Combinatorial Libraries Research Articles

Occurrence and Synthesis Pathways of (Suspected) Genotoxic α,β-Unsaturated Carbonyls in Chocolate and Other Commercial Sweet Snacks.

α,β-Unsaturated carbonyls are highly reactive and described as structural alerts for genotoxicity. Ten of them (either commercially available or synthesized here by combinatorial chemistry) were first investigated throughout the chocolate-making process by solvent-assisted flavor evaporation (SAFE) coupled to GC-MS/SIM. Monitored α,β-unsaturated aldehydes were formed during chocolate production, primarily through aldol condensation of Strecker aldehydes triggered by bean roasting. Notably, levels of 2-phenylbut-2-enal (up to 399 μg·kg-1) and 5-methyl-2-phenylhex-2-enal (up to 216 μg·kg-1) increased up to 40-fold. Dry conching caused evaporation of α,β-unsaturated carbonyls, while wet conching partially restored or increased their levels due to cocoa butter addition. Further analyses showed that α,β-unsaturated aldehydes also occurred in most commercial sweet snacks (up to 16 μg·kg-1), although often at lower concentrations than in roasted cocoa or derived chocolates. In the end, none of the monitored α,β-unsaturated aldehydes did raise a health concern compared to current maximum use levels (2-5 mg·kg-1). On the other hand, much higher levels of genotoxic furan-2(5H)-one were found in crepe and cake samples (up to 4.3 mg·kg-1).

Non-prime- and prime-side profiling of Pro-Pro endopeptidase specificity using synthetic combinatorial peptide libraries and mass spectrometry.

A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), possess the unique ability to hydrolyze proline-proline bonds in proteins. Since a protease's function is largely determined by its substrate specificity, methods that can extensively characterize substrate specificity are valuable tools for protease research. Previously, we achieved an in-depth characterization of PPEP prime-side specificity. However, PPEP specificity is also determined by the non-prime-side residues in the substrate. To gain a more complete insight into the determinants of PPEP specificity, we characterized the non-prime- and prime-side specificity of various PPEPs using a combination of synthetic combinatorial peptide libraries and mass spectrometry. With this approach, we deepened our understanding of the P3-P3' specificities of PPEP-1 and PPEP-2, while identifying the endogenous substrate of PPEP-2 as the most optimal substrate in our library data. Furthermore, by employing the library approach, we investigated the altered specificity of mutants of PPEP-1 and PPEP-2. Additionally, we characterized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displays a PPEP-1-like prime-side specificity, which was substantiated by the experimental data. Intriguingly, another putative PPEP from Clostridioides difficile, CD1597, did not display Pro-Pro endoproteolytic activity. Collectively, we characterized PPEP specificity in detail using our robust peptide library method and, together with additional structural information, provide more insight into the intricate mechanisms that govern protease specificity.

Correlation of protein binding pocket properties with hits’ chemistries used in generation of ultra-large virtual libraries

Although the size of virtual libraries of synthesizable compounds is growing rapidly, we are still enumerating only tiny fractions of the drug-like chemical universe. Our capability to mine these newly generated libraries also lags their growth. That is why fragment-based approaches that utilize on-demand virtual combinatorial libraries are gaining popularity in drug discovery. These à la carte libraries utilize synthetic blocks found to be effective binders in parts of target protein pockets and a variety of reliable chemistries to connect them. There is, however, no data on the potential impact of the chemistries used for making on-demand libraries on the hit rates during virtual screening. There are also no rules to guide in the selection of these synthetic methods for production of custom libraries. We have used the SAVI (Synthetically Accessible Virtual Inventory) library, constructed using 53 reliable reaction types (transforms), to evaluate the impact of these chemistries on docking hit rates for 40 well-characterized protein pockets. The data shows that the virtual hit rates differ significantly for different chemistries with cross coupling reactions such as Sonogashira, Suzuki–Miyaura, Hiyama and Liebeskind–Srogl coupling producing the highest hit rates. Virtual hit rates appear to depend not only on the property of the formed chemical bond but also on the diversity of available building blocks and the scope of the reaction. The data identifies reactions that deserve wider use through increasing the number of corresponding building blocks and suggests the reactions that are more effective for pockets with certain physical and hydrogen bond-forming properties.

