Scanning Mutagenesis Research Articles

Enzymatic DNA Synthesis by Engineering Terminal Deoxynucleotidyl Transferase

Template-free enzymatic approaches are considered the most promising solution for next-generation artificial DNA synthesis. However, the development of these technologies has been hampered by the lack of efficient enzymes specialized for stepwise nucleotide addition. By combining evolutionary analysis, high-throughput mutagenesis scanning, and rational design, we identified a terminal deoxynucleotidyl transferase from Zonotrichia albicollis (ZaTdT) and reshaped its catalytic cavity to better accommodate 3′-ONH2-modified nucleotides. The catalytic activity of the engineered ZaTdT for 3′-ONH2-dNTPs is 3 orders of magnitude higher than that of the commonly used mammalian TdT. The engineered ZaTdT enables highly efficient single-nucleotide extension of the growing oligonucleotide chain with an average stepwise yield of 98.7%, which makes it practical for de novo enzymatic DNA synthesis.

Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation.

Co-stimulation is critical to the function of chimeric antigen receptor (CAR) T-cells. Previously, we demonstrated that dual co-stimulation can be effectively harnessed by a parallel (p)CAR architecture in which a CD28-containing second generation CAR is co-expressed with a 4-1BB containing chimeric co-stimulatory receptor (CCR). When compared to linear CARs, pCAR-engineered T-cells elicit superior anti-tumor activity in a range of pre-clinical models. Since CD19 is the best validated clinical target for cellular immunotherapy, we evaluated a panel of CD19-specific CAR and pCAR T-cells in this study. First, we generated a panel of single chain antibody fragments (scFvs) by alanine scanning mutagenesis of the CD19-specific FMC63 scFv (VH domain) and these were incorporated into second generation CD28+CD3ζ CARs. The resulting panel of CAR T-cells demonstrated a broad range of CD19 binding ability and avidity for CD19-expressing tumor cells. Each scFv-modified CAR was then converted into a pCAR by co-expression of an FMC63 scFv-targeted CCR with a 4-1BB endodomain. When compared to second generation CARs that contained an unmodified or mutated FMC63 scFv, each pCAR demonstrated a significant enhancement of tumor re-stimulation potential and IL-2 release, reduced exhaustion marker expression and enhanced therapeutic efficacy in mice with established Nalm-6 leukemic xenografts. These data reinforce the evidence that the pCAR platform delivers enhanced anti-tumor activity through effective provision of dual co-stimulation. Greatest anti-tumor activity was noted for intermediate avidity CAR T-cells and derived pCARs, raising the possibility that effector to target cell avidity is an important determinant of efficacy.

Engineering the regiocomplementarity of an epoxide hydrolase from Rhodotorula paludigena by means of computer-aided design for the scale-up enantioconvergent hydrolysis of racemic m-nitrostyrene oxide

E. coli/rpeh, a transformed E. coli strain expressing RpEH, showed higher EH regiocomplementarity for (S)- and (R)-m-nitrostyrene oxide (mNSO). To more effectively prepare (R)-m-nitrophenyl-ethane-1,2-diol (mNPED) via hydrolysis of rac-mNSO, the regioselectivity coefficients of RpEH, especially its αS for (S)-mNSO, were further elevated by microtuning its substrate-binding pocket (SBP). By means of computer-aided design, eight residues lining the SBP of RpEH were identified, and subjected to valine scanning mutagenesis. Two mutants, RpEHY254V and RpEHL360V, were selected, hydrolyzing rac-mNSO into (R)-mNPED with enhanced eep values of 92.0% and 94.8%. Then, both Tyr254 and Leu360 were subjected to partial site-saturation mutagenesis. After screening, the best mutant RpEHL360C was obtained, whose αS and βR (96.3% and 98.6%) were larger than those (92.2% and 95.2%) of RpEH. The enantioconvergent hydrolysis of 600 mM rac-mNSO was carried out using 200 mg/mL wet cells of E. coli/rpehL360C at 20 °C for 2.5 h in 50 mL 30% Tween-20/phosphate buffer (100 mM, pH 7.0), producing (R)-mNPED with 93.4% eep and 95.7% yield. Furthermore, the analysis by molecular docking (MD) simulation revealed that, compared with RpEH, RpEHL360C more regiopreferentially attacks both the Cα in the oxirane ring of (S)-mNSO and Cβ of (R)-mNSO.

