Loldf proteins or amino acid fragments and uses thereof

Abstract

The present application belongs to the field of biological medicine, and relates to recombinant LolDF protein or amino acid fragment and application thereof. Specifically, it relates to Acinetobacter baumannii isolated recombinant LolDF protein or amino acid fragment and LolDF protein complex, a preparation method of the recombinant LolDF protein complex, a screening method of anti-Acinetobacter baumannii drugs, a reagent for screening anti-Acinetobacter baumannii drugs, and application of the reagent in antibacterial drug activity analysis, structure analysis or computer drug design or drug screening, and a use of norgestrel in preparation of anti-Acinetobacter baumannii drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to LolDF protein or amino acid fragments and their applications. Background Technology

[0002] Infectious diseases are a leading cause of morbidity and mortality worldwide, with bacterial infections playing a significant role. Gram-negative bacteria dominate clinical isolates. The pathogen spectrum of Gram-negative bacteria is diverse, with Acinetobacter spp. being a notable group. Acinetobacter spp. It contains 55 species, mainly including Acinetobacter calcifera (Callic acid Acinetobacter). A. calcoaceticus Acinetobacter ruvidii ( A. lwoffi Acinetobacter baumannii ( A. baumanii Acinetobacter hemolyticus ( A. haemolytius Acinetobacter jumbo (), A. junii ) and Acinetobacter johnsonii ( A. johnsonii ), etc. Acinetobacter baumannii ( Acinetobacter baumannii Acinetobacter baumannii (AB), also known as Acinetobacter baumannii, is a Gram-negative bacterium with extremely strong vitality and widespread presence in nature. It is a common member of the Acinetobacter genus in hospital-acquired infections, typically causing bacteremia, pneumonia, meningitis, peritonitis, endocarditis, as well as urinary tract and skin infections. Currently, due to the overuse of antibiotics, Acinetobacter baumannii has developed drug resistance, becoming "multidrug-resistant Acinetobacter baumannii," and the resistance rate is showing an increasing trend year by year.

[0003] Traditional drug screening strategies require lengthy research and development processes, incurring significant costs and wasting substantial resources. The development of new drugs often lags behind the emergence and spread of drug-resistant pathogens, highlighting the urgent need for methods to screen for drugs against Acinetobacter baumannii. Summary of the Invention

[0004] This invention provides a method for screening, designing, and optimizing the LolDF protein complex of Acinetobacter baumannii, including key sites, as well as methods for the identification, design, and optimization of antimicrobial inhibitors. The "Lol system" refers to a class of proteins found in Gram-negative bacteria and represents an effective way to bypass Gram-negative bacterial defenses. The LolDF complex of Acinetobacter baumannii is a structurally unique (composed of LolD and the fusion protein LolF) and functionally essential lipoprotein transporter. This invention fills a gap in the understanding of the structure and mechanism of this disease, providing greater possibilities for screening more anti-Acinetobacter baumannii drugs.

[0005] Specifically, On one hand, the present invention provides a recombinant LolDF protein or amino acid fragment isolated from Acinetobacter baumannii, characterized in that it contains specific amino acid sites: Val44, Val47, Phe51, Leu260, Ala263, Ile264, Glu267, Lys268, Leu270, Leu359, Ala362, Tyr363, Phe364, and Leu368.

[0006] In some embodiments, the amino acid sequence of the recombinant LolDF protein or amino acid fragment is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0007] The Acinetobacter baumannii LolDF protein complex is characterized by being formed by the homodimerization of two LolD subunits and two LolF subunits.

