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1.
Protein Expr Purif ; 225: 106583, 2025 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-39168394

RESUMO

In recombinant protein purification, differences in isoelectric point (pI)/surface charge and hydrophobicity between the product and byproducts generally form the basis for separation. For bispecific antibodies (bsAbs), in many cases the physicochemical difference between product and byproducts is subtle, making byproduct removal considerably challenging. In a previous report, with a bsAb case study, we showed that partition coefficient (Kp) screening for the product and byproducts under various conditions facilitated finding conditions under which effective separation of two difficult-to-remove byproducts was achieved by anion exchange (AEX) chromatography. In the current work, as a follow-up study, we demonstrated that the same approach enabled identification of conditions allowing equally good byproduct removal by mixed-mode chromatography with remarkably improved yield. Results from the current and previous studies proved that separation factor determination based on Kp screening for product and byproduct is an effective approach for finding conditions enabling efficient and maximum byproduct removal, especially in challenging cases.


Assuntos
Anticorpos Biespecíficos , Proteínas Recombinantes , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Humanos
2.
Methods Mol Biol ; 2834: 181-193, 2025.
Artigo em Inglês | MEDLINE | ID: mdl-39312166

RESUMO

The discovery of molecular toxicity in a clinical drug candidate can have a significant impact on both the cost and timeline of the drug discovery process. Early identification of potentially toxic compounds during screening library preparation or, alternatively, during the hit validation process is critical to ensure that valuable time and resources are not spent pursuing compounds that may possess a high propensity for human toxicity. This report focuses on the application of computational molecular filters, applied either pre- or post-screening, to identify and remove known reactive and/or potentially toxic compounds from consideration in drug discovery campaigns.


Assuntos
Biologia Computacional , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Bibliotecas de Moléculas Pequenas , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/toxicidade , Humanos , Descoberta de Drogas/métodos , Biologia Computacional/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Desenho de Fármacos , Toxicologia/métodos
3.
Methods Mol Biol ; 2834: 293-301, 2025.
Artigo em Inglês | MEDLINE | ID: mdl-39312171

RESUMO

The development of novel drug candidates is a current challenge in pharmacology where therapeutic benefits must exceed side effects. Toxicology testing is therefore a fundamental step in drug discovery research. Herein, we describe the first line of toxicology testing program, consisting in cell-based high-throughput screening assays, which have the advantage of being easy, rapid, cheap, and reproducible while providing quantitative information. We illustrate MTT and Crystal Violet assays, two common colorimetric tests able to assess both cytostatic and cytotoxic effects, respectively, of a drug candidate. MTT assay allows evaluation of cellular metabolic activity, by which cell viability can be inferred; Crystal Violet staining is directly correlated with attached viable cells, thus allowing direct assessment of cell survival and death. Therefore, combination of the two methodologies represents a useful tool for predicting drug sensitivity and efficacy, the first milestones in pre-clinical toxicology workflow.


Assuntos
Sobrevivência Celular , Avaliação Pré-Clínica de Medicamentos , Violeta Genciana , Ensaios de Triagem em Larga Escala , Sais de Tetrazólio , Testes de Toxicidade , Testes de Toxicidade/métodos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Avaliação Pré-Clínica de Medicamentos/métodos , Sais de Tetrazólio/química , Ensaios de Triagem em Larga Escala/métodos , Animais , Colorimetria/métodos , Tiazóis/toxicidade
4.
J Nanobiotechnology ; 22(1): 672, 2024 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-39497197

RESUMO

BACKGROUND: The efficacy of mRNA-based vaccines and therapies relies on lipid nanoparticles (LNPs) as carriers to deliver mRNA into cells. The chemical structure of ionizable lipids (ILs) within LNPs is crucial in determining their delivery efficiency. RESULTS: In this study, we synthesized 623 alkyne-bearing ionizable lipids using the A3 coupling reaction and assessed their effectiveness in mRNA delivery. ILs with specific structural features-18-carbon alkyl chains, a cis-double bond, and ethanolamine head groups-demonstrated superior mRNA delivery capabilities. Variations in saturation, double bond placement, and chain length correlated with decreased efficacy. Alkynes positioned adjacent to nitrogen atoms in ILs reduced the acid dissociation constant (pKa) of LNPs, thereby hindering mRNA delivery efficiency. Conversion of alkynes to alkanes significantly enhanced mRNA delivery of ILs both in vitro and in vivo. Moreover, combining optimized ILs with cKK-E12 yields synergistic LNPs that showed markedly augmented mRNA expression levels in vivo. CONCLUSIONS: Overall, our study provides insights into the structure-function relationships of ILs, providing a foundation for the rational design of ILs to enhance the efficacy of LNPs in mRNA delivery.


