatch-induced host responses (e.g. thrombosis, hyperplasia), be biodegradable, and demonstrate switch-like shape recovery near body temperature with favorable biocompatibility. Combinatorial approaches are essential in optimizing SMP material properties for a particular application. In this study, a new class of thermo-responsive SMPs with pendant, photocrosslinkable allyl groups, x%poly(ε-caprolactone)-co-y%(α-allyl carboxylate ε-caprolactone) (x%PCL-y%ACPCL), are created in a robust, facile manner with readily tunable material properties. Thermomechanical and shape memory properties can be drastically altered through subtle changes in allyl composition. Molecular weight and gel content can also be altered in this combinatorial format to fine-tune material properties. Materials exhibit highly elastic, switch-like shape recovery near 37°C. Endothelial compatibility is comparable to tissue culture polystyrene (TCPS) and 100%PCL in vitro and vascular compatibility is demonstrated in vivo in a murine model of hindlimb ischemia, indicating promising suitability for vascular applications. Statement of SignificanceWith the ongoing thrust to make surgeries minimally-invasive, it is prudent to develop new biomaterials that are highly compatible and effective in this workflow. Thermo-responsive shape memory polymers (SMPs) have great potential for minimally-invasive applications because SMP medical devices (e.g. stents, grafts) can fit into small-bore minimally-invasive surgical devices and recover their functional shape when deployed in the body. To realize their potential, it is imperative to devise combinatorial approaches that enable optimization of mechanical, SM, and cellular responses for a particular application. In this study, a new class of thermo-responsive SMPs is created in a robust, facile manner with readily tunable material properties. Materials exhibit excellent, switch-like shape recovery near body temperature and promising biocompatibility for minimally-invasive vascular applications.

Pendant allyl crosslinking as a tunable shape memory actuator for vascular applications

Thermo-responsive shape memory polymers (SMPs) can be programmed to fit into small-bore incisions and recover their functional shape upon deployment in the body. This property is of significant interest for developing the next generation of minimally-invasive medical devices. To be used in such applications, SMPs should exhibit adequate mechanical strengths that minimize adverse compliance mismatch-induced host responses (e.g. thrombosis, hyperplasia), be biodegradable, and demonstrate switch-like shape recovery near body temperature with favorable biocompatibility. Combinatorial approaches are essential in optimizing SMP material properties for a particular application. In this study, a new class of thermo-responsive SMPs with pendant, photocrosslinkable allyl groups, x%poly(ε-caprolactone)-co-y%(α-allyl carboxylate ε-caprolactone) (x%PCL-y%ACPCL), are created in a robust, facile manner with readily tunable material properties. Thermomechanical and shape memory properties can be drastically altered through subtle changes in allyl composition. Molecular weight and gel content can also be altered in this combinatorial format to fine-tune material properties. Materials exhibit highly elastic, switch-like shape recovery near 37°C. Endothelial compatibility is comparable to tissue culture polystyrene (TCPS) and 100%PCL in vitro and vascular compatibility is demonstrated in vivo in a murine model of hindlimb ischemia, indicating promising suitability for vascular applications. Statement of SignificanceWith the ongoing thrust to make surgeries minimally-invasive, it is prudent to develop new biomaterials that are highly compatible and effective in this workflow. Thermo-responsive shape memory polymers (SMPs) have great potential for minimally-invasive applications because SMP medical devices (e.g. stents, grafts) can fit into small-bore minimally-invasive surgical devices and recover their functional shape when deployed in the body. To realize their potential, it is imperative to devise combinatorial approaches that enable optimization of mechanical, SM, and cellular responses for a particular application. In this study, a new class of thermo-responsive SMPs is created in a robust, facile manner with readily tunable material properties. Materials exhibit excellent, switch-like shape recovery near body temperature and promising biocompatibility for minimally-invasive vascular applications.