[0008] SEQ ID NO: 1: Lolf: 1-411 MFKPISLYIGLRYTRARRSNHFISFIALVSMVGLTLGVAVLITVLSVMNGFDRELKNRVLGMVPQATVSSTQILTDWPELVKRVENHPHVTGVAPFTQLQGM LTAQGQVAGIMVTGIDPKYEKNVSIIQNHIVAGSLDSLKKGEFGIVLGKDMADSLGLRLNDSVTLVLPEATPSPAGVVPRFKRFKVVGIFSVGAEVDSMVGYI ALYDASTLLRLPDGAQGVRLKLDDIFAAPQVADDIVKNLPSNFYATNWTYTHGNLFNAIQMEKTLVGLLLVLIIVVAAFNIVSSLVMVVTDKKSDIAILRTLG ASPSMITKIFMVQGTVIGVIGTVAGTVLGVILALTISDIISWFNNVLGLNLFDAYFVHYLPSYLRWQDVTIIVIVSLLLSFLATIYPALRAAKVQPAEALRYE SEQ ID NO: 2: Transmembrane Helix 1 (TM1): 44-59: VLSVMNGFDRELKNRV SEQ ID NO: 3: Transmembrane Helix 2 (TM2): 253-274: WTYTHGNLFNAIQMEKTLVGLL SEQ ID NO: 4: Shoulder ring (SL): 354-370: VLGLNLFDAYFVHYLPS The nucleotide corresponding to SEQ ID NO: 1 is SEQ ID NO: 5. The nucleotide corresponding to SEQ ID NO: 2 is SEQ ID NO: 6. GTACTTTCTGTAATGAACGGTTTCGACCGCGAATTAAAAAATCGTGTT The nucleotide corresponding to SEQ ID NO: 3 is SEQ ID NO: 7. TGGACCTATACACATGGCAACTTATTTAATGCCATCCAAATGGAAAAAACATTAGTCGGTTTATTG The nucleotide corresponding to SEQ ID NO: 4 is SEQ ID NO: 8. GTTCTCGGACTTAATCTGTTCGATGCCTATTTTGTACATTACTTACCTTCT SEQ ID NO: 9: LolD MSKVVLEAKDIYKHFDDGKSKVEVIKGLSLQVEAGQFVSIVGASGSGKSTLLHVLGGLDQPTKGQVFLNGQRFDNLGEAERGFQRNQYLGFVYQFHHLLPEFTALENVAMPLMLRAD SQYKSVKAQAEYLLDRVGLSHRMDHKPGELSGGERQRVALARALVTKPAVVLADEPTGNLDRKTAVGIFELLTDLKKELNMAMLIVTHDEQLAQAADSILHMEDGLWVNGSHHHHHH On the other hand, the present invention also provides a LolDF protein complex comprising the LolDF protein or amino acid fragments described in the present invention, a membrane scaffold protein, and a target lipid, wherein the molar ratio of the LolDF protein or fragments, the membrane scaffold protein, and the target lipid is 1:(1.5~3):(50~200).

[0009] In some embodiments, the molar ratio of the LolDF protein or fragment, the membrane scaffold protein, and the target lipid is 1:2:110.

[0010] In some embodiments, the membrane scaffold protein is MSP1D1, MSP1E3D1, MSP1E1D1, MSP1E2D1, MSP1D1ΔH5, MSP2N2, or MSP2N3; and the target lipid is POPG, DMPC, DPPC, POPC, DSPC, DPhPC, or DOPC.

[0011] In some embodiments, the membrane scaffold protein is MSP1D1; the target lipid is POPG.

[0012] On the other hand, the present invention also provides an application of the isolated recombinant LolDF protein or amino acid fragment or the LolDF protein complex described in the present invention in antibacterial drug activity analysis, computer drug design, or drug screening.

[0013] In some embodiments, the drug is an anti-Gram-negative bacterial drug.

[0014] In some embodiments, the drug is an anti-Acinetobacter drug, specifically an anti-Acinetobacter baumannii drug.

[0015] In some embodiments, the drug is a steroid compound.

[0016] In some embodiments, the drug is norprogesterone or an analogue thereof.

[0017] On the other hand, the present invention also provides a method for preparing a recombinant LolDF protein complex, comprising: (1) Construction of expression vector and induction of expression: The genome of Acinetobacter baumannii was extracted, the complete LolDF operon was amplified and cloned into the expression vector pQlinkN, and a histidine tag was introduced at its C-terminus; the vector was transformed into host cells Escherichia coli, and the expression of the target protein was induced by IPTG under low temperature conditions; (2) Extraction and purification of transmembrane proteins: The host cells were lysed and centrifuged to obtain total cell membrane precipitate; the cell membrane was solubilized using a buffer containing detergent; and then high-purity LolDF transmembrane proteins were obtained by affinity chromatography and gel filtration chromatography in sequence. (3) Construction of nanolipid disk recombinant system: The purified LolDF protein, membrane scaffold protein and target lipid were mixed in a predetermined molar ratio; after incubation, detergent adsorption medium was added to remove free detergent and promote the self-assembly of the complex. Finally, the complex was separated and purified by gel filtration chromatography to obtain a highly active LolDF complex embedded in the nanolipid disk.