Assuntos
Lipídeos , Nanopartículas , RNA Mensageiro , RNA Mensageiro/genética , Lipídeos/química , Animais , Camundongos , Nanopartículas/química , Humanos , Alcinos/química , Técnicas de Transferência de Genes , Feminino , Lipossomos
5.
J Agric Food Chem ; 2024 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-39495102

RESUMO

In the field of food processing, enzymes play a pivotal role in improving product quality and flavor, and extending shelf life. However, the exposure of traditional food enzymes to high temperatures during processing often leads to a decrease in activity or even inactivation, limiting the effectiveness of their application under high-temperature conditions. Therefore, the modification of thermostability and activity of enzymes to adapt to extreme conditions through protein engineering has become a key way to improve the efficiency and economic benefits of industrial production. Directed evolution and semirational design strategies in the laboratory have proven to be broadly applicable frameworks for biochemical researchers in the food field, including those who are beginners. In this review, we systematically summarize semirational design strategies and high-throughput screening strategies, and introduce some intuitive computer simulation software to improve the thermostability and enzyme activity of food enzymes. The application of these strategies and techniques provides a comprehensive guide for the optimization of food enzymes. In addition, the latest hot topics of genetically engineered food enzymes in the field of application are discussed.

6.
Enzyme Microb Technol ; 182: 110531, 2024 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-39486155

RESUMO

Polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), hold notable significance due to their pharmaceutical relevance. Obtaining PUFAs from diverse sources like vegetables, fish oils, and algae poses challenges due to the mixed fatty acid (FA) composition. Therefore, focusing on particular FAs necessitates purification and resolution processes. To address this, we propose a continuous assay for screening lipases selective for ethyl EPA (E-EPA) or ethyl DHA (E-DHA). Utilizing microplate spectrophotometry, the method enables quantification of liberated fatty acids from ethyl esters (E-EPA or E-DHA). This involves assessing enzyme selectivity by measuring the release of FAs through p-nitrophenolate protonation, either separately for each substrate or in competition with a reference substrate, resorufin acetate. Ten lipases underwent screening, revealing Burkholderia cepacia lipase's (BCL) preference for ethyl DHA hydrolysis (E-EPA/E-DHA = 0.82 ± 0.07 and the lipase selectivity ratio (S) for E-EPA/E-DHA = 0.13 ± 0.04) and Candida antarctica lipase B's (CALB) high specific activity towards both E-EPA and E-DHA (531.14 ± 37.76 and 281.79 ± 2.79 U/mg, respectively) and E-EPA preference (E-EPA/E-DHA = 1.86 ± 0.15 and S E-EPA/E-DHA = 2.59±0.15). Candida rugosa recombinant isoform 4 (rCRLip4) and commercial Candida rugosa lipase (CRL) exhibited significant preference for E-EPA hydrolysis (E-EPA/E-DHA = 2.18 ±0.51 and 2.26 ±0.36, respectively; and S E-EPA/E-DHA = 7.59 ± 1.59 and 7.88 ± 2.13, respectively). Docking analyses of rCRLip4, BCL, and CALB demonstrated no statistically significant differences in activation energies or distances to the catalytic serine; however, they agreed with the experimental results. These findings suggest potential mutagenesis or directed evolution strategies for CALB to enhance E-EPA selectivity, with rCRLip4 emerging as a promising candidate for further investigation. This assay offers a valuable tool for identifying lipases with desired substrate selectivity, with broad implications for pharmaceutical and biotechnological applications.