Combinatorial Generation of Complexity by Redox Enzymes in the Chaetoglobosin A Biosynthesis

Redox enzymes play a central role in generating structural complexity during natural product biosynthesis. In the postassembly tailoring steps, redox cascades can transform nascent chemical scaffolds into structurally complex final products. Chaetoglobosin A (1) is biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase. It belongs to the chaetoglobosin family of natural products, comprising many analogs having different degrees of oxidation introduced during their biosynthesis. We report here the determination of the complete biosynthetic steps leading to the formation of 1 from prochaetoglobosin I (2). Each oxidation step was elucidated using Chaetomium globosum strains carrying various combinations of deletion of the three redox enzymes, one FAD-dependent monooxygenase, and two cytochrome P450 oxygenases, and in vivo biotransformation of intermediates by heterologous expression of the three genes in Saccharomyces cerevisiae. Five analogs were identified in this study as intermediates formed during oxidization of 2 to 1 by those redox enzymes. Furthermore, a stereochemical course of each oxidation step was clearly revealed with the absolute configurations of five intermediates determined from X-ray crystal structure. This approach allowed us to quickly determine the biosynthetic intermediates and the enzymes responsible for their formation. Moreover, by addressing the redox enzymes, we were able to discover that promiscuity of the redox enzymes allowed the formation of a network of pathways that results in a combinatorial formation of multiple intermediate compounds during the formation of 1 from 2. Our approach should expedite elucidation of pathways for other natural products biosynthesized by many uncharacterized enzymes of this fungus.

Core-Pricing in Large Multi-Object Auctions: A Market Design for Selling TV-Ads

Motivated by markets for television advertising time-slots, we introduce a new combinatorial auction format, including a general method for approximating core-selecting payments for hard problems. Given the size and complexity of ad markets in practice, an enumerative XOR bidding language (widely discussed in the literature and used in recent government spectrum auctions) grows too quickly to be practical. We therefore propose a compact bidding language for coverage or demographic reach, and investigate the resulting winner determination (a form of multi-knapsack problem) using numerical experiments. For realistic instances of the problem, very good solutions can be found relatively quickly, though closing the integrality gap to find marginally better solutions or prove optimality can take a prohibitively large amount of time. Our subsequent adaptation of the core-selecting payment technique to this environment of nearly-optimal optimization results is a new, practically viable paradigm by which competitive market outcomes can be achieved despite computational limitations.

Expanded combinatorial formation of porphyrin macrocycles in aqueous solution containing vesicles. A prebiotic model

The role of combinatorial processes in the origin of life remains relatively unexplored. In a chemical model for the possible prebiogenesis of tetrapyrrole macrocycles reported previously, a tandem combinatorial reaction of two diones (substituents = methyl, acetic acid) and two aminoketones (substituents = ethyl, propanoic acid) afforded up to 538 porphyrins (upon oxidation of the corresponding porphyrinogens). The reaction was performed at a 1 : 1 ratio of hydrophobic and hydrophilic substituents in each pool of reactants, and the resulting porphyrins partitioned in ∼1 : 1 ratio between aqueous solution and phosphatidylcholine vesicle membranes. Here, a change in the ratio of hydrophobic and hydrophilic substituents of the [2 × 2] reaction gave corresponding changes in the polarity profile of the resulting porphyrins (3.5–9.0% yield). Reaction of four diones and four aminoketones (bearing hydrophilic or hydrophobic substituents) in the presence of lipid vesicles followed by photooxidation afforded porphyrins in 8.7% yield. The resulting porphyrins partitioned in ∼1 : 1 ratio between phosphatidylcholine vesicles and aqueous solution, as observed previously for the [2 × 2] reaction. Both the aqueous fraction and the vesicles fraction were photochemically active as evidenced by the fluorescence quantum yield (Φf ∼ 0.1). Software (PorphyrinViLiGe) for virtual library generation indicates that the [4 × 4] reaction affords up to 131 464 porphyrins. The relative insensitivity of physicochemical properties (partitioning, photoactivity) toward combinatorial expansion may be a valuable yet unappreciated attribute for prebiotic functionality.

