Synthesis Of Combinatorial Libraries Research Articles

Exploring potential biomarkers and lead molecules in gastric cancer by network biology, drug repurposing and virtual screening strategies.

Gastric cancer poses a significant global health challenge, necessitating innovative approaches for biomarker discovery and therapeutic intervention. This study employs a multifaceted strategy integrating network biology, drug repurposing, and virtual screening to elucidate and expand the molecular landscape of gastric cancer. We identified and prioritized key genes implicated in gastric cancer by utilizing data from diverse databases and text-mining techniques. Network analysis underscored intricate gene interactions, emphasizing potential therapeutic targets such as CTNNB1, BCL2, TP53, etc, and highlighted ACTB among the top hub genes crucial in disease progression. Drug repurposing on 626 FDA-approved drugs for digestive system-related cancers revealed Norgestimate and Nimesulide as likely top candidates for gastric cancer, validated by molecular docking and dynamics simulations. Further, combinatorial synthesis of scaffold libraries derived from known chemotypes generated 56,160 virtual compounds, of which 76 new compounds were prioritized based on promising binding affinities and interactions at critical residues. Hotspot residue analysis identified GLU 214 and others as essential for ligand binding stability, enhancing compound efficacy and specificity. These findings support the therapeutic potential of targeting beta-actin protein in gastric cancer treatment, suggesting a future for further experimental validation and clinical translation. In conclusion, this study highlights the potential of repurposable drugs and virtual screening which can be used in combination with existing anti-gastric cancer drugs for gastric cancer therapy, emphasizing the role of computational methodologies in drug discovery.

GGAssembler: Precise and economical design and synthesis of combinatorial mutation libraries.

Golden Gate assembly (GGA) can seamlessly generate full-length genes from DNA fragments. In principle, GGA could be used to design combinatorial mutation libraries for protein engineering, but creating accurate, complex, and cost-effective libraries has been challenging. We present GGAssembler, a graph-theoretical method for economical design of DNA fragments that assemble a combinatorial library that encodes any desired diversity. We used GGAssembler for one-pot in vitro assembly of camelid antibody libraries comprising >105 variants with DNA costs <0.007$ per variant and dropping significantly with increased library complexity. >93% of the desired variants were present in the assembly product and >99% were represented within the expected order of magnitude as verified by deep sequencing. The GGAssembler workflow is, therefore, an accurate approach for generating complex variant libraries that may drastically reduce costs and accelerate discovery and optimization of antibodies, enzymes and other proteins. The workflow is accessible through a Google Colab notebook at https://github.com/Fleishman-Lab/GGAssembler.

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation.

Macrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol-containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol-containing elements and used them for the combinatorial synthesis of a 2,688-member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X-ray structure analysis of macrocycle-target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation

AbstractMacrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol‐containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol‐containing elements and used them for the combinatorial synthesis of a 2,688‐member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X‐ray structure analysis of macrocycle‐target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Combinatorial design of ionizable lipid nanoparticles for muscle-selective mRNA delivery with minimized off-target effects

Ionizable lipid nanoparticles (LNPs) pivotal to the success of COVID-19 mRNA (messenger RNA) vaccines hold substantial promise for expanding the landscape of mRNA-based therapies. Nevertheless, the risk of mRNA delivery to off-target tissues highlights the necessity for LNPs with enhanced tissue selectivity. The intricate nature of biological systems and inadequate knowledge of lipid structure–activity relationships emphasize the significance of high-throughput methods to produce chemically diverse lipid libraries for mRNA delivery screening. Here, we introduce a streamlined approach for the rapid design and synthesis of combinatorial libraries of biodegradable ionizable lipids. This led to the identification of iso-A11B5C1, an ionizable lipid uniquely apt for muscle-specific mRNA delivery. It manifested high transfection efficiencies in muscle tissues, while significantly diminishing off-targeting in organs like the liver and spleen. Moreover, iso-A11B5C1 also exhibited reduced mRNA transfection potency in lymph nodes and antigen-presenting cells, prompting investigation into the influence of direct immune cell transfection via LNPs on mRNA vaccine effectiveness. In comparison with SM-102, while iso-A11B5C1’s limited immune transfection attenuated its ability to elicit humoral immunity, it remained highly effective in triggering cellular immune responses after intramuscular administration, which is further corroborated by its strong therapeutic performance as cancer vaccine in a melanoma model. Collectively, our study not only enriches the high-throughput toolkit for generating tissue-specific ionizable lipids but also encourages a reassessment of prevailing paradigms in mRNA vaccine design. This study encourages rethinking of mRNA vaccine design principles, suggesting that achieving high immune cell transfection might not be the sole criterion for developing effective mRNA vaccines.