7.
Eur J Med Chem ; 280: 116948, 2024 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-39437576

RESUMO

Fibroblast activation protein (FAP) has been extensively studied as a cancer biomarker for decades. Recently, small-molecule FAP inhibitors have been widely adopted as a targeting moiety of experimental theranostic radiotracers. Here we present a fast qPCR-based analytical method allowing FAP inhibition screening in a high-throughput regime. To identify clinically relevant compounds that might interfere with FAP-targeted approaches, we focused on a library of FDA-approved drugs. Using the DNA-linked Inhibitor Antibody Assay (DIANA), we tested a library of 2667 compounds within just a few hours and identified numerous FDA-approved drugs as novel FAP inhibitors. Among these, prodrugs of cephalosporin antibiotics and reverse transcriptase inhibitors, along with one elastase inhibitor, were the most potent FAP inhibitors in our dataset. In addition, by employing FAP DIANA in the quantification mode, we were able to determine FAP concentrations in human plasma samples. Together, our work expands the repertoire of FAP inhibitors, analyzes the potential interference of co-administered drugs with FAP-targeting strategies, and presents a sensitive and low-consumption ELISA alternative for FAP quantification with a detection limit of 50 pg/ml.

8.
Cell Rep Methods ; 4(10): 100882, 2024 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-39437714

RESUMO

The application of CRISPR-Cas systems to genome editing has revolutionized experimental biology and is an emerging gene and cell therapy modality. CRISPR-Cas systems target off-target regions within the human genome, which is a challenge that must be addressed. Phages have evolved anti-CRISPR proteins (Acrs) to evade CRISPR-Cas-based immunity. Here, we engineer an Acr (AcrIIA4) to increase the precision of CRISPR-Cas-based genome targeting. We developed an approach that leveraged (1) computational guidance, (2) deep mutational scanning, and (3) highly parallel DNA repair measurements within human cells. In a single experiment, ∼10,000 Acr variants were tested. Variants that improved editing precision were tested in additional validation experiments that revealed robust enhancement of gene editing precision and synergy with a high-fidelity version of Cas9. This scalable high-throughput screening framework is a promising methodology to engineer Acrs to increase gene editing precision, which could be used to improve the safety of gene editing-based therapeutics.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , Genoma Humano/genética , Células HEK293 , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Reparo do DNA
9.
ACS Infect Dis ; 2024 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-39438291

RESUMO

To cause infection, bacterial pathogens must overcome host immune factors and barriers to nutrient acquisition. Reproducing these aspects of host physiology in vitro has shown great promise for antibacterial drug discovery. When used as a bacterial growth medium, human serum replicates several aspects of the host environment, including innate immunity and iron limitation. We previously reported that a high-throughput chemical screen using serum as the growth medium enabled the discovery of novel growth inhibitors overlooked by conventional screens. Here, we report that a subset of compounds from this high-throughput serum screen display an unexpected growth enhancing phenotype and are enriched for synthetic siderophores. We selected 35 compounds of diverse chemical structure and quantified their ability to enhance bacterial growth in human serum. We show that many of these compounds chelate iron, suggesting they were acting as siderophores and providing iron to the bacteria. For two different pharmacophores represented among these synthetic siderophores, conjugation to the ß-lactam antibiotic ampicillin imparted iron-dependent enhancement in antibacterial activity. Conjugation of the most potent growth-enhancing synthetic siderophore with the monobactam aztreonam produced MLEB-22043, a broad-spectrum antibiotic with significantly improved activity against Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. This synthetic siderophore-monobactam conjugate uses multiple TonB-dependent transporters for uptake into P. aeruginosa. Like aztreonam, MLEB-22043 demonstrated activity against metallo-ß-lactamase expressing bacteria, and, when combined with the ß-lactamase inhibitor avibactam, was active against clinical strains coexpressing the NDM-1 metallo-ß-lactamase and serine ß-lactamases. Our work shows that human serum is an effective bacterial growth medium for the high-throughput discovery of synthetic siderophores, enabling the development of novel Trojan Horse antibiotics.

10.
Artigo em Inglês | MEDLINE | ID: mdl-39440967

RESUMO

Ammonia (NH3) is a vital chemical compound in industry and agriculture, and the electrochemical nitrogen reduction reaction (eNRR) is considered a promising approach for NH3 synthesis. However, the development of eNRR faces the challenge of high overpotential and low Faradaic efficiency. In this work, graphyne (GY) is anchored by 3d, 4d, and 5d dual transition metal atoms to form diatomic catalysts (DACs) and is roundly investigated as an electrocatalyst for eNRR via density functional theory calculations. Due to the protrusion of anchored metal atoms, the active sites of GY are better exposed compared to other substrates, exhibiting higher activity. Through four-step hierarchical high-throughput screening (ΔG*N2 < 0 eV, ΔG*N2 â†’ *N2H < 0.4 eV, ΔG*NH2 â†’ *NH3 < 0.4 eV, and ΔG*N2 < ΔG*H), the number of selected catalysts in each step is 325, 240, 145, and 20, respectively. Considering a series of factors, including stability, initial potential, and selectivity, 13 kinds of eligible catalysts are identified. Optimal eNRR paths studies show that the best catalyst Mn2@GY features no onset potential. For the three catalysts (Mn2@GY, Ir2@GY, and RhOs@GY), the onset potentials of the most favorable eNRR pathways are -0.07, -0.12, and -0.17 V, respectively. The excellent catalytic activity can be credited to the effective charge transfer and orbital interaction between the active site and adsorbed N2. Our work demonstrates the significance of DACs for ammonia synthesis and provides a paradigm for the study of DACs even for other important reactions.