Computationally engineered ACE2 decoy binds with nanomolar affinity with the SARS-CoV-2 spike protein

Even with the availability of vaccines, therapeutic options for COVID-19 still remain highly desirable, especially in hospitalized patients with moderate or severe disease. Soluble ACE2 (sACE2) is a promising therapeutic candidate that neutralizes SARS CoV-2 infection by acting as a decoy. By using computational mutagenesis, we designed a number of sACE2 derivatives carrying 3 to 4 mutations. The top predicted sACE2 decoy based upon the in silico mutagenesis scan was subjected to molecular dynamics and free energy calculations for further validation.

Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most common cause of cystic fibrosis. The F508 residue is located on nucleotide-binding domain 1 (NBD1) in contact with the cytosolic extensions of the transmembrane helices, in particular intracellular loop 4 (ICL4). To investigate how absence of F508 at this interface impacts the CFTR protein, we carried out a mutagenesis scan of ICL4 by introducing second-site mutations at 11 positions in cis with F508del. Using an image-based fluorescence assay, we measured how each mutation affected membrane proximity and ion-channel function. The scan strongly validated the effectiveness of R1070W at rescuing F508del defects. Molecular dynamics simulations highlighted two features characterizing the ICL4/NBD1 interface of F508del/R1070W-CFTR: flexibility, with frequent transient formation of interdomain hydrogen bonds, and loosely stacked aromatic sidechains (F1068, R1070W, and F1074, mimicking F1068, F508, and F1074 in WT CFTR). F508del-CFTR displayed a distorted aromatic stack, with F1068 displaced toward the space vacated by F508, while in F508del/R1070F-CFTR, which largely retained F508del defects, R1070F could not form hydrogen bonds and the interface was less flexible. Other ICL4 second-site mutations which partially rescued F508del-CFTR included F1068M and F1074M. Methionine side chains allow hydrophobic interactions without the steric rigidity of aromatic rings, possibly conferring flexibility to accommodate the absence of F508 and retain a dynamic interface. These studies highlight how both hydrophobic interactions and conformational flexibility might be important at the ICL4/NBD1 interface, suggesting possible structural underpinnings of F508del-induced dysfunction.

Escherichia coli alanyl-tRNA synthetase maintains proofreading activity and translational accuracy under oxidative stress

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that synthesize aminoacyl-tRNAs to facilitate translation of the genetic code. Quality control by aaRS proofreading and other mechanisms maintains translational accuracy, which promotes cellular viability. Systematic disruption of proofreading, as recently demonstrated for alanyl-tRNA synthetase (AlaRS), leads to dysregulation of the proteome and reduced viability. Recent studies showed that environmental challenges such as exposure to reactive oxygen species can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. We found that while oxidation leads to modification of several residues in Escherichia coli AlaRS, unlike in other aaRSs, this does not affect proofreading activity against the noncognate substrates serine and glycine and only results in a 1.6-fold decrease in efficiency of cognate Ala-tRNAAla formation. Mass spectrometry analysis of oxidized AlaRS revealed that the critical proofreading residue in the editing site, Cys666, and three methionine residues (M217 in the active site, M658 in the editing site, and M785 in the C-Ala domain) were modified to cysteine sulfenic acid and methionine sulfoxide, respectively. Alanine scanning mutagenesis showed that none of the identified residues were solely responsible for the change in cognate tRNAAla aminoacylation observed under oxidative stress, suggesting that these residues may act as reactive oxygen species “sinks” to protect catalytically critical sites from oxidative damage. Combined, our results indicate that E. coli AlaRS proofreading is resistant to oxidative damage, providing an important mechanism of stress resistance that helps to maintain proteome integrity and cellular viability.

Development of Cobalt-Binding Peptide Chelate from Human Serum Albumin: Cobalt-Binding Properties and Stability.