Expeditious Discovery of Small-Molecule Thermoresponsive Ionic Liquid Materials: A Review.

Ionic liquids (ILs) are a class of low-melting molten salts (<100 °C) constituted entirely of ions, and their research has gained tremendous attention in line with their remarkably growing applications (>124,000 publications dated 30 August 2023 from the Web of ScienceTM). In this review, we first briefly discussed the recent developments and unique characteristics of ILs and zwitterionic liquids (ZILs). Compared to molecular solvents and other conventional organic compounds, (zwitter) ionic liquids carry negligible volatility and are potentially recyclable and reusable. For structures, both ILs and ZILs can be systematically tailor-designed and engineered and are synthetically fine-tunable. As such, ionic liquids, including chiral, supported, task-specific ILs, have been widely used as powerful ionic solvents as well as valuable additives and catalysts for many chemical reactions. Moreover, ILs have demonstrated their value for use as polymerase chain reaction (PCR) enhancers for DNA amplification, chemoselective artificial olfaction for targeted VOC analysis, and recognition-based affinity extraction. As the major focus of this review, we extensively discussed that small-molecule thermoresponsive ILs (TILs) and ZILs (zwitterionic TILs) are new types of smart materials and can be expeditiously discovered through the structure and phase separation (SPS) relationship study by the combinatorial approach. Using this SPS platform developed in our laboratory, we first depicted the rapid discovery of N,N-dialkylcycloammonium and 1,3,4-trialkyl-1,2,3-triazolium TILs that concomitantly exhibited LCST (lower critical solution temperature) phase transition in water and displayed biochemically attractive Tc values. Both smart IL materials were suited for applications to proteins and other biomolecules. Zwitterionic TILs are ZILs whose cations and anions are tethered together covalently and are thermoresponsive to temperature changes. These zwitterionic TIL materials can serve as excellent extraction solvents, through temperature change, for biomolecules such as proteins since they differ from the common TIL problems often associated with unwanted ion exchanges during extractions. These unique structural characteristics of zwitterionic TIL materials greatly reduce and may avoid the denaturation of proteins under physiological conditions. Lastly, we argued that both rational structural design and combinatorial library synthesis of small-molecule TIL materials should take into consideration the important issues of their cytotoxicity and biosafety to the ecosystem, potentially causing harm to the environment and directly endangering human health. Finally, we would concur that before precise prediction and quantitative simulation of TIL structures can be realized, combinatorial chemistry may be the most convenient and effective technology platform to discover TIL expeditiously. Through our rational TIL design and combinatorial library synthesis and screening, we have demonstrated its power to discover novel chemical structures of both TILs and zwitterionic TILs. Undoubtedly, we will continue developing new small-molecule TIL structures and studying their applications related to other thermoresponsive materials.

Pushing the Boundaries of Diversity: Innovative Design and Synthesis of Hydrazone-Based Dynamic Combinatorial Libraries of Macrocycles

Pushing the Boundaries of Diversity: Innovative Design and Synthesis of Hydrazone-Based Dynamic Combinatorial Libraries of Macrocycles

A review on high-throughput development of high-entropy alloys by combinatorial methods

High-entropy alloys (HEAs) are an emerging class of alloys with multi-principal elements that greatly expands the compositional space for advanced alloy design. Besides chemistry, processing history can also affect the phase and microstructure formation in HEAs. The number of possible alloy compositions and processing paths gives rise to enormous material design space, which makes it challenging to explore by traditional trial-and-error approaches. This review highlights the progress in combinatorial high-throughput studies towards rapid prediction, manufacturing, and characterization of promising HEA compositions. This review begins with an introduction to HEAs and their unique properties. Then, this review describes high-throughput computational methods such as machine learning that can predict desired alloy compositions from hundreds or even thousands of candidates. The next section of this review presents advances in combinatorial synthesis of material libraries by additive manufacturing for efficient development of high-performance HEAs at bulk scale. The final section of this review discusses the high-throughput characterization techniques that are used to accelerate the material property measurements for systematic understanding of the composition-processing-structure-property relationships in combinatorial HEA libraries.