[0018] In some embodiments, the histidine tag is a short peptide consisting of 6-10 histidine residues.

[0019] In some embodiments, Escherichia coli BL21(DE3) is selected.

[0020] In some embodiments, the low temperature condition is 15 °C to 20 °C, specifically 18 °C.

[0021] In some embodiments, the detergent is Triton X-100, DDM, Digitonin, or Tween 20. DDM is preferred.

[0022] In some embodiments, the membrane scaffold protein is MSP1D1, MSP1E3D1, MSP1E1D1, MSP1E2D1, MSP1D1ΔH5, MSP2N2, or MSP2N3. MSP1D1 is preferred.

[0023] In some embodiments, the target lipid is POPG, DMPC, DPPC, POPC, DSPC, DPhPC, or DOPC. POPG is preferred.

[0024] In some embodiments, the detergent adsorption medium is Bio-Beads, Amberlite, DetergentOUT, or Pierce HiPPR. Bio-Beads are preferred.

[0025] In some embodiments, the molar ratio of LolDF protein, membrane scaffold protein and target lipid is 1:2:110.

[0026] On the other hand, the present invention also provides a method for screening drugs against Acinetobacter baumannii, using the method described in the present invention to isolate recombinant LolDF protein or amino acid fragments or LolDF protein complexes.

[0027] On the other hand, the present invention also provides a reagent for screening drugs against Acinetobacter baumannii, wherein the isolated recombinant LolDF protein or amino acid fragment or the LolDF protein complex described in the present invention is contained.

[0028] On the other hand, the present invention also provides the use of norogestrol in the preparation of anti-Acinetobacter baumannii drugs.

[0029] Detailed description Certain embodiments of the present invention will now be described in detail. The present invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials described herein can be used to practice the present invention. The present invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0030] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0031] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0032] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0033] In the following content, all numbers disclosed herein, whether or not they use words such as "approximately" or "about," are approximate values. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a number with a value of N is disclosed, any numbers with values ​​of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" indicates addition or subtraction.

[0034] The term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention, but does not exclude other aspects.

[0035] The amino acid numbering used herein follows the following rule: unless otherwise explicitly stated in the context, the amino acid numbering for a specific position in the LolF protein is based on the complete amino acid sequence of Acinetobacter baumannii LolF shown in SEQ ID NO: 1 in the sequence listing of this application, and this number includes the N-terminal initiating methionine (Met1).

[0036] When this invention refers to LolF homologous sequences from other species, or corresponding amino acid residues in artificially modified variants and mutant sequences, the position number refers to the position in which the target sequence matches the corresponding residue in SEQ ID NO: 1 in terms of spatial structure or sequence alignment after optimal global alignment with SEQ ID NO: 1. For example, if a variant has a deletion of an amino acid at the N-terminus, causing position 50 of its absolute sequence to actually correspond to position 51 of SEQ ID NO: 1, then this position of the variant is still referred to as position 51 herein.

[0037] Membrane scaffold proteins, core membrane scaffold tools in nanodisk technology, provide an ideal environment for the study of the structure and function of membrane proteins. These include, but are not limited to: MSP1D1, MSP1E3D1, MSP1E1D1, MSP1E2D1, MSP1D1ΔH5, MSP2N2, and MSP2N3. Preferably, the membrane scaffold protein is MSP1D1 (membrane scaffold protein 1D1).

[0038] The target lipid, mimicking the physiological environment of the bacterial membrane, includes, but is not limited to: 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dispalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPG), 1,2-distearatel-sn-glycerol-3-phosphocholine (DSPC), 1,2-diphydanoyl-sn-glycerol-3-phosphocholine (DPhPC), and 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). Preferably, the target lipid is POPG (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylglycerol).

[0039] pQlinkN is a member of the pQlink system, a prokaryotic expression vector specifically designed for multi-gene co-expression. It can load multiple genomes onto the same plasmid, enabling their simultaneous expression in a single E. coli cell.

