CN115737621A - Benzyl isothiocyanate targeted binding protein and application thereof - Google Patents

Abstract

The invention discloses benzyl isothiocyanate targeted binding protein and application thereof, wherein on the basis of proteomics data, by using modified proteomics thinking for reference, a unique modification site for BITC treatment is identified by screening a protein locus point with the same offset as a drug molecule, 53 benzyl isothiocyanate targeted binding proteins in staphylococcus aureus are comprehensively analyzed, and 99 action modification sites are related in total, so that a molecular basis is provided for development and application of a BITC targeted anticancer drug or a targeted antibacterial agent.

Description

Benzyl isothiocyanate targeted binding protein and application thereof

Technical Field

The invention belongs to the technical field of biology and new medicines, and particularly belongs to benzyl isothiocyanate targeted binding protein and application thereof.

Background

Benzyl isothiocyanate BITC, with the molecular weight of 149.21g/mol and the molecular formula of C8H7NS, is lipophilic, insoluble in water, is one of the most studied isothiocyanate molecules, and exists in cruciferous vegetables, such as watercress, cabbage, cauliflower, kale and broccoli. Early studies found that benzyl isothiocyanate exhibited the same bactericidal effect against the standard staphylococcus aureus strain ATCC25923 and two clinical MRSA strains, with MICs of 10 μ g/mL. In mammals, BITC is metabolized in the liver by the degradation and binding of glutathione-S-transferase and glutamyl transpeptidase, respectively. It is reported that 62% of BITC is excreted in urine in the form of mercapturic acid. In addition to antibacterial, antifungal and anti-inflammatory properties, BITC also has clinical anticancer effects. A large number of animal experiments and epidemic researches show that benzyl isothiocyanate has a strong effect of inhibiting the growth of tumor cells, and has the effect of inhibiting the growth of lung cancer cells, liver cancer cells, prostate cancer cells, esophageal cancer cells, leukemia cells and the like. Phase II clinical studies conducted by the cancer center of Minnesota university in the United states on the effect of benzyl isothiocyanate on preventing lung cancer of healthy smoking people find that benzyl isothiocyanate can reduce the activity of carcinogens after tobacco metabolism. The plant and its natural product have the potential of inhibiting cancer, and can reduce the risk of cancer. Several previous studies have shown that daily ingestion of cruciferous vegetables helps to avoid the development of cancer. ITCs present in cruciferous vegetables have superior anti-cancer properties and inhibit cell proliferation.

Staphylococcus aureus is a food-borne pathogenic bacterium, causing food contamination that may occur in different kinds of food resources, and producing enterotoxin that may cause toxic shock and various autoimmune diseases. The hazards that staphylococcus aureus presents to food safety and public health are not negligible. The contamination of multidrug-resistant staphylococcus aureus has become a global concern about food safety and public health, and the research and development of novel antibacterial agents and the exploration of novel control technologies have been reluctant.

The activity range of the bactericide is wider than that of antibiotics. Therefore, antimicrobial products with significant antimicrobial activity against gram-negative bacteria can be a valuable source of therapeutic agents for controlling specific infections by this bacterium. Some of these hydrolysates are more effective than traditional antibiotics in inhibiting the growth of pathogenic microorganisms, such as Benzyl Isothiocyanate (BITC). BITC is one of the most important plant-derived bioactive components of Isothiocyanate (ITCs) and has antibacterial property. BITC is reported to inhibit the growth of pathogens. Early studies found that benzyl isothiocyanate has the same bactericidal effect on a standard staphylococcus aureus strain ATCC25923 and two clinical MRSA strains (methicillin-resistant strains), but the target action protein is unclear.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides 53 benzyl isothiocyanate targeted binding proteins and application thereof, wherein on the basis of proteomics data, a modified proteomics thought is used for reference, a unique modification site for BITC treatment is identified by screening a protein site group point with the same offset as a drug molecule, 53 benzyl isothiocyanate targeted binding proteins in staphylococcus aureus are comprehensively analyzed, and 99 action modification sites are involved in total, so that a molecular basis is provided for the development and application of a BITC targeted anticancer drug or a targeted antibacterial agent.

In order to achieve the purpose, the invention provides the following technical scheme:

finalization and post-replenishment

Compared with the prior art, the invention at least has the following beneficial effects:

the invention provides benzyl isothiocyanate targeted binding protein and application thereof, wherein 88 differential proteins are identified based on the inhibition effect of proteomics analysis BITC on staphylococcus aureus, wherein 76 proteins are down-regulated and 12 proteins are up-regulated. Bioinformatics analysis, proteomics identification and the like reveal that the synergistic effect of multiple targets promotes the bactericidal effect of the BITC on staphylococcus aureus. Comprehensively resolving 53 covalent binding proteins of benzyl isothiocyanate in staphylococcus aureus, wherein 99 action modification sites are involved, and providing a molecular basis for BITC targeted development and application; the proteomics-based action target identification method can indiscriminately identify unknown action targets of compounds in a complex biological system, has the advantages of high reproducibility, high accuracy and the like, gradually becomes an important means for finding the action targets of the compounds at present, and greatly promotes the target identification of active molecules; benzyl Isothiocyanate (BITC) is active essential oil derived from cruciferous plants, is commonly used for food preservation and freshness preservation, has antibacterial and antitumor activity, has great drug development value, and meets the requirement of environmental protection.