Radioactive isotopes are used as drugs or contrast agents in the medical field after being conjugated with chelates such as DOTA, NOTA, DTPA, TETA, CyDTA, TRITA, and DPDP. The N-terminal sequence of human serum albumin (HSA) is known as a metal binding site, such as for Co2+, Cu2+, and Ni2+. For this study, we designed and synthesized wAlb12 peptide from the N-terminal region of HSA, which can bind to cobalt, to develop a peptide-based chelate. The wAlb12 with a random coil structure tightly binds to the Co(II) ion. Moreover, the binding property of wAlb12 toward Co(II) was confirmed using various spectroscopic experiments. To identify the binding site of wAlb12, the analogs were synthesized by alanine scanning mutagenesis. Among them, H3A and Ac-wAlb12 did not bind to Co(II). The analysis of the binding regions confirmed that the His3 and α-amino group of the N-terminal region are important for Co(II) binding. The wAlb12 bound to Co(II) with Kd of 75 μM determined by isothermal titration calorimetry when analyzed by a single-site binding model. For the use of wAlb12 as a chelate in humans, its cytotoxicity and stability were investigated. Trypsin stability showed that the wAlb12 − Co(II) complex was more stable than wAlb12 alone. Furthermore, the cell viability analysis showed wAlb12 and wAlb12 + Co(II) to be non-toxic to the Raw 264.7 and HEK 293T cell lines. Therefore, a hot radioactive isotope such as cobalt-57 will have the same effect as a stable isotope cobalt. Accordingly, we expect wAlb12 to be used as a peptide chelate that binds with radioactive isotopes.

Selectivity mechanism of BCL-XL/2 inhibition through in silico investigation.

The BCL-XL protein is among the most important members of the anti-apoptotic subfamily of the BCL-2 protein family, and is currently a promising new target for anti-tumor drug research. However, the BCL-XL/2 proteins have similar structures and functions, which could lead to undesirable side effects because of inhibitors that can bind to both BCL-XL and BCL-2. Therefore, it is crucial to expound on the structural basis of the selective mechanism towards BCL-XL/2 inhibition. In the current study, we employed hybrid computational methods including molecular docking and dynamics simulation, MM/GBSA energy calculation, alanine scanning mutagenesis and Hirshfeld surface analysis to comprehensively reveal the selectivity mechanism towards BCL-XL/2 from multiple perspectives, revealing the significant effects of the BCL-XL residues SER106 and LEU108 as well as the BCL-2 residue ASP103 on the inhibitory selectivity. Overall, our findings provide useful references for the rational design of BCL-XL/2 selective inhibitors with better affinity.

Engineering Proteins by Combining Deep Mutational Scanning and Yeast Display.

Protein engineering using display platforms such as yeast display and phage display has allowed discovery of proteins with therapeutic and industrial applications. Antibodies and T cell receptors developed for therapeutic applications are often engineered by constructing libraries of mutations in loops of five to ten residues called complementarity determining regions that are in proximity to the antigen. In the past decade, deep mutational scanning has become a powerful tool in a protein engineer's toolbox, as it allows one to compare the impact of all 20 amino acids at each position, across the length of the protein. Thus, a single experiment can provide a sequence-activity landscape with information about hotspots or suboptimal binding sites in the original proteins. These residues or regions may be overlooked by engineering methods that are driven solely by structures or directed evolution of error-prone PCR libraries. Here, we describe experimental methods to engineer proteins by combining yeast display and deep mutational scanning mutagenesis, using T cell receptors as an example.

Learning Strategies in Protein Directed Evolution.

Synthetic biology is a fast-evolving research field that combines biology and engineering principles to develop new biological systems for medical, pharmacological, and industrial applications. Synthetic biologists use iterative "design, build, test, and learn" cycles to efficiently engineer genetic systems that are reliable, reproducible, and predictable. Protein engineering by directed evolution can benefit from such a systematic engineering approach for various reasons. Learning can be carried out before starting, throughout or after finalizing a directed evolution project. Computational tools, bioinformatics, and scanning mutagenesis methods can be excellent starting points, while molecular dynamics simulations and other strategies can guide engineering efforts. Similarly, studying protein intermediates along evolutionary pathways offers fascinating insights into the molecular mechanisms shaped by evolution. The learning step of the cycle is not only crucial for proteins or enzymes that are not suitable for high-throughput screening or selection systems, but it is also valuable for any platform that can generate a large amount of data that can be aided by machine learning algorithms. The main challenge in protein engineering is to predict the effect of a single mutation on one functional parameter-to say nothing of several mutations on multiple parameters. This is largely due to nonadditive mutational interactions, known as epistatic effects-beneficial mutations present in a genetic background may not be beneficial in another genetic background. In this work, we provide an overview of experimental and computational strategies that can guide the user to learn protein function at different stages in a directed evolution project. We also discuss how epistatic effects can influence the success of directed evolution projects. Since machine learning is gaining momentum in protein engineering and the field is becoming more interdisciplinary thanks to collaboration between mathematicians, computational scientists, engineers, molecular biologists, and chemists, we provide a general workflow that familiarizes nonexperts with the basic concepts, dataset requirements, learning approaches, model capabilities and performance metrics of this intriguing area. Finally, we also provide some practical recommendations on how machine learning can harness epistatic effects for engineering proteins in an "outside-the-box" way.