[0040] IPTG (isopropyl-β-D-thiogalactoside) is a chemical inducer widely used to induce the expression of exogenous proteins in prokaryotic cells such as Escherichia coli (E. coli).

[0041] Detergents are amphiphilic molecules possessing both hydrophilic and hydrophobic structures, used to dissolve and extract membrane proteins from cell membranes. Depending on the application scenario, this invention preferably uses nonionic detergents, including but not limited to Triton X-100, DDM, Digitonin, and Tween 20.

[0042] The "descaling agent adsorption medium" is used to remove and replace the descaling agent, effectively solving the interference of the descaling agent on downstream experiments. The descaling agent adsorption medium used in the embodiments of this invention can be selected from Bio-Beads, Amberlite, DetergentOUT, and Pierce HiPPR.

[0043] The amino acid fragments discussed in this article can be obtained using prokaryotic expression systems, such as *E. coli*. Gene synthesis and amplification: Based on known nucleotide sequences, full-length DNA is chemically synthesized or amplified by PCR from existing templates. Cloning into expression vectors: The sequence is inserted into plasmids containing strong promoters (e.g., T7, lac), ribosome binding sites (RBS), and tags (e.g., His-tag). Transformation: The recombinant plasmid is transformed into *E. coli* expression strains (e.g., BL21(DE3)). Induction of expression: Inducers such as IPTG are added, and the cells are cultured at a suitable temperature for several hours. Cell disruption and purification: Cells are disrupted by sonication or high-pressure chromatography, and the target protein is purified by affinity chromatography (e.g., Ni-NTA). Validation: The amino acid sequence is confirmed by SDS-PAGE, Western blot, or mass spectrometry. The amino acid fragments discussed in this article (<50 amino acids) can also be directly chemically synthesized using solid-phase peptide synthesis. Attached Figure Description

[0044] Figure 1 The results of LolDF protein extraction and purification, and SDS-PAGE identification of the LolDF complex after assembly and purification are shown. Figure 1 a represents LolDF protein. Figure 1 b is a LolDF complex.

[0045] Figure 2 This refers to the interactions between Abaucin and surrounding amino acid sites after the Abaucin molecule is bound to the LolF pocket, including hydrogen bonds and hydrophobic interactions.

[0046] Figure 3 The image shows a cryo-electron density map of LolDF of Acinetobacter baumannii, and the binding site of the Abaucin molecule found in the electron density map, which is bound in the pocket described in the patent.

[0047] Figure 4Cryo-electron microscopy (cryo-EM) studies of single-particle LolDF: a. Representative cryo-EM images of LolDF combined with Abaucin. b. Two-dimensional class average maps of cryo-EM particle images. c. Three-dimensional classification and refinement of cryo-EM particle images. d. Local resolution estimation of the final cryo-EM image of LolDF combined with two Abaucin molecules. e. Angular distribution of particle images used for the final three-dimensional reconstruction of LolDF combined with two Abaucin molecules. f. Local resolution of the final cryo-EM image of LolDF combined with four Abaucin molecules. g. Angular distribution of particle images used for the final three-dimensional reconstruction of LolDF combined with four Abaucin molecules. h. Fourier shell correlation curve of LolDF combined with two Abaucin molecules. The final overall resolution was estimated based on the gold standard FSC=0.143 criterion. j. Comparison of cryo-EM density maps and model superpositions of LolDF combined with two and four Abaucin molecules.

[0048] Figure 5 This is a local 3D structure of the transmembrane region of the target protein, containing transmembrane helices (TM1 and TM2) of adjacent subunits (orange and pink). The green box highlights a specific loop region (SL) located above the transmembrane region, which constitutes a potential inhibitor-binding pocket or a key structural interaction interface.

[0049] Figure 6 To investigate the enzyme activity with and without Abaucin after mutating the relevant sites for Abaucin binding, we aimed to demonstrate that amino acid residues E267, F364, and L368 are key sites for the Abaucin inhibitor to bind to the AbLolDF protein and exert its targeted inhibitory effect.