Abyssomicins from the South China Sea Deep‐Sea Sediment Verrucosispora sp.: Natural Thioether Michael Addition Adducts as Antitubercular Prodrugs

Tuberculosis (TB) is a leading cause of death in the world today, and is exacerbated by the prevalence of multi- (MDR-TB), extensively (XDR-TB), and totally (TDR-TB) drug resistant strains. Despite the threat to human health, existing frontline TB therapeutics remain constrained to a handful of vintage antibiotics prescribed in a combinatorial format to achieve efficacy. The current shortfall in antitubercular drugs demands urgent attention, to develop new antibiotics effective against all strains of tuberculosis.

Different design - different cost: An empirical analysis of combinatorial public procurement bidding of road maintenance

This paper is an empirical analysis of first-price sealed-bid procurement auctions in Sweden, with and without combinatorial bidding. The data comprises procurement auctions of identical contracts (road resurfacing) with identical bidders conducted under the same time period (2009-2011) in two different regions in Sweden. Given the comparison of the suppliersʼ offered price per tons of asphalt, we cannot reject the hypothesis of identical distribution of standalone bids generated in both types of auction. The distribution of package bids within the combinatorial format is significantly lower than the distribution of standalone bids within the non-combinatorial format, suggesting substantial cost reduction of allowing package bidding. Also, within the combinatorial format, our analysis of data indicates higher costs when packages are predetermined by the purchaser rather than chosen freely by the suppliers.

Eukaryotic genomes may exhibit up to 10 generic classes of gene promoters

BackgroundThe main function of gene promoters appears to be the integration of different gene products in their biological pathways in order to maintain homeostasis. Generally, promoters have been classified in two major classes, namely TATA and CpG. Nevertheless, many genes using the same combinatorial formation of transcription factors have different gene expression patterns. Accordingly, we tried to ask ourselves some fundamental questions: Why certain genes have an overall predisposition for higher gene expression levels than others? What causes such a predisposition? Is there a structural relationship of these sequences in different tissues? Is there a strong phylogenetic relationship between promoters of closely related species?ResultsIn order to gain valuable insights into different promoter regions, we obtained a series of image-based patterns which allowed us to identify 10 generic classes of promoters. A comprehensive analysis was undertaken for promoter sequences from Arabidopsis thaliana, Drosophila melanogaster, Homo sapiens and Oryza sativa, and a more extensive analysis of tissue-specific promoters in humans. We observed a clear preference for these species to use certain classes of promoters for specific biological processes. Moreover, in humans, we found that different tissues use distinct classes of promoters, reflecting an emerging promoter network. Depending on the tissue type, comparisons made between these classes of promoters reveal a complementarity between their patterns whereas some other classes of promoters have been observed to occur in competition. Furthermore, we also noticed the existence of some transitional states between these classes of promoters that may explain certain evolutionary mechanisms, which suggest a possible predisposition for specific levels of gene expression and perhaps for a different number of factors responsible for triggering gene expression. Our conclusions are based on comprehensive data from three different databases and a new computer model whose core is using Kappa index of coincidence.ConclusionsTo fully understand the connections between gene promoters and gene expression, we analyzed thousands of promoter sequences using our Kappa Index of Coincidence method and a specialized Optical Character Recognition (OCR) neural network. Under our criteria, 10 classes of promoters were detected. In addition, the existence of “transitional” promoters suggests that there is an evolutionary weighted continuum between classes, depending perhaps upon changes in their gene products.

ChemInform Abstract: Metal‐Free Synthesis of 2‐Substituted (N, O, C) Benzothiazoles via an Intramolecular C—S Bond Formation.