Enabling biocatalysis in high-concentration organic cosolvent by enzyme gate engineering.

Biocatalysis in high-concentration organic solvents (OSs) offers many advantages, but realizing this process remains a huge challenge. An R-selective ω-amine transaminase variant (AcATAM2 ) exhibited high activity toward 50 g/L pro-sitagliptin ketone 1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione (PTfpB). However, AcATAM2 displayed unsatisfactory organic-cosolvent resistance against high-concentration dimethyl sulfoxide (DMSO), which is required to enhance the solubility of the hydrophobic substrate PTfpB. Located in the substrate-binding tunnel, enzyme gates are structural elements that undergo reversible conformational transitions, thus affecting the accessibility of the binding pocket to solvent molecules. Depending on the conformation of the enzyme gates, one can define an open or closed conformation on which the enzyme activity in OSs may depend. To enhance the DMSO resistance of AcATAM2 , we identified the beneficial residues at the "enzyme gate" region via computational analysis, alanine scanning, and site-saturation mutagenesis. Two beneficial variants, namely, AcATAM2F56D and AcATAM2F56V , not only displayed improved enzyme activity but also exhibited enhanced DMSO resistance (the half-life value increased from 25.71 to 42.49 h under 60% DMSO). Molecular dynamic simulations revealed that the increase in DMSO resistance was mainly caused by the decrease in the number of DMSO molecules in the substrate-binding pocket. Moreover, in the kilogram-scale experiment, the conversion of 80 g/L substrate was increased from 50% (AcATAM2 ) to 85% (M2F56D in 40% DMSO) with a high e.e. of >99% within 24 h.

A stepwise mutagenesis approach using histidine and acidic amino acid to engineer highly pH-dependent protein switches.

Antibody-based drugs can be highly toxic, because they target normal tissue as well as tumor tissue. The pH value of the extracellular microenvironments around tumor tissues is lower than that of normal tissues. Therefore, antibodies that engage in pH-dependent binding at slightly acidic pH are crucial for improving the safety of antibody-based drugs. Thus, we implemented a stepwise mutagenesis approach to engineering pH-dependent antibodies capable of selective binding in the acidic microenvironment in this study. The first step involved single-residue histidine scanning mutagenesis of the antibody's complementarity-determining regions to prescreen for pH-dependent mutants and identify ionizable sensitive hot-spot residues that could be substituted by acidic amino acids to obtain pH-dependent antibodies. The second step involved single-acidic amino acid residue substitutions of the identified residues and the assessment of pH-dependent binding. We identified six ionizable sensitive hot-spot residues using single-histidine scanning mutagenesis. Nine pH-dependent antibodies were isolated using single-acidic amino acid residue mutagenesis at the six hot-spot residue positions. Relative to wild-type anti-CEA chimera antibody, the binding selectivity of the best performing mutant was improved by approximately 32-fold according to ELISA and by tenfold according to FACS assay. The mutant had a high affinity in the pH range of 5.5–6.0. This study supports the development of pH-dependent protein switches and increases our understanding of the role of ionizable residues in protein interfaces. The stepwise mutagenesis approach is rapid, general, and robust and is expected to produce pH-sensitive protein affinity reagents for various applications.

Combining oligomer build-up with alanine scanning to determine the flocculation protein mutants for enhancing oligosaccharide binding

ABSTRACT Oligosaccharide binding on protein plays an important role in cellular signalling and recognition. Protein mutation can alter the binding strength and thus regulate the relevant processes. In this work, we investigated the interactions between the mannose oligosaccharides and the wild type and mutant forms of yeast flocculation protein Flo1p from Saccharomyces cerevisiae. The binding strength of an oligosaccharide chain was estimated using molecular dynamics simulation and MM/GBSA binding free energy calculation, via the build-up of oligosaccharide from monosaccharide to disaccharide, trisaccharide and tetrasaccharide. At the protein-disaccharide stage, several mutation positions were identified via alanine scanning mutagenesis, leading to nine mutants, from which two mutants Q117N and Q117R were selected with significantly stronger binding free energies relative to the wild type protein. Furthermore, the two mutants also show enhanced binding at the trisaccharide and tetrasaccharide stages. Conformational stability and shape change were analysed to help understand the protein-oligosaccharide interactions. This study also presented a computational example to estimate the oligosaccharide binding strength using its terminal sugar residues.