[0050] Figure 7 The addition of phenorgestrel can induce a sharp decrease in the activity of the target protein ATPase, exhibiting extremely high inhibitory efficiency. After the addition of 50 µM abaucin, the enzyme activity of LolDF decreased to about 50%, while after the addition of 50 µM phenorgestrel, the enzyme activity of LolDF could be reduced to about 70%, showing a stronger inhibitory effect. Detailed Implementation

[0051] The implementation schemes of this application will be described in detail below with reference to specific embodiments. However, those skilled in the art will understand that the following embodiments are only for illustrating this application and should not be regarded as limiting the scope of this application. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased commercially.

[0052] I. Preparation of LolDF protein complex 1. Expression vector construction and induction of expression: The genome of Acinetobacter baumannii was extracted, the complete lolDF operon was amplified and cloned into the expression vector pQlinkN, and a hexahistine tag was introduced at its C-terminus; the vector was transformed into host cells Escherichia coli BL21(DE3), and the expression of the target protein was induced by IPTG under low temperature (18 °C).

[0053] 2. Extraction and purification of transmembrane proteins: The host cells were lysed and centrifuged to obtain total cell membrane precipitate; the cell membrane was solubilized using a buffer containing detergent (1% DDM); subsequently, high-purity LolDF transmembrane protein was obtained by affinity chromatography and gel filtration chromatography. Figure 1 a).

[0054] 3. Construction of the Nanodisc Recombinant System: Purified LolDF protein, membrane scaffold protein (such as MSP1D1), and target lipid (such as POPG) were mixed in a predetermined molar ratio (preferably 1:2:110); after incubation, detergent adsorption medium (such as Bio-Beads) was added to remove free detergent, promoting the self-assembly of the complex. Finally, the complex was purified by gel filtration chromatography to obtain a highly active LolDF complex embedded in a nanoliposome disk. Figure 1 b).

[0055] II. Structural analysis of the LolDF protein complex 1. Sample preparation and data collection: The nanolipid disk-LolDF complex was prepared into a cryo-mesh, and high-resolution cryo-electron microscopy image data were collected using transmission electron microscopy and direct electron detector; a variety of conformations of sample systems were constructed, including a nucleotide-free closed state, a vanadate-captured fully closed state, and a ligand-bound open state.

[0056] 2. 3D Reconstruction and Pocket Positioning: Image processing software was used to perform motion correction and 3D classification reconstruction on the images. In the reconstructed high-resolution electron density map, the precise binding position of the ligand was determined by identifying non-protein electron densities between the periplasmic TMDs that do not belong to the native amino acid main side chain of the protein and match the size and shape of the target ligand molecule. Figure 3 () Figure 4 ).

[0057] 3. Atomic Model Construction: An initial homology model was constructed using known homologous transporter structures as templates. The spatial constraints of ligand molecules were fitted to the aforementioned non-protein electron density. The specific spatial coordinates and interaction network between each amino acid site in the binding pocket and the ligand molecules were determined through real-space refinement.

[0058] The center coordinates of the grid box were determined based on the location of key amino acids in the LolDF active pocket; the grid size was set to 10 Å × 10 Å × 10 Å; the grid spacing was set to 0.375 Å to ensure coverage of the main binding site and its adjacent potential sub-binding regions, thereby achieving sufficient sampling of the dual-ligand binding space. Figure 5 ).

[0059] III. Site-directed mutagenesis and activity verification of the key binding site of the target protein AbLolDF. 1. Construction of mutant expression vectors Primer design and amplification: Using an expression plasmid containing the wild-type AbLolDF gene as a template, specific mutation primers were designed. Site-directed mutagenesis was used to mutate isoleucine I264, glutamate E267, leucine L270, tyrosine Y363, phenylalanine F364, and leucine L368 in the AbLolDF sequence to alanine (A).

[0060] Sequencing verification: The amplified products were transformed into cloning hosts, and single clones were selected for Sanger sequencing. After the sequencing results were correctly aligned, six point mutation recombinant expression plasmids, namely I264A, E267A, L270A, Y363A, F364A, and L368A, were obtained.

[0061] 2. Expression of mutant proteins and nanodisc assembly Protein expression and crude extraction: The correctly sequenced plasmids and wild-type plasmids were transformed into *E. coli* expression hosts, respectively. After IPTG induction, bacterial cells were collected and lysed, and cell membrane components were separated and extracted by ultracentrifugation. Membrane proteins were dissolved from the lipid bilayer using the detergent DDM and preliminarily purified by nickel affinity chromatography (Ni-NTA).