Optimization of G-Quadruplex Ligands through a SAR Study Combining Parallel Synthesis and Screening of Cationic Bis(acylhydrazones).

G-quadruplexes (G4s), secondary structures adopted by guanine-rich DNA and RNA sequences, are implicated in numerous biological processes and have been suggested as potential drug targets. Accordingly, there is an increasing interest in developing high-throughput methods that allow the generation of congeneric series of G4-targeting molecules ("ligands") and investigating their interactions with the targets. We have developed an operationally simple method of parallel synthesis to generate "ready-to-screen" libraries of cationic acylhydrazones, a motif that we have previously identified as a promising scaffold for potent, biologically active G4 ligands. Combined with well-established screening techniques, such as fluorescence melting, this method enables the rapid synthesis and screening of combinatorial libraries of potential G4 ligands. Following this protocol, we synthesized a combinatorial library of 90 bis(acylhydrazones) and screened it against five different nucleic acid structures. This way, we were able to analyze the structure-activity relationships within this series of G4 ligands, and identified three novel promising ligands whose interactions with G4-DNAs of different topologies were studied in detail by a combination of several biophysical techniques, including native mass spectrometry, and molecular modeling.

Multi-spray pyrolysis for combinatorial synthesis of materials libraries and their high-throughput screening: Application to Li-ion conducting electrolytes

We present a multi-head spray pyrolysis system and its application in high-throughput combinatorial synthesis for research of solid Li-ion conductors. Each spraying nozzle is fed with a separate precursor solution. The overlap of areas that are sprayed leads to unprecedented composition flexibility of the films obtained after pyrolysis. Thus, a library with a continuous composition spread of a Li-La-P-O model system is formed. The Li-ion conduction was determined on 169 cells of the library, using high throughput impedance measurements in a controlled environment. While the activation energies that were found were relatively small, Li-ion conduction was still low. This low mobility is hypothesized to originate from the sub-optimal occupation of Li sites in the non-stoichiometric materials' lattices, and/or porosity and tortuosity issues, which in turn, reduces their effective concentration and contribution to ion transport. In addition, porosity and tortuosity in sprayed electrolyte causes random orientation of the grains and grain boundaries in the solid followed by the random diffusion scattering of Li-ions and low Li-ions conductivity, despite low apparent activation energy of conduction.

Multi-Component Sequential Synthesis of Dihydroorotic Acid-Based Amphiphilic Molecules

AbstractAn efficient multicomponent sequential process, which occurs in mild condition has been exploited for the synthesis of systematically modified amphiphilic molecules where the cationic head is tethered to a lipophilic tail through a dihydroorotic acid linker. The process is operatively simple, high yielding, and flexible. Such a strategy could impact combinatorial synthesis of wide libraries of amphiphilic molecules to be tested as transfection agents and/or as antimicrobials.

Structural characterisation of Cu-Zr thin film combinatorial libraries with synchrotron radiation at the limit of crystallinity

We report for the first-time combinatorial synthesis of thin film metallic glass libraries via magnetron co-sputtering at the limit of crystallinity. Special care was taken to prepare extremely pure CuZr films (1–2 µm thickness) with large compositional gradients (Cu18.2Zr81.8 to Cu74.8Zr25.2) on X-ray transparent polymer substrates in high-vacuum conditions. Combined mapping of atomic structure (synchrotron radiation) and chemical composition (X-ray fluorescence spectroscopy) revealed that over the entire composition range, covering multiple renowned glass formers, two phases are present in the film. Our high-resolution Synchrotron approach identified the two phases as: untextured amorphous Cu51Zr14 (cluster size 1.3 nm) and textured, nanocrystalline α-Zr (grain size 1–5 nm). Real space HR-STEM analyses of a representative composition substantiate our XRD results. Determined cluster and grain sizes are below the resolution limit of conventional laboratory-scale X-ray diffractometers. The presented phase mixture is not permitted in the Cu-Zr phase diagram and contrary to existing literature. The phase ratio follows a linear trend with amorphous films on the Cu-rich side and increasing amounts of α-Zr with increasing Zr content. While cluster size and composition of the amorphous phase remain constant thorough the compositional gradient, crystallite size and texture of the nanocrystalline α-Zr change as a function of Zr content.