Drawings

FIG. 1 is a CshA mass spectrum; FIG. 2 is a CshB mass spectrum; FIG. 3 is an Lgt mass spectrum; FIG. 4 is a mass spectrum of transformation initiation factor IF-2; FIG. 5 is a CapG mass spectrum; FIG. 6 is a chromatogram of Cyclic-di-AMP; fig. 7 is an Ldh mass spectrum; FIG. 8 is a Pyk mass spectrum; FIG. 9 is an EzrA mass spectrum; FIG. 10 is an ABC transporter mass spectrum; FIG. 11 is a mass spectrum of Aminoglycoside 6' -N-Acetyltransferase; FIG. 12 is a Udk mass spectrum; FIG. 13 is an Aur mass spectrum; FIG. 14 is a HslU mass spectrum; FIG. 15 is a ThrE mass spectrum; FIG. 16 is a PhiSLT ORF401-like protein mass spectrum; FIG. 17 is a Phi PV83 orf 19-like protein mass spectrum; FIG. 18 is a Yyce mass spectrum; FIG. 19 is an Aldehydehydrogenic B mass spectrum; FIG. 20 is a LysR substrate binding domain protein mass spectrum; FIG. 21 is an agcS _2 mass spectrum; FIG. 22 is a mass spectrum of a Dihydrolipoamide acetyltransferase component; FIG. 23 is an Exotoxin mass spectrum; FIG. 24 is a bshC mass spectrum; FIG. 25 is a Ribosol protein L7Ae mass spectrum; FIG. 26 is a hemN mass spectrum; FIG. 27 is an infC mass spectrum; FIG. 28 is a menE-1 mass spectrum; FIG. 29 is a Toxin, beta-gram domain protein mass spectrum; FIG. 30 is a CobB mass spectrum; FIG. 31 is a nadE mass spectrum; FIG. 32 is a dapE mass spectrum; FIG. 33 is a cbiX mass spectrum; FIG. 34 is a ysdC mass spectrum; FIG. 35 shows a gltB _1 mass spectrum; FIG. 36 is a srmB mass spectrum; FIG. 37 is an argF mass spectrum; FIG. 38 is a mass spectrum of N-acetyltransferase domain-stabilizing protein; FIG. 39 is a DNA-3-methylalandine glycosylase mass spectrum; FIG. 40 is an mfd mass spectrum; FIG. 41 is a Lipoprotein mass spectrum; FIG. 42 is a gyrA _1 mass spectrum; FIG. 43 is an NADH dehydrogenase mass spectrum; FIG. 44 is a format dehydrogenation, alpha subBunit mass spectrum; FIG. 45 is a LysR family transpositional regulator mass spectrum; FIG. 46 is a mass spectrum of a reactive membrane protein; FIG. 47 is an entC mass spectrum; FIG. 48 is an aroC _3 mass spectrum; FIG. 49 is a uvrA _3 mass spectrum; FIG. 50 is a glcB _2 mass spectrum; FIG. 51 is an irtA mass spectrum; FIG. 52 is a Superfamily I DNA/RNA helicase protein mass spectrum; FIG. 53 is a Collagen adhesin mass spectrum.

Detailed Description

The invention is further described with reference to the following figures and detailed description.

The method for obtaining the BITC anti-staphylococcus aureus target protein comprises the following steps:

s1, adding a BITC solution into a staphylococcus aureus culture solution (1x107 CFU/mL) cultured to a logarithmic growth phase to enable the final concentration to reach 40 mu g/mL, and taking a group without adding BITC as a control group;

s2, culturing the sample in a shaking incubator at 37 ℃ and 200r/min for 2h, then centrifuging at 4 ℃ for 5min at 6000Xg, washing the obtained thalli precipitate for 3 times in a 1.5mL centrifugal separation tube by PBS (10mM, pH 7.4), quickly freezing by liquid nitrogen, and storing in a refrigerator at-80 ℃;

s3, respectively carrying out 3 repeated experiments on the control group and the treatment group;

s4 samples were ground to a powder in liquid nitrogen, suspended in lysis buffer containing 10mM 1, 4-Dithiothreitol (DTT) and ice-cold Acetone (AC), the suspension was incubated at-20 ℃ for 2h, then centrifuged at 4500Xg at 4 ℃ for 5min, the pellet was resuspended with 10mM DTT and ice-cold AC, and further centrifuged at 4500Xg at 4 ℃ for 5min, the pellet was collected and dissolved in lysis buffer.