11.
J Pestic Sci ; 49(3): 148-158, 2024 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-39398507

RESUMO

Approximately 3000 Bacillus thuringiensis (Bt) isolates were screened to discover novel three-domain (3D) Cry proteins active against Helicoverpa zea (corn earworm). From 400 active isolates found during the primary screening, Cry1Ac and Cry2A, which are known to be active against H. zea, were removed using multiplex-primer PCR and high-throughput column chromatography. This process reduced the number of active cultures to 48. DNA segments encoding Domain III of these 48 cultures were amplified by PCR and sequenced. Sequencing revealed two novel Cry1B-type Domain IIIs. Further sequencing of the flanking regions of these domains revealed that one was part of Cry1Bj (GenBank: KT952325). However, the other Domain III lacked Domains I and II. Instead, this Domain III was associated with two open reading frames, ORF1 and ORF2. ORF1 was identified as an ATP-binding protein, and ORF2 as an ATPase, suggesting that Bt exchanges Domain III among homologous Cry proteins.

12.
J Microbiol Biotechnol ; 34(11): 1-10, 2024 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-39403724

RESUMO

Vibrio vulnificus, an opportunistic human pathogen, employs biofilm formation as a key survival and virulence mechanism. BrpT, a transcriptional regulator, is essential for V. vulnificus biofilm development by regulating the expression of biofilm-related genes. In this study, we aimed to identify a small molecule inhibitor of BrpT to combat V. vulnificus biofilm formation. High-throughput screening of 7,251 compounds using an Escherichia coli reporter strain carrying the arabinose-inducible brpT gene and a BrpT-activated promoter fused to the luxCDABE operon identified a hit compound, BTI (BrpT Inhibitor). BTI potently inhibited BrpT activity in V. vulnificus (EC50 of 6.48 µM) without affecting bacterial growth or host cell viability. Treatment with BTI significantly reduced the expression of the BrpT regulon and impaired biofilm formation and colony rugosity in V. vulnificus, thus increasing its susceptibility to antibiotics. In vitro biochemical analyses revealed that BTI directly binds to BrpT and inhibits its transcriptional regulatory activity. The identification of BTI as a specific inhibitor of BrpT that effectively diminishes V. vulnificus biofilm formation provides a promising foundation for the development of novel anti-biofilm strategies, with the potential to address the growing challenge of antibiotic resistance and improve the treatment of biofilm-associated infections.

13.
J Biomol Struct Dyn ; : 1-19, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39403785

RESUMO

The enzyme Asparaginyl Endopeptidase (AEP) is associated with proteinopathy-related pathologies such as Alzheimer's disease (AD) and Frontal Temporal Dementia (FTD). The onset of pathologies by AEP is due to cleaved fragments forming protein aggregates resulting in neurodegeneration. Unfortunately, there are no clinically approved small molecule inhibitors for AEP, and therefore, it serves as an unmet medical need for the design and development of potential novel small molecules. In developing potential inhibitors for proteolytic activity, a structured approach utilizing structure-based computer-aided drug design (SB-CADD) parameters was employed. This involved virtual high throughput screening (vHTS) across various CNS-focused databases enriched with diverse functionality. We identified the top sixty ligands based on the glide XP-docking score out of 10 million ligands. The free binding energy was then calculated using MM-GBSA for all top selected molecules which resulted in discovering that AEPI-1 to AEPI-6 (Asparaginyl Endopeptidase inhibitors) displayed high affinity towards the catalytic triad. Further investigation determined that all top six hits form stable complexes during 50 ns molecular dynamic simulations. We also observed that AEPI-2 demonstrated the highest stability within the binding pockets. Post-MD analyses such as DCCM, PCA, PDF, and ADMET properties were also evaluated. By bridging all the observations, we observed these six molecules occupy the active site of the ß-helix (ß1, ß3, and ß4) of the S1 pocket and additional binding sites in α1 and ß5, suggesting its suitability as a potential candidate for drug discovery against Asparaginyl Endopeptidase.