Identification of B Cell and T Cell Epitopes Using Synthetic Peptide Combinatorial Libraries.

This article presents a combinatorial library method that consists of the synthesis and screening of mixture-based synthetic combinatorial libraries of peptide molecules to identify B and T cell epitopes. The protocols employ peptide libraries to identify peptides recognized by MAbs and T cells. The first protocol uses a positional scanning peptide library made up of hexapeptides to identify antigenic determinants recognized by MAbs. The 120 mixtures in the hexapeptide library are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide library to identify T cell peptide ligands. The 200 mixtures of the decapeptide library are tested for their ability to induce T cell activation. Support protocols cover optimization of the assay conditions for each MAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide library, along with deconvolution approaches. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Screening peptide library for antibody inhibition Basic Protocol 2: Screening a peptide library to identify CD4+ Or CD8+ T cell ligands Support Protocol 1: Optimizing antigen and antibody concentrations for screening assay Support Protocol 2: Preparing a positional scanning peptide library.

Facile and High‐Yielding Synthesis of C(3)‐Aminoisoindolinones Under Metal Catalyst‐Free Conditions

AbstractC(3)‐Aminoisoindolinones have attracted the attention of medicinal chemists due to their unique and stable N−C−N substructure and possibilities for facile and scalable synthesis of combinatorial libraries. We discovered that para‐toluenesulfonic acid (PTSA), an organic protic acid, efficiently catalyzes condensation of isoindolinone‐3‐ols with primary and secondary amines to furnish C(3) N‐substituted isoindolinones in high yields. The N−C bond formation reaction is highly efficient and experimentally simple. The method replaces known transition metal catalysts involving Cu, Pt, Pd or Au. It works efficiently for both intra‐ and inter‐molecular variants. Furthermore, it is amenable for synthesis of C(3)‐aminoisoindolinones with free N(2) via photolytic deprotection of the 2‐nitrobenzyl group. We have demonstrated application of the method for synthesis of a variety of medicinally relevant bicyclic and tricyclic C(3)‐aminoisoindolinones.

Peptide Dendrimers: From Enzyme Models to Antimicrobials and Transfection Reagents.

Aiming at studying cooperativity effects between amino acids in easily accessible protein models, we have explored the chemistry of peptide dendrimers, which we obtain as pure products by solid-phase peptide synthesis using a branching diamino acid such as lysine at every second or third position in a peptide sequence, followed by reverse-phase HPLC purification. This article reviews discoveries driven by combinatorial library synthesis and screening, including enantioselective esterase and aldolase enzyme models, cobalamin binding and peroxidase dendrimers, glycopeptide dendrimer biofilm inhibitors and their X-ray crystal structures as complexes with lectins, antimicrobial peptide dendrimers active against multidrug resistant Gram-negative bacteria, and transfection reagents for siRNA and CRISPR-Cas9 plasmid DNA. Latest developments include cheminformatics and artificial intelligence for exploring the peptide chemical space, and the principle of stereorandomization to understand the role of peptide chirality in activity.

Structural and optical properties of amorphous Si\u2013Ge\u2013Te thin films prepared by combinatorial sputtering

The lack of order in amorphous chalcogenides offers them novel properties but also adds increased challenges in the discovery and design of advanced functional materials. The amorphous compositions in the Si–Ge–Te system are of interest for many applications such as optical data storage, optical sensors and Ovonic threshold switches. But an extended exploration of this system is still missing. In this study, magnetron co-sputtering is used for the combinatorial synthesis of thin film libraries, outside the glass formation domain. Compositional, structural and optical properties are investigated and discussed in the framework of topological constraint theory. The materials in the library are classified as stressed-rigid amorphous networks. The bandgap is heavily influenced by the Te content while the near-IR refractive index dependence on Ge concentration shows a minimum, which could be exploited in applications. A transition from a disordered to a more ordered amorphous network at 60 at% Te, is observed. The thermal stability study shows that the formed crystalline phases are dictated by the concentration of Ge and Te. New amorphous compositions in the Si–Ge–Te system were found and their properties explored, thus enabling an informed and rapid material selection and design for applications.