[0062] Nanodisc reassembly: In order to test protein activity under conditions closest to physiological environment, purified wild-type AbLolDF and its mutants were mixed and assembled with membrane scaffold protein and target lipid in the aforementioned optimized molar ratio (1:2:110).

[0063] Product purification: After assembly, unassembled contaminating proteins, empty nanodiscs and free lipids were removed by molecular sieve chromatography, and the homogeneous nanodisc component containing the target protein was collected for subsequent enzyme activity testing.

[0064] 3. ATPase enzyme activity assay and inhibition experiment Experimental grouping: The purified and assembled wild-type AbLolDF and the 6 mutant proteins were each divided into two equal groups: Experimental group (+Abaucin): Added with a predetermined concentration of Abaucin inhibitor.

[0065] Control group (-Abaucin): No inhibitor added (an equal amount of buffer was added as a blank control).

[0066] Reaction initiation and detection: Add appropriate amounts of ATP and Mg to the reaction buffer. 2+ Initiate the hydrolysis reaction. After incubation at a constant temperature for a specific time, measure the absorbance at a specific wavelength using the molybdate colorimetric method, and calculate the amount of inorganic phosphate (Pi) released by the reaction.

[0067] Activity calculation: Enzyme activity unit is defined as the number of moles of inorganic phosphate produced per minute by the hydrolysis of one mole of target protein (mol of Pi min). -1 mol -1 ).

[0068] 4. Experimental Results and Data Analysis Performance of wild-type and non-critical site mutants: As shown in the figure, the ATPase activity of wild-type AbLolDF was significantly decreased after the addition of Abaucin (**** indicates p < 0.0001). Similarly, the enzyme activity of I264A, L270A, and Y363A mutants was still significantly inhibited after the addition of Abaucin (****). Figure 6 This indicates that the I264, L270, and Y363 sites are not key binding sites for Abaucin to exert its inhibitory effect, and that mutations in these sites do not affect drug binding and inhibition.

[0069] Performance of key binding site mutants: Notably, the ATPase activity of the three mutants E267A, F364A, and L368A after the addition of Abaucin was not statistically significantly different (ns) compared with the untreated control group. This indicates that after mutations at these three amino acid sites, Abaucin loses its inhibitory ability against AbLolDF.

[0070] Conclusion: Experimental results demonstrate that amino acid residues E267, F364, and L368 are key sites for the inhibitor Abaucin to bind to AbLolDF protein and exert its targeted inhibitory effect.

[0071] III. Drug Screening 1. Construction of a drug screening platform A structural biology-based drug screening platform was developed for the efficient identification of candidate compounds with potential antibacterial activity. The platform consists of a data processing module, a molecular docking module, and a compound screening and optimization module, all integrated through a unified computational workflow.

[0072] The data processing module is used to standardize the compound library, including desalting, structural normalization and SMILES format standardization, and pre-filtering molecules that do not conform to basic medicinal chemistry rules.

[0073] The structure evaluation module employs molecular docking methods to assess the target binding ability of candidate compounds. By calculating the binding free energy between the ligand and the receptor protein, it achieves a quantitative evaluation of the binding ability. The screening and optimization module further combines medicinal chemistry rules, structural diversity analysis, and similarity assessment to perform multi-layer filtering and ranking of candidate compounds, thereby obtaining a high-quality set of candidate molecules.

[0074] 2. Virtual Screening Process This invention employs a multi-stage virtual screening strategy to screen the compound library step by step, including AI prediction screening, molecular docking screening, and compound attribute constraint screening, in order to improve the hit rate and structural diversity of candidate compounds.

[0075] (1) Compound library screening and pretreatment First, the compound library underwent structural normalization, including the removal of inorganic salts, standardization of charge states, and standardization of SMILES representation. Subsequently, the compounds were preliminarily filtered, retaining those with molecular weights in the range of 180–600 Da to ensure reasonable drug development potential.

[0076] (2) Molecular docking screening For pretreated compounds, molecular docking calculations were performed using AutoDock GPU to evaluate their binding affinity to target proteins. The docking calculations were based on the Lamarckian Genetic Algorithm (LGA) for conformational search, and the binding affinity was evaluated using the AutoDock4 empirical free energy scoring function.