Molecular simulations reveal the impact of RAMP1 on ligand binding and dynamics of calcitonin gene-related peptide receptor (CGRPR) heterodimer

Calcitonin gene-related peptide receptor (CGRPR) is a heterodimer protein complex consisting of a class-B G protein-coupled receptor (GPCR) named calcitonin receptor-like receptor (CLR) and an accessory protein, receptor activity modifying protein type 1 (RAMP1). Here in this study, with several molecular modeling approaches and molecular dynamics (MD) simulations, the structural and dynamical effects of RAMP1 on the binding of small molecule CGRPR inhibitors (namely rimegepant and telcagepant) to the CGRPR extracellular ectodomain complex site (site 1) and transmembrane binding site (site 2) are investigated. Results showed that although these molecules stay stable at site 1, they can also bind to site 2, which may be interpreted as non-specificity of the ligands, however, most of these interactions at transmembrane binding site are not sustainable or are weak. Furthermore, to examine the site 2 for gepant binding, different in silico experiments (i.e., alanine scanning mutagenesis, SiteMap, ligand decomposition binding free energy analyses) are also conducted and the results confirmed the putative binding pocket (site 2) of the gepants at the CGRPRs.

Precise location of two novel linear epitopes on the receptor-binding domain surface of MERS-CoV spike protein recognized by two different monoclonal antibodies

Middle East respiratory syndrome coronavirus (MERS-CoV) is a coronavirus which can cause severe human respiratory diseases with a fatality rate of almost 36%.In this study, we report the generation, characterization and epitope mapping of several monoclonal antibodies against the spike receptor-binding domain (RBD) of MERS-CoV. Two monoclonal antibodies (4C7 and 6E8) that can react with linearized RBD have been selected for subsequent identification of RBD mAb-binding epitopes. Two distinct novel linear epitopes, 423FTCSQIS429 and 546SPLEGGGWL554,were precisely located at the outermost surface of RBD by dot-blot hybridization and ELISAs. Multiple sequence alignment analysis showed that these two peptides were highly conserved. Alanine (A)-scanning mutagenesis demonstrated that residues 423F, 428I, and 429S are the crucial residues for the linear epitope 423FTCSQIS429 while residues 548L, 550G, 553W, 554L for epitope 546SPLEGGGWL554. These findings may be helpful for further understanding of the function of RBD protein and the development of subsequent diagnosis and detection methods.

Rapid characterization of spike variants via mammalian cell surface display

SummaryThe SARS-CoV-2 spike protein is a critical component of vaccines and a target for neutralizing monoclonal antibodies (nAbs). Spike is also undergoing immunogenic selection with variants that increase infectivity and partially escape convalescent plasma. Here, we describe Spike Display, a high-throughput platform to rapidly characterize glycosylated spike ectodomains across multiple coronavirus-family proteins. We assayed ∼200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by 13 nAbs. An alanine scan of all five N-terminal domain (NTD) loops highlights a public epitope in the N1, N3, and N5 loops recognized by most NTD-binding nAbs. NTD mutations in variants of concern B.1.1.7 (alpha), B.1.351 (beta), B.1.1.28 (gamma), B.1.427/B.1.429 (epsilon), and B.1.617.2 (delta) impact spike expression and escape most NTD-targeting nAbs. Finally, B.1.351 and B.1.1.28 completely escape a potent ACE2 mimic. We anticipate that Spike Display will accelerate antigen design, deep scanning mutagenesis, and antibody epitope mapping for SARS-CoV-2 and other emerging viral threats.