Quadruplex DNA-guided ligand selection from dynamic combinatorial libraries of acylhydrazones.

Dynamic combinatorial libraries of acylhydrazones were prepared from diacylhydrazides and several cationic or neutral aldehydes in the presence of 5-methoxyanthranilic acid catalyst. Pull-down experiments with magnetic beads functionalized with a G-quadruplex (G4)-forming oligonucleotide led to the identification of putative ligands, which were resynthesized or emulated by close structural analogues. G4-binding properties of novel derivatives were assessed by fluorimetric titrations, mass spectrometry and thermal denaturation experiments, giving evidence of strong binding (Kd < 10 nM) for two compounds.

High-Throughput Exploration of Metal Vanadate Thin-Film Systems (M-V-O, M = Cu, Ag, W, Cr, Co, Fe) for Solar Water Splitting: Composition, Structure, Stability, and Photoelectrochemical Properties.

Combinatorial synthesis and high-throughput characterization of thin-film materials libraries enable to efficiently identify both photoelectrochemically active and inactive, as well as stable and instable systems for solar water splitting. This is shown on six ternary metal vanadate (M-V-O, M = Cu, Ag, W, Cr, Co, Fe) thin-film materials libraries, fabricated using combinatorial reactive magnetron cosputtering with subsequent annealing in air. By means of high-throughput characterization of these libraries correlations between composition, crystal structure, photocurrent density, and stability of the M-V-O systems in different electrolytes such as acidic, neutral and alkaline media were identified. The systems Cu-V-O and Ag-V-O are stable in alkaline electrolyte and exhibited photocurrents of 170 and 554 μA/cm2, respectively, whereas the systems W-V-O, Cr-V-O, and Co-V-O are not stable in alkaline electrolyte. However, the Cr-V-O and Co-V-O systems showed an enlarged photoactive region in acidic electrolyte, albeit with very low photocurrents (<10 μA/cm2). Complete data sets obtained from these different screening sets, including information on nonpromising systems, lays groundwork for their use to predict new systems for solar water splitting, for example, by machine learning.

DNA-Encoded Chemistry: Drug Discovery from a Few Good Reactions.

Click chemistry, proposed nearly 20 years ago, promised access to novel chemical space by empowering combinatorial library synthesis with a "few good reactions". These click reactions fulfilled key criteria (broad scope, quantitative yield, abundant starting material, mild reaction conditions, and high chemoselectivity), keeping the focus on molecules that would be easy to make, yet structurally diverse. This philosophy bears a striking resemblance to DNA-encoded library (DEL) technology, the now-dominant combinatorial chemistry paradigm. This review highlights the similarities between click and DEL reaction design and deployment in combinatorial library settings, providing a framework for the design of new DEL synthesis technologies to enable next-generation drug discovery.

Methylation of Methyl 4-Hydroxy-2-thioxo-1,2-dihydroquinoline-3-carboxylate: Synthetic, Crystallographic, and Molecular Docking Studies.

Consecutive alkylation of 4-hydroxy-2-thioxo-1,2-dihydroquinoline-3-carboxylate by CH3I has been investigated to establish regioselectivity of the reaction for reliable design and synthesis of combinatorial libraries. In the first stage, the product of S-methylation-methyl 4-hydroxy-2-(methylthio)quinoline-3-carboxylate was obtained. The subsequent alkylation with CH3I led to the formation of both O- and N-methylation products mixture-methyl 4-methoxy-2-(methylthio)quinoline-3-carboxylate and methyl 1-methyl-2-(methylthio)-4-oxo-1,4-dihydroquinoline-3-carboxylate with a predominance of O-methylated product. The structure of synthesized compounds was confirmed by means of elemental analysis, 1H-NMR, 13C-NMR, LC/MS, and single-crystal X-ray diffraction. The quantum chemical calculations of geometry and electron structure of methyl 4-hydroxy-2-(methylthio)quinoline-3-carboxylate’s anion were carried out. According to molecular docking simulations, the studied compounds can be considered as potent inhibitors of Hepatitis B Virus replication. Experimental in vitro biological studies confirmed that studied compounds demonstrated high inhibition of HBV replication in 10 µM concentration.