[0077] To simulate the potential multi-site binding characteristics of the target protein, this invention employs a uniform grid box to cover the target binding region during docking and extends it to adjacent spatial regions to fully sample potential dual-ligand binding pockets. A dual-ligand binding pocket refers to a binding space within the target protein consisting of two spatially adjacent or partially overlapping sub-binding regions, capable of simultaneously accommodating two independent small molecule ligands and forming a stable binding conformation.

[0078] The location of the binding pocket is determined based on key functional amino acid residues of the LolDF protein, including but not limited to conserved residues involved in substrate recognition and transport. The spatial distribution of these key residues defines the active center region and serves as the basis for setting the coordinates of the grid box center.

[0079] The specific parameter settings are as follows: The center coordinates of the grid box were determined based on the location of key amino acids in the LolDF active pocket (to supplement the key amino acid location); the grid size was set to 60 Å × 60 Å × 60 Å; the grid spacing was set to 0.375 Å to ensure coverage of the main binding site and its adjacent potential sub-binding region, thereby achieving sufficient sampling of the dual-ligand binding space.

[0080] The selection criteria are as follows: • Binding affinity range: ≤ -6 kcal / mol; • Retain ligand conformations that can stably bind within the target binding pocket and have a reasonable spatial conformation; • Cluster analysis was performed on docking conformations based on an RMSD threshold of 2.0 Å, and the lowest energy conformation was selected as the representative conformation; • The binding conformation must be compatible with the space of the dual-ligand binding pocket, i.e. there should be no significant spatial conflict or severe steric hindrance.

[0081] (3) Screening constrained by drug chemical properties Compounds that pass the docking screening undergo further drug property filtering and are evaluated according to Lipinski's Rule of Five, with a maximum of one rule violation allowed. Compounds are also required to contain at least one hydrogen bond donor (HBD) or hydrogen bond acceptor (HBA) to ensure they possess the necessary target-target interaction capabilities.

[0082] (4) Screening for structural diversity and novelty The Murcko scaffold skeleton splitting method was used to classify candidate compounds into structural categories. Only the compound with the highest docking score was retained in each skeleton category to enhance the structural diversity of the candidate set.

[0083] Subsequently, the Tanimoto similarity coefficient was calculated based on the ECFP4 fingerprint, and the candidate compounds were compared with a library of known antibiotic structures for structural similarity. When the similarity coefficient was below 0.3, the compound was considered to have high structural novelty.

[0084] 3. Screening Results and Discovery of Active Compounds Through a multi-stage virtual screening process and rigorous evaluation, this invention successfully identified a candidate molecule with significant LolDF-targeting binding ability—norgestimate—from a massive compound library. Norgestimate is a clinically approved oral progestin, traditionally used primarily for female contraception and hormone replacement therapy. This invention breaks through its original indication limitations, revealing for the first time the novel biological activity of norgestimate in antibacterial infection, particularly as an inhibitor of the LolDF homodimeric lipoprotein transporter of Acinetobacter baumannii.

[0085]

[0086] Norprogesterone 4. Inhibitory effect of von Willebrand on LolDF ATPase activity Subsequent in vitro biochemical functional validation results further demonstrated that vonorgestrel exhibited a significant and potent targeted inhibitory effect on the LolDF protein of Acinetobacter baumannii. Specifically, in the ATPase hydrolysis activity test targeting the LolDF complex, the addition of vonorgestrel induced a sharp decrease in the ATPase activity of the target protein, exhibiting extremely high inhibitory efficiency. Figure 7 After the addition of 50 µM Abaucin, the enzyme activity of LolDF decreased to about 50%, while after the addition of 50 µM Norprogesterone, the enzyme activity of LolDF could be reduced to about 70%, showing a stronger inhibitory effect.

[0087] This extremely strong ATPase activity inhibition can directly cut off the key energy source required by LolDF in the process of extracting and transporting lipoproteins in the inner membrane, thereby effectively blocking the lipoprotein transport cycle necessary for pathogenicity and survival. This core biochemical validation result fully demonstrates that hornogest has excellent targeted inhibitory function against LolDF, showing its great potential for development into a novel specific antibacterial drug.