Identification and characterization of a nonbiological small-molecular mimic of a Zika virus conformational neutralizing epitope

Antigenic similarities between Zika virus (ZIKV) and other flaviviruses pose challenges to the development of virus-specific diagnostic tools and effective vaccines. Starting with a DNA-encoded one-bead-one-compound combinatorial library of 508,032 synthetic, non-natural oligomers, we selected and characterized small molecules that mimic ZIKV epitopes. High-throughput fluorescence-activated cell sorter-based bead screening was used to select molecules that bound IgG from ZIKV-immune but not from dengue-immune sera. Deep sequencing of the DNA from the "Zika-only" beads identified 40 candidate molecular structures. A lead candidate small molecule "CZV1-1" was selected that correctly identifies serum specimens from Zika-experienced patients with good sensitivity and specificity (85.3% and 98.4%, respectively). Binding competition studies of purified anti-CZV1-1 IgG against known ZIKV-specific monoclonal antibodies (mAbs) showed that CZV1-1 mimics a nonlinear, neutralizing conformational epitope in the domain III of the ZIKV envelope. Purified anti-CZV1-1 IgG neutralized infection of ZIKV in cell cultures with potencies comparable to highly specific ZIKV-neutralizing mAbs. This study demonstrates an innovative approach for identification of synthetic non-natural molecular mimics of conformational virus epitopes. Such molecular mimics may have value in the development of accurate diagnostic assays for Zika, as well as for other viruses.

Novel Alaninamide Derivatives with Drug-like Potential for Development as Antiseizure and Antinociceptive Therapies─In Vitro and In Vivo Characterization.

In the present study, a series of original alaninamide derivatives have been designed applying a combinatorial chemistry approach, synthesized, and characterized in the in vivo and in vitro assays. The obtained molecules showed potent and broad-spectrum activity in basic seizure models, namely, the maximal electroshock (MES) test, the 6 Hz (32 mA) seizure model, and notably, the 6 Hz (44 mA) model of pharmacoresistant seizures. Most potent compounds 26 and 28 displayed the following pharmacological values: ED50 = 64.3 mg/kg (MES), ED50 = 15.6 mg/kg (6 Hz, 32 mA), ED50 = 29.9 mg/kg (6 Hz, 44 mA), and ED50 = 34.9 mg/kg (MES), ED50 = 12.1 mg/kg (6 Hz, 32 mA), ED50 = 29.5 mg/kg (6 Hz, 44 mA), respectively. Additionally, 26 and 28 were effective in the ivPTZ seizure threshold test and had no influence on the grip strength. Moreover, lead compound 28 was tested in the PTZ-induced kindling model, and then, its influence on glutamate and GABA levels in the hippocampus and cortex was evaluated by the high-performance liquid chromatography (HPLC) method. In addition, 28 revealed potent efficacy in formalin-induced tonic pain, capsaicin-induced pain, and oxaliplatin- and streptozotocin-induced peripheral neuropathy. Pharmacokinetic studies and in vitro ADME-Tox data proved favorable drug-like properties of 28. The patch-clamp recordings in rat cortical neurons showed that 28 at a concentration of 10 μM significantly inhibited fast sodium currents. Therefore, 28 seems to be an interesting candidate for future preclinical development in epilepsy and pain indications.

Accelerating ionizable lipid discovery for mRNA delivery using machine learning and combinatorial chemistry.

To unlock the full promise of messenger (mRNA) therapies, expanding the toolkit of lipid nanoparticles is paramount. However, a pivotal component of lipid nanoparticle development that remains a bottleneck is identifying new ionizable lipids. Here we describe an accelerated approach to discovering effective ionizable lipids for mRNA delivery that combines machine learning with advanced combinatorial chemistry tools. Starting from a simple four-component reaction platform, we create a chemically diverse library of 584 ionizable lipids. We screen the mRNA transfection potencies of lipid nanoparticles containing those lipids and use the data as a foundational dataset for training various machine learning models. We choose the best-performing model to probe an expansive virtual library of 40,000 lipids, synthesizing and experimentally evaluating the top 16 lipids flagged. We identify lipid 119-23, which outperforms established benchmark lipids in transfecting muscle and immune cells in several tissues. This approach facilitates the creation and evaluation of versatile ionizable lipid libraries, advancing the formulation of lipid nanoparticles for precise mRNA delivery.

In-Electrospray source Hydrogen/Deuterium exchange coupled to multistage fragmentation for the investigation of the protonation and fragmentation pathways of gas phase ions.