An efficient, economical, and convenient method was developed for the preparation of 2-substituted (N, O, C) benzothiazoles from N′-substituted-N-(2-halophenyl)thioureas, O′-substituted-N-(2-halophenyl) carbamothioates, or N-(2-halophenyl) thioamides via a base-promoted cyclization in dioxane without any transition metal. A one-pot variant combining the synthesis of the thiourea and the cyclization was also demonstrated. High yields were obtained, and a variety of functional groups were tolerated under these conditions. Transition-metal-free, mild reactive conditions, wide application scope, and shorter reaction times make this method superior to the reported methods for the synthesis of 2-substituted benzothiazoles and suitable for combinatorial format.

Metal-Free Synthesis of 2-Substituted (N, O, C) Benzothiazoles via an Intramolecular C−S Bond Formation

An efficient, economical, and convenient method was developed for the preparation of 2-substituted (N, O, C) benzothiazoles from N'-substituted-N-(2-halophenyl)thioureas, O'-substituted-N-(2-halophenyl) carbamothioates, or N-(2-halophenyl) thioamides via a base-promoted cyclization in dioxane without any transition metal. A one-pot variant combining the synthesis of the thiourea and the cyclization was also demonstrated. High yields were obtained, and a variety of functional groups were tolerated under these conditions. Transition-metal-free, mild reactive conditions, wide application scope, and shorter reaction times make this method superior to the reported methods for the synthesis of 2-substituted benzothiazoles and suitable for combinatorial format.

An Experimental Test of Flexible Combinatorial Spectrum Auction Formats

This paper reports laboratory experiments that evaluate the performance of a flexible package bidding format developed by the FCC, in comparison with other combinatorial formats. In general, the interest of policy makers in combinatorial auctions is justified by the laboratory data. When value complementarities are present, package bidding yields improved performance. We find clear differences among the combinatorial auction formats both in terms of efficiency and seller revenue, however. Notably, the combinatorial clock provides the highest revenue. The FCC's flexible package bidding format performed worse than the alternatives, which is one of the main reasons why it was not implemented. (JEL D44, H82)

The class E floral homeotic protein SEPALLATA3 is sufficient to loop DNA in ‘floral quartet’-like complexes in vitro

The organs of a eudicot flower are specified by four functional classes, termed class A, B, C and E, of MADS domain transcription factors. The combinatorial formation of tetrameric complexes, so called ‘floral quartets’, between these classes is widely believed to represent the molecular basis of floral organ identity specification. As constituents of all complexes, the class E floral homeotic proteins are thought to be of critical relevance for the formation of floral quartets. However, experimental support for tetrameric complex formation remains scarce. Here we provide physico-chemical evidence that in vitro homotetramers of the class E floral homeotic protein SEPALLATA3 from Arabidopsis thaliana bind cooperatively to two sequence elements termed ‘CArG boxes’ in a phase-dependent manner involving DNA looping. We further show that the N-terminal part of SEPALLATA3 lacking K3, a subdomain of the protein–protein interactions mediating K domain, and the C-terminal domain, is sufficient for protein dimerization, but not for tetramer formation and cooperative DNA binding. We hypothesize that the capacity of class E MADS domain proteins to form tetrameric complexes contributes significantly to the formation of floral quartets. Our findings further suggest that the spacing and phasing of CArG boxes are important parameters in the molecular mechanism by which floral homeotic proteins achieve target gene specificity.

The future of microfluidic assays in drug development

Background: Microfluidic technology emerges as a convenient route to applying automated and reliable assays in a high-throughput manner with low cost. Objective: This review aims to answer questions related to the capabilities and potential applications of microfluidic assays that can benefit the drug development process and extends an outlook on its future trends. Methods: This article reviews recent publications in the field of microfluidics, with an emphasis on novel applications for drug development. Results/conclusion: Microfluidics affords unique capabilities in sample preparation and separation, combinatorial synthesis and array formation, and incorporating nanotechnology for more functionalities. The pharmaceutical industry, facing challenges from limited productivity and accelerated competition, can thus greatly benefit from applying new microfluidic assays in various drug development stages, from target screening and lead optimization to absorption distribution metabolism elimination and toxicity studies in preclinical evaluations, diagnostics in clinical trials and drug formulation and manufacturing process optimization.