S5 protein was quantified by Bradford method, diluted 5-fold with 100mM triethylammonium bicarbonate (TEAB), trypsin, 1:50 (w/w) digestion. The peptide fragments were desalted using a Strata X C18 column (Phenomenex inc., CA, USA) and dried using a vacuum centrifuge, frozen at 80 ℃ and used for quantitative proteomics analysis.

S6, analyzing the inhibiting effect of the BITC on the staphylococcus aureus based on proteomics, and carrying out protein quantitative analysis on a staphylococcus aureus sample to identify 88 differential proteins in total, wherein 76 proteins are down-regulated and 12 proteins are up-regulated.

S7, in order to further screen the BITC modified protein sites, protein sites with the same molecular weight offset as that of a drug molecule are respectively searched for a BITC processing group and a control group through library searching software maxquant, unique modified sites of the BITC processing group are identified through screening the protein sites with the same offset as that of the drug molecule, and 99 action sites are identified in total, wherein 53 proteins are involved, and the method specifically comprises the following steps: cshA protein, cshB protein, prolipoprotein diacetylglyceryl transfer enzyme protein, transfer initiation factor IF-2 protein, capG protein, cyclic-di-AMP protein, ldh protein, pyk protein, ezrA protein, ABC transporter protein, amino lysoside 6' -N-acetyl transfer enzyme protein, udk protein, aur protein, hslU protein, thrE protein, phiSLT ORF401-like protein, phi PV83 ORF 19-like protein, yyceI protein, aldehydehydogenase B protein, lysR substrate binding domain protein, agcS-2 protein, dihydrolipoamide acyl transfer enzyme protein, exoxent protein, sosmC protein, riboprotein, ribosyl transfer enzyme protein, mN protein, toxin C1 protein, beta-grandip domain protein, cobB protein, nadE protein, dapE protein, cbiX protein, ysdC protein, gltB _1 protein, srmB protein, argF protein, N-acetyltransferase domain-binding protein, DNA-3-methyleneglycosylase protein, mfd protein, lipoprotein protein, gyrA _1 protein, NADH dehydrogenase protein, formate dehydrogenase, alpha subentry protein, lysR family transmembrane regulator protein, reactive pure membrane protein, entC protein, aroC _3 protein, uvrA _3 protein, glcB _2 protein, irtA protein, superfamily I DNA/RNase protein, collagen family protein.

Specifically, the NCBI search code of the 1.CshA protein is VTY19355.1; the NCBI search code of the CshB protein is VTY31152.1; the NCBI search code of the protein diacylglycerol transferase protein is CAG9962757.1; 4.NCBI search code of transformation initiation factor IF-2 protein is KXA36252.1; NCBI search code of CapG protein SCS10250.1; NCBI search code of cyclic-di-AMP protein is BCN95209.1; the NCBI search code for ldh protein is EJU82459.1;8. NCBI search code of Pyk protein is AZL91690.1; 9.NCBI search code of EzrA protein is AZL91709.1; the NCBI search code of ABC transporter protein is CAC7089859.1; the NCBI search code of the amino-glycoside 6' -N-Acetyltra-transferase protein is SCS52960.1; the NCBI search code for udk protein is EFW32356.1; the NCBI search code of Aur protein is AZL92545.1; the NCBI search code of HslU protein is AZL91250.1; 15.NCBI search code of ThrE protein is AZL90811.1; 169HISLT ORF401-like protein has NCBI accession code OBY02146.1; the NCBI search code of phi PV83 orf 19-like protein is PHX24470.1; the NCBI search code for the 18 yci protein is UKS35359.1; the NCBI search code of the Aldehydehydrogene B protein is SCS45302.1; the NCBI search code for lysr substrate binding domain protein is KXA 397781.1; NCBI search code for agcs _2protein KXA40614.1; the NCBI search code of the dihydrolipoamide acetyltransferase component protein is CAG42804.1; the NCBI search code of the Exotoxin protein is AZL91819.1; the NCBI search code of the bshC protein is AZL91176.1; the NCBI accession code of Ribosol protein L7Ae protein is OBY01910.1; the NCBI search code of the hemN protein is UKS36683.1; NCBI search code for infc protein is OBY01036.1; the NCBI search code of the menE _1protein is EIK32941.1; the NCBI search code of Toxin, beta-gradp domain protein is EFW31165.1; NCBI search code for cobb protein EJU82514.1; the NCBI search code of nadE protein is BBA 245631.1; the NCBI accession number for the dapE protein is CPM20293.1; the NCBI search code of the cbix protein is CPL97512.1; the NCBI search code of the ysdC protein is CPL70611.1; NCBI search code for gltb _1protein BAR12687.1; the NCBI search code of srmB protein is KFD31251.1; NCBI search code for argf protein CPL88181.1; the NCBI search code of the N-acetyltransferase domain-contacting protein is TXO06693.1; the NCBI search code of the DNA-3-methyaddine saccharosidase protein is EFW31974.1; the NCBI search code of the mfd protein is EIK25642.1; the NCBI search code of the 41Lipoprotein protein is BBA23099.1; the NCBI search code of the gyrA _1protein is KKI64673.1; the NCBI search code of the NADH dehydrogenase protein is OBY02024.1; the NCBI search code of format dehydrogenation, alpha subBunit protein is KXA35290.1; the NCBI search code of the LysR family translational regulator protein is OBY02041.1; the NCBI search code of the Putative membrane protein is AEW65823.1; NCBI search code for entc protein KFD32374.1; the NCBI search code of aroC _3protein is OBY00605.1; the NCBI search code for uvra _3protein is EIK35378.1; NCBI search code for glcb _2protein SCS40645.1; NCBI search code of irta protein KFD31171.1; the NCBI search code of Superfamily I DNA/RNAhelicase protein is CCW20441.1; the NCBI search code of the Collagen adhisin protein is AAA20874.1;