Directed Evolution of a Plant Glycosyltransferase for Chemo- and Regioselective Glycosylation of Pharmaceutically Significant Flavonoids

Glycosyltransferases have attracted increasing interest for the ability to construct glycosylated molecules in a facile way. However, promiscuous chemoselectivity and poor regioselectivity hinder their widespread application in the synthetic field, especially in the pharmaceutical area. Here, a plant glycosyltransferase, MiCGT, was engineered by directed evolution to catalyze the glycosylation of flavonoids, which opens the door to pharmaceutical applications. Combining an alanine scan and iterative saturation mutagenesis, mutants with enhanced chemo- and regioselectivity and significantly improved activities toward seven different flavonoids were evolved, and two glycosylated products were prepared on a large scale. The best quadruple mutant VFAH enables strict 3-O glycosylation selectivity and a 120-fold activity enhancement toward the model substrate quercetin relative to the wild type (WT). Moreover, the crystal structures of the WT and mutant VFAH were obtained, a breakthrough of its kind in plant glycosyltransferase research. The origin of substrate specificity and regioselectivity was elucidated by combining the experimental data with the unique structure information. We anticipate that this work will aid future protein engineering of this type of enzyme.

Analysis of Hendra Virus Fusion Protein N-Terminal Transmembrane Residues.

Hendra virus (HeV) is a zoonotic enveloped member of the family Paramyoxviridae. To successfully infect a host cell, HeV utilizes two surface glycoproteins: the attachment (G) protein to bind, and the trimeric fusion (F) protein to merge the viral envelope with the membrane of the host cell. The transmembrane (TM) region of HeV F has been shown to have roles in F protein stability and the overall trimeric association of F. Previously, alanine scanning mutagenesis has been performed on the C-terminal end of the protein, revealing the importance of β-branched residues in this region. Additionally, residues S490 and Y498 have been demonstrated to be important for F protein endocytosis, needed for the proteolytic processing of F required for fusion. To complete the analysis of the HeV F TM, we performed alanine scanning mutagenesis to explore the residues in the N-terminus of this region (residues 487–506). In addition to confirming the critical roles for S490 and Y498, we demonstrate that mutations at residues M491 and L492 alter F protein function, suggesting a role for these residues in the fusion process.

Identification of a B-Cell Epitope in the VP3 Protein of Senecavirus A.

Senecavirus A (SVA) is a member of the genus Senecavirus of the family Picornaviridae. SVA-associated vesicular disease (SAVD) outbreaks have been extensively reported since 2014–2015. Characteristic symptoms include vesicular lesions on the snout and feet as well as lameness in adult pigs and even death in piglets. The capsid protein VP3, a structural protein of SVA, is involved in viral replication and genome packaging. Here, we developed and characterized a mouse monoclonal antibody (mAb) 3E9 against VP3. A motif 192GWFSLHKLTK201 was identified as the linear B-cell epitope recognized by mAb 3E9 by using a panel of GFP-tagged epitope polypeptides. Sequence alignments show that 192GWFSLHKLTK201 was highly conserved in all SVA strains. Subsequently, alanine (A)-scanning mutagenesis indicated that W193, F194, L196, and H197 were the critical residues recognized by mAb 3E9. Further investigation with indirect immunofluorescence assay indicated that the VP3 protein was present in the cytoplasm during SVA replication. In addition, the mAb 3E9 specifically immunoprecipitated the VP3 protein from SVA-infected cells. Taken together, our results indicate that mAb 3E9 could be a powerful tool to work on the function of the VP3 protein during virus infection.

Identification of raloxifene as a novel α-glucosidase inhibitor using a systematic drug repurposing approach in combination with cross molecular docking-based virtual screening and experimental verification

α-Glucosidase is a promising target for the treatment of diabetes. Drug repurposing can increase the chances of discovering an active inhibitor. Therefore, this study aimed to identify potential α-glucosidase inhibitor using drug repurposing and in silico strategies. We identified critical amino acid residues of the three α-glucosidase proteins. Based on cross molecular docking studies of three α-glucosidase proteins and drugs in the FDA database, we screened hits with the favorable binding affinities and modes targeting the three proteins. Subsequently, an in vitro activity assay showed that raloxifene was an excellent inhibitor of α-glucosidase. Moreover, molecular dynamics simulations of raloxifene and three proteins were performed to assess the stability of the protein-hit systems in physiological conditions and clarify protein-hit interactions. We also performed the binding free energy calculation, Hirshfeld surface and alanine scanning mutagenesis analyses. These results demonstrated that binding between raloxifene and the three proteins was stable, and the critical amino acid residues of the three proteins formed stable contacts with raloxifene. The molecular mechanisms agree well with its activity, reinforcing that raloxifene is a candidate α-glucosidase inhibitor. Our study smoothes the path for the development of novel a-glucosidase inhibitors with high efficacy and low toxicity for the treatment of diabetes.