[0088] The screening platform of this invention not only verified the feasibility of discovering new targets for pathogens based on structural biology, but also successfully implemented the drug development strategy of "repurposing old drugs" by hitting noprogesterone.

[0089] Given that norgestrel is a marketed drug with extremely detailed and mature clinical human pharmacokinetic (PK) and pharmacodynamic (PD) data, as well as a clear safety evaluation profile, its secondary development or structural modification as a novel antibacterial lead compound can directly bypass the lengthy early toxicology and safety assessment stages in traditional new drug development, significantly shortening the development cycle and substantially reducing the risks associated with drug development. This discovery provides a novel therapeutic framework with significant translational value for addressing the increasingly severe crisis of multidrug-resistant (MDR) Gram-negative bacterial infections, possessing significant public health implications and substantial economic value.

[0090] This invention is not limited to the above embodiments, meaning it does not imply that the invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, all fall within the protection and disclosure scope of this invention. Furthermore, it should be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, this invention will not further describe all possible combinations.

Claims

1. A method for isolating recombinant LolDF protein or amino acid fragments from Acinetobacter baumannii, characterized in that, Contains specific amino acid sites: Val44, Val47, Phe51, Leu260, Ala263, Ile264, Glu267, Lys268, Leu270, Leu359, Ala362, Tyr363, Phe364, Leu368.

2. The method for isolating recombinant LolDF protein or amino acid fragments from Acinetobacter baumannii according to claim 1, characterized in that, The amino acid sequence of the recombinant LolDF protein or amino acid fragment is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

3. A LolDF protein complex comprising the LolDF protein or amino acid fragment of claim 1, a membrane scaffold protein, and a target lipid, wherein the molar ratio of the LolDF protein or fragment, the membrane scaffold protein, and the target lipid is 1:(1.5~3):(50~200); preferably 1:2:

110.

4. The LolDF protein complex according to claim 3, characterized in that, The membrane scaffold protein is MSP1D1, MSP1E3D1, MSP1E1D1, MSP1E2D1, MSP1D1ΔH5, MSP2N2, or MSP2N3; the target lipid is POPG, DMPC, DPPC, POPC, DSPC, DPhPC, or DOPC.

5. The application of the isolated recombinant LolDF protein or amino acid fragment as described in claim 1 or 2, or the LolDF protein complex as described in claim 3 or 4, in antimicrobial drug activity analysis, computer-aided drug design, or drug screening.

6. The application according to claim 5, wherein the drug is an anti-Acinetobacter baumannii drug; preferably, the drug is a steroidal compound; preferably, the drug is phenorphine or an analogue thereof.

7. A method for preparing a recombinant LolDF protein complex, comprising: (1) Construction of expression vector and induction of expression: The genome of Acinetobacter baumannii was extracted, the complete LolDF operon was amplified and cloned into the expression vector pQlinkN, and a histidine tag was introduced at its C-terminus; the vector was transformed into host cells Escherichia coli, and the expression of the target protein was induced by IPTG under low temperature conditions; (2) Extraction and purification of transmembrane proteins: The host cells were lysed and centrifuged to obtain total cell membrane precipitate; the cell membrane was solubilized using a buffer containing detergent; High-purity LolDF transmembrane protein was then obtained by affinity chromatography and gel filtration chromatography in sequence. (3) Construction of nanolipid disk recombinant system: The purified LolDF protein, membrane scaffold protein and target lipid were mixed in a predetermined molar ratio; after incubation, detergent adsorption medium was added to remove free detergent and promote the self-assembly of the complex. Finally, the complex was separated and purified by gel filtration chromatography to obtain a highly active LolDF complex embedded in the nanolipid disk.

8. A method for screening drugs against Acinetobacter baumannii, characterized in that, The method of isolating and recombinant LolDF protein or amino acid fragments as described in claim 1 or 2, or the LolDF protein complex as described in any one of claim 3 or 4.

9. A reagent for screening drugs against Acinetobacter baumannii, comprising the isolated recombinant LolDF protein or amino acid fragment as described in claim 1 or 2, or the LolDF protein complex as described in any one of claims 3 or 4.

10. Use of von Willebrand in the preparation of drugs against Acinetobacter baumannii.

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