Identification of molecules in complex natural matrices relies on matching the fragmentation spectra of ions under investigation and the spectra acquired for the corresponding analytical standards. Currently, there are many databases of experimentally measured tandem mass spectrometry spectra (such as NIST, MzCloud, and Metlin), and considerable progress has been made in the development of software for predicting tandem mass spectrometry fragments in silico using combinatorial, machine learning, and quantum chemistry approaches (such as MetFrag, CFM-ID, and QCxMS). However, the electrospray ionization molecules can be ionized at different sites (protonated or deprotonated), and the fragmentation spectra of such ions are different. Here, we are using the combination of the in-ESI source hydrogen/deuterium exchange reaction and MSn fragmentation for the investigation of the fragmentation pathways for different protomers of organic molecules. It is shown that the distribution of the deuterium in the fragment ions reflects the presence of different protomers. For several molecules, the distribution of deuterium was traced up to the MS5 level of fragmentation revealing many unusual and unexpected effects. For example, we investigated the loss of HF from the ciprofloxacin and norfloxacin ions and observed that for ions protonated at -COOH group, the eliminating hydrogen always comes from -NH group. When ions are protonated at another site, the elimination of hydrogen with a probability of 30% occurs from the -NH group, and with a probability of 70%, it originates from other sites on the molecule. Such effects were not described previously. Quantum chemical simulation was used for the verification of the protonated structures and simulation of the corresponding fragmentation spectra.

Bionic synthesis of novel monoterpenoid indole alkaloid-like analogs with tyrosinase inhibition activity based on loganetin

A series of novel monoterpenoid indole alkaloid (MIA)-like analogs (1–24) were synthesized by using a biomimetic synthetic strategy and combinatorial chemistry. The tyrosinase inhibitory activities and antioxidant properties of these compounds were assessed through tyrosinase inhibition assay, DPPH and ABTS free radicals scavenging assays, respectively. The results showed that most synthesized compounds (2, 4–8, 10–12, 14–17, 19) exhibited significant inhibitory activities against both monophenolase and diphenolase. In particular, compound 14, with an IC50 value of 0.18 ± 0.02 mM, displayed approximately 72-fold stronger monophenolase inhibitory activity than that of loganetin with an IC50 value of 12.76 ± 1.00 mM. Similarly, compound 17 exhibited the best diphenolase inhibitory activity (IC50, 0.30 ± 0.09 mM), which was approximately 47 times higher than that of loganetin (IC50, 14.05 ± 0.47 mM). Additionally, six compounds (3, 13–17) showed significant in vitro antioxidant activities. Compound 17 exhibited the most promising ABTS radical scavenging activity with an IC50 value of 2.90 ± 0.07 μM, while compound 15 exhibited the highest DPPH free radical scavenging capacity with an IC50 value of 17.59 ± 0.21 μM. The structure-activity relationships analysis revealed that the substituents at C-5 and C-11 in the tryptamine moiety might significantly impact antioxidant activity. Furthermore, the detailed interactions and binding modes of the most potent derivative toward tyrosinase were elucidated by a molecular docking study, which indicated that 14 and 17 might exert their inhibitory effects by binding to key amino acid residues (His 85 and His 263) at the tyrosinase active site.

Functionalization of Supramolecular Polymers by Dynamic Covalent Boroxine Chemistry.

Molecular scaffolds that enable the combinatorial synthesis of new supramolecular building blocks are promising targets for the construction of functional molecular systems. Here, we report a supramolecular scaffold based on boroxine that enables the formation of chiral and ordered 1D supramolecular polymers, which can be easily functionalized for circularly polarized luminescence. The boroxine monomers are quantitatively synthesized in situ, both in bulk and in solution, from boronic acid precursors and cooperatively polymerize into 1D helical aggregates stabilized by threefold hydrogen-bonding and π-π stacking. We then demonstrate amplification of asymmetry in the co-assembly of chiral/achiral monomers and the co-condensation of chiral/achiral precursors in classical and in situ sergeant-and-soldiers experiments, respectively, showing fast boronic acid exchange reactions occurring in the system. Remarkably, co-condensation of pyrene boronic acid with a hydrogen-bonding chiral boronic acid results in chiral pyrene aggregation with circularly polarized excimer emission and g-values in the order of 10-3. Yet, the electron deficiency of boron in boroxine makes them chemically addressable by nucleophiles, but also sensitive to hydrolysis. With this sensitivity in mind, we provide first insights into the prospects offered by boroxine-based supramolecular polymers to make chemically addressable, functional, and adaptive systems.