14.
Biochem J ; 481(20): 1475-1495, 2024 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-39400295

RESUMO

The cytosolic glutathione-degrading enzyme, ChaC1, is highly up-regulated in several cancers, with the up-regulation correlating to poor prognosis. The ability to inhibit ChaC1 is therefore important in different pathophysiological situations, but is challenging owing to the high substrate Km of the enzyme. As no inhibitors of ChaC1 are known, in this study we have focussed on this goal. We have initially taken a computational approach where a systemic structure-based virtual screening was performed. However, none of the predicted hits proved to be effective inhibitors. Synthetic substrate analogs were also not inhibitory. As both these approaches targeted the active site, we shifted to developing two high-throughput, robust, yeast-based assays that were active site independent. A small molecule compound library was screened using an automated liquid handling system using these screens. The hits were further analyzed using in vitro assays. Among them, juglone, a naturally occurring naphthoquinone, completely inhibited ChaC1 activity with an IC50 of 8.7 µM. It was also effective against the ChaC2 enzyme. Kinetic studies indicated that the inhibition was not competitive with the substrate. Juglone is known to form adducts with glutathione and is also known to selectively inhibit enzymes by covalently binding to active site cysteine residues. However, juglone continued to inhibit a cysteine-free ChaC1 variant, indicating that it was acting through a novel mechanism. We evaluated different inhibitory mechanisms, and also analogues of juglone, and found plumbagin effective as an inhibitor. These compounds are the first inhibitor leads against the ChaC enzymes using a robust yeast screen.


Assuntos
Inibidores Enzimáticos , Glutationa , Ensaios de Triagem em Larga Escala , Naftoquinonas , Saccharomyces cerevisiae , Humanos , Ensaios de Triagem em Larga Escala/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Naftoquinonas/farmacologia , Naftoquinonas/química , Naftoquinonas/metabolismo , Glutationa/metabolismo , Glutationa/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Cinética , Domínio Catalítico
15.
J Biomol Struct Dyn ; : 1-17, 2024 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-39413038

RESUMO

Enterococcus faecalis is commonly found in the GI tract of humans and animals. It causes various infections, especially in hospital environments, and shows growing antibiotic resistance. This study utilized a subtractive proteomics approach to find out the potential drug targets in E. faecalis. Unique metabolic pathways were analysed and compared to the host to minimize adverse effects. Among twenty nine pathogenic specific and seventy three host-pathogen common pathways identified using the KEGG database, sixty seven essential proteins were found through the DEG BLAST search. PSORTB predicted that forty cytoplasmic proteins could be suitable as druggable targets. Further analysis identified fourteen proteins with virulence properties using the VFDB BLAST. Among these, seven proteins with more than ten antigenic sites were subjected to DrugBank BLAST, identifying three novel and four existing drug targets. One of the crucial drug targets, MurM, was selected due to its critical role in peptidoglycan biosynthesis. The reason for selecting MurM is crucial for addressing antibiotic resistance, disrupting bacterial cell wall synthesis, and attaining selective antimicrobial activity. MurM belongs to the mixed αß class with two functional domains. The possible binding site residues of MurM are Trp31, Lys35, Trp38, Arg215, and Tyr219. Virtual screening identified potential lead candidates for MurM, and four were selected based on their physiochemical, pharmacokinetic, and structural properties. This study provides valuable insights into identifying and analysing a potential drug target, the MurM protein, and its inhibitors in E. faecalis V583.