Functionalization of Supramolecular Polymers by Dynamic Covalent Boroxine Chemistry

AbstractMolecular scaffolds that enable the combinatorial synthesis of new supramolecular building blocks are promising targets for the construction of functional molecular systems. Here, we report a supramolecular scaffold based on boroxine that enables the formation of chiral and ordered 1D supramolecular polymers, which can be easily functionalized for circularly polarized luminescence. The boroxine monomers are quantitatively synthesized in situ, both in bulk and in solution, from boronic acid precursors and cooperatively polymerize into 1D helical aggregates stabilized by threefold hydrogen‐bonding and π–π stacking. We then demonstrate amplification of asymmetry in the co‐assembly of chiral/achiral monomers and the co‐condensation of chiral/achiral precursors in classical and in situ sergeant‐and‐soldiers experiments, respectively, showing fast boronic acid exchange reactions occurring in the system. Remarkably, co‐condensation of pyrene boronic acid with a hydrogen‐bonding chiral boronic acid results in chiral pyrene aggregation with circularly polarized excimer emission and g‐values in the order of 10−3. Yet, the electron deficiency of boron in boroxine makes them chemically addressable by nucleophiles, but also sensitive to hydrolysis. With this sensitivity in mind, we provide first insights into the prospects offered by boroxine‐based supramolecular polymers to make chemically addressable, functional, and adaptive systems.

Small protein blockers of human IL-6 receptor alpha inhibit proliferation and migration of cancer cells

BackgroundInterleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target.MethodsAn albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking.ResultsWe demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells.ConclusionWe report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.

Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging

BackgroundProgrammed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging.MethodsWe designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1.ResultsBinding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7–99.3% and in vitro stability in human serum after 120 min was in the range 94.6–98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors.ConclusionsUsing directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.Graphical

Base-Filling in Double-Helical Nucleic Acids.

Base-filling, i. e., post-synthetic furnishing of an oligonucleotide scaffold with base moieties or their analogues, is an interesting alternative to the conventional approach of sequential coupling of building blocks (modified or otherwise). Reversible attachment of the base moieties is particularly attractive as it allows the use of dynamic combinatorial chemistry and usually leads to higher fidelity. This concept article summarizes the various backbones and coupling reactions used for base-filling over the past fifteen years, discusses the impact of base stacking and pairing on efficiency and fidelity and highlights potential and realized applications.

Multicomponent Click Reaction: An Indispensable Tool for Easy Access of Functionalized 1,2,3‐Triazoles

AbstractMulticomponent reactions are operationally simple and display a significant role in diverse chemical modification by reducing reaction times as well as additional steps involved. In this review, we highlighted the impact of multi‐component reactions in assistance with modular Click chemistry to develop a library of triazole‐appended scaffolds including 1,2,3‐triazole‐fused heterocycles, glycoconjugates, macrocycles as well as in the combinatorial synthesis of differently functionalized triazoles along with mechanistic insights with a diverse range of applications in the field of medicinal chemistry.

I2−catalyzed cascade annulation of 2-(pyridin−2−yl)acetate derivatives with N,N−dimethylenamine ketones: Site-selective synthesis of functionalized indolizines

A novel synthesis method was developed for developing functionalized indolizines. In this reaction, 2-(pyridin-2-yl)acetate derivatives and N,N-dimethylenamine ketones were used as substrates. All these processes were enabled by heating a mixture of starting materials in toluene and in the presence of I2 under air. Cascade reaction resulted in the formation of two bonds and cleavage of one bond in one pot. Consequently, a number of functionalized indolizines were produced via a cascade reaction. This protocol can be used to synthesize functionalized unsymmetrical 2,4-diarylpyridines. Moreover, it is suitable for combinatorial and parallel syntheses of indolizines derivatives in a one-pot reaction and it is not a tedious multi-step reaction.

Use of Phage Display and Other Molecular Display Methods for the Development of Monobodies.