16.
ACS Synth Biol ; 2024 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-39425622

RESUMO

Transcription factors (TFs) are a promising therapeutic target for a multitude of diseases. TFs perform their cellular roles by participating in multiple specific protein-protein interactions. For example, homo- or heterodimerization of some TFs controls DNA binding, while interactions between TFs and components of basal transcriptional machinery or chromatin modifiers can also be critical. While, in theory, small molecules could be used to disrupt specific protein-protein interfaces required for TF function, in practice, it is difficult to identify small molecules with the necessary specificity and efficacy, likely due to the extensive protein-protein interfaces that often underlie TF function. However, in contrast to small molecules, peptides have the potential to provide both the specificity and efficacy required to disrupt such interfaces. Here, we identified ∼15 peptides that inhibit the proliferation of leukemia cells using a high-throughput pooled screen of a library of 80-mer protein regions (peptides) derived from human nuclear-localized proteins. The antiproliferative peptides were enriched for regions known to be involved in specific TF dimerization, including the basic leucine zipper (bZIP) domain family. One of these bZIP domains, JDP2;bZIP_1, from the TF JDP2, was the top antiproliferative peptide, reducing the proliferation of K562 cells by 2-fold. JDP2;bZIP_1 inhibited AP-1 transcriptional activity and phenocopied JDP2 overexpression, suggesting that the peptide affected proliferation through a native JDP2 mechanism. Unexpectedly, given the strong conservation of the bZIP domain, residues outside of the annotated dimerization domain were critical for the peptide's antiproliferative potency. The peptide-mediated antiproliferative effect initiated erythrocyte differentiation in K562 cells and increased G0/G1 cells across multiple cell line models. We also found that many of the antiproliferative peptides identified in this study, including JDP2;bZIP_1, did not require a nuclear localization signal to function, a potential benefit for delivering these peptides in therapeutic applications.

17.
STAR Protoc ; 5(4): 103404, 2024 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-39427309

RESUMO

Tracking the localization and proximal interaction partners of endogenous proteins provides valuable functional insight. Here, we present a protocol for CRISPR-based endogenous protein tagging in mammalian cells. We describe steps for endogenously tagging human TSC22D2 and MAP4, including designing Cas9 and Cas12a guides for knockin, modularized repair template design and cloning, and procedures for lipid transfection and electroporation. This protocol accommodates Cas nucleases in plasmid expression or ribonucleoprotein complex (RNP) formats. This "endo-tagging" approach offers flexibility and broad applicability. For complete details on the use and execution of this protocol, please refer to Xiao et al.1.

18.
STAR Protoc ; 5(4): 103405, 2024 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-39427312

RESUMO

Murine trophoblast organoids present a more balanced array of trophoblast subtypes, rendering them a suitable platform for CRISPR-Cas9-based screening. Here, we present a protocol for the derivation and culture of murine trophoblast organoids from trophoblast stem cells or placentae. We describe steps for establishing and differentiating murine trophoblast organoids, the characterization of trophoblast organoids in both conditions, the generation of focused single guide RNA (sgRNA) libraries, and the subsequent screening using those libraries in murine trophoblast organoids. For complete details on the use and execution of this protocol, please refer to Mao et al.1.

19.
Cell Syst ; 15(10): 898-910.e5, 2024 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-39383860

RESUMO

De novo protein design explores uncharted sequence and structure space to generate novel proteins not sampled by evolution. A main challenge in de novo design involves crafting "designable" structural templates to guide the sequence searches toward adopting target structures. We present a convolutional variational autoencoder that learns patterns of protein structure, dubbed Genesis. We coupled Genesis with trRosetta to design sequences for a set of protein folds and found that Genesis is capable of reconstructing native-like distance and angle distributions for five native folds and three novel, the so-called "dark-matter" folds as a demonstration of generalizability. We used a high-throughput assay to characterize the stability of the designs through protease resistance, obtaining encouraging success rates for folded proteins. Genesis enables exploration of the protein fold space within minutes, unrestricted by protein topologies. Our approach addresses the backbone designability problem, showing that small neural networks can efficiently learn structural patterns in proteins. A record of this paper's transparent peer review process is included in the supplemental information.


Assuntos
Aprendizado Profundo , Dobramento de Proteína , Proteínas , Proteínas/química , Redes Neurais de Computação , Conformação Proteica , Modelos Moleculares , Algoritmos
20.
STAR Protoc ; 5(4): 103361, 2024 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-39369385

RESUMO

Acquired resistance to oncogene-targeted therapies is the major driver of mortality for patients with cancer. Here, we present a 6-to-16-week assay to model the development of acquired resistance in adherent and suspension cancer cell lines. We describe steps for determining therapeutic dose, assaying acquired resistance, and testing combination therapies. This protocol is a high-throughput, cost-effective, and scalable method to model acquired drug resistance to established and newly developed therapies. For complete details on the use and execution of this protocol, please refer to Sealover et al.1 and Theard et al.2.

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