Synthetic binding proteins are human-made binding proteins that use non-antibody proteins as the starting scaffold. Molecular display technologies, such as phage display, enable the construction of large combinatorial libraries and their efficient sorting and, thus, are crucial for the development of synthetic binding proteins. Monobodies are the founding system of a set of synthetic binding proteins based on the fibronectin type III (FN3) domain. Since the original report in 1998, the monobody and related FN3-based systems have steadily been refined, and current methods are capable of rapidly generating potent and selective binding molecules to even challenging targets. The FN3 domain is small (∼90 amino acids) and autonomous and is structurally similar to the conventional immunoglobulin (Ig) domain. Unlike the Ig domain, however, the FN3 lacks a disulfide bond but is highly stable. These attributes of FN3 present unique opportunities and challenges in the design of phage and other display systems, combinatorial libraries, and library sorting strategies. This article reviews key technological innovations in the establishment of our monobody development pipeline, with an emphasis on phage display methodology. These give insights into the molecular mechanisms underlying molecular display technologies and protein-protein interactions, which should be broadly applicable to diverse systems intended for generating high-performance binding proteins.

Lab on a chip (LOC) platform for drug screening against the intracellular forms of Trypanosoma cruzi

Trypanosoma cruzi is the etiological agent of Chagas disease, endemic in Latin America with recent spreading to non-endemic areas due to human migration. The low efficacy and substantial prevalence of side effects of the approved medications benznidazole (BZN) and nifurtimox observed mainly during the chronic phase of Chagas disease, when most patients receive their diagnosis, demonstrate the need to find new therapeutic compounds to address this neglected global health disease. Here, we developed a platform for screening the antitrypanosomal activity of compounds over intracellular amastigotes using microfluidics, LOC devices, and advanced image analysis algorithms. This platform allows the simultaneous testing of six different concentrations of a compound and the automatic and high-precision processing of the images obtained during the experimental phase, providing quickly and easily a multitude of statistical data for the screening of new drugs. To assess the developed platform, we analyzed the effects of BZN as a reference drug and compared the dose–response curves with those obtained with a flow cytometry-based method, obtaining similar IC50 results. Furthermore, a collection of compounds obtained by chemical synthesis from a combinatorial library was tested over intracellular amastigotes in infected Vero cells.

Synthesis and biological evaluation of orally active anti-Trypanosoma agents

In previous studies, we developed anti-trypanosome tubulin inhibitors with promising in vitro selectivity and activity against Human African Trypanosomiasis (HAT). However, for such agents, oral activity is crucial. This study focused on further optimizing these compounds to enhance their ligand efficiency, aiming to reduce bulkiness and hydrophobicity, which should improve solubility and, consequently, oral bioavailability. Using Trypanosoma brucei brucei cells as the parasite model and human normal kidney cells and mouse macrophage cells as the host model, we evaluated 30 new analogs synthesized through combinatorial chemistry. These analogs have fewer aromatic moieties and lower molecular weights than their predecessors. Several new analogs demonstrated IC50s in the low micromolar range, effectively inhibiting trypanosome cell growth without harming mammalian cells at the same concentration. We conducted a detailed structure–activity relationship (SAR) analysis and a docking study to assess the compounds’ binding affinity to trypanosome tubulin homolog. The results revealed a correlation between binding energy and anti-Trypanosoma activity. Importantly, compound 7 displayed significant oral activity, effectively inhibiting trypanosome cell proliferation in mice.

Epitaxial oxide ionotronics: Interfaces and oxygen vacancies

Epitaxial oxide ionotronics: Interfaces and oxygen vacancies

Binary ZnY porous materials through integrated high-throughput approach

Porous materials, characterized by their important applications in catalysis, energy storage, and conversion, are predominantly derived from binary alloy systems due to the compositional complexity and the limited availability of phase diagrams for multi-component alloys. In this work, supergravity-induced solidification, a high-throughput approach, is employed to construct ternary MgZnY combinatorial libraries in a near-equilibrium state. This method utilizes the differences in densities and melting points among metal phases, where denser phases with higher melting points solidify and migrate towards the bottom, while lighter phases precipitate successively as the temperature decreases, resulting in samples with compositional and structural gradients along the centrifugal direction. Subsequently, a high-throughput vapor phase dealloying technique is developed based on the above bulk gradient samples, selectively volatilizing elements with high saturated vapor pressures and leaving behind residual elements that evolve into porous structures. Through the integrated high-throughput approach, we have successfully identified multiple precursor alloys capable of constructing intermetallic nanoporous Zn2Y1 (atomic percentage, at.%) with diverse morphologies. Furthermore, we have meticulously fabricated several alloys using the aforementioned high-throughput techniques. These alloys demonstrate consistent nanoporous compositions, underscoring the efficacy of our high-throughput approaches. This integrated high-throughput method significantly simplifies the exploration and development of porous materials, offering an innovative route to exploit the capabilities of multi-component alloy systems in a broad range of applications.