example 1

The CshA protein can protect specific mrna or sRNAs from being degraded, and the survival rate of the staphylococcus aureus under the induction of toxin is improved; cshB is essential for staphylococcus aureus fatty acid homeostasis. From fig. 1, it is known that a BITC molecule exists in S (serine) at position 479, T (threonine) at position 480, K (lysine) at position 481, or K (lysine) at position 482 of the CshA protein. FIG. 2 shows that T (threonine) at 395 of CshB protein has a BITC molecule. RNA molecules are involved in a number of cellular processes. Due to their relative simplicity (consisting of only 4 distinct nucleic acid building blocks) and the molecular crowding found in the cytoplasm of cells, RNA is prone to unwanted intermolecular and intramolecular interactions. To prevent these interactions, each cell expresses proteins that contribute to the normal functioning of the RNA molecule. One major class of proteins is DEAD-box RNA helicases. DEAD-box RNA helicase is a ubiquitous enzyme consisting of a highly conserved helicase core, which contains two RecA-like domains. Within these two domains, 12 signature sequence motifs have been identified. These motifs are involved in ATP and RNA binding, ATP hydrolysis, and communication between different sites. In addition to the conserved helicase core, most DEAD-box proteins contain variable N-or C-terminal extensions. CshA is the largest of the four RNA helicases, with a specific domain at its C-terminus. CshA and the RNA helicase CshB are involved in cold adaptation. Both proteins are localized around the nucleoid, a pattern reminiscent of the ribosome or Cold Shock Protein (CSP). Further, fluorescence resonance energy transfer analysis is carried out by using CshB and cold shock protein B (CspB), and the interaction between the two proteins is proved, so that the function of the CshB in cold adaptation is supported. In studying the growth of individual mutants, different functions of DEAD-box helicases have been demonstrated. Deletion of either one of the helicase encoding genes CshA, cshB resulted in the cold sensitive phenotype. Clearly, helicases cannot be substituted for each other. Similar results were obtained with the gram-positive pathogens listeria monocytogenes and bacillus cereus. In both organisms, the absence of homologues of bacillus subtilis CshA, cshB resulted in a cold sensitive phenotype. In contrast, one previous study found that CshA was not necessary at low temperatures, and had the combined lethality of CshB mutations even at ambient temperatures. Differences may be due to the use of different vectors or different genetic backgrounds.

Example 2

Lgt bacterial lipoproteins have a wide range of important biological functions such as maintenance of the cell envelope structure, insertion and stabilization of outer membrane proteins, nutrient uptake, trafficking, adhesion, invasion and virulence. As can be seen from FIG. 3, a BITC molecule exists in T (threonine) at position 276 or R (arginine) at position 277 of prolipotein diacylgyl transferase protein. Although lipoproteins vary in structure, function, and origin, most of these lipoproteins contain N-acyldiacylglycero-cysteine as their N-terminal amino acid. The biosynthetic pathway of lipoproteins involves three reactions in gram-positive and gram-negative bacteria. Since this pathway has been shown to affect the survival of G-, these enzymes provide good potential targets for the development of future antibiotics with a broad spectrum of action.

Example 3

Transformation initiation factor IF-2 it can be seen from FIG. 4 that a BITC molecule is present in T (threonine) at position 41 or S (serine) at position 42 of the transformation initiation factor IF-2 protein. Initiation of bacterial protein synthesis or translation proceeds along a multistep pathway, with assembly of the 30s Initiation Complex (IC) being the beginning position of the pathway. Assembly of a 30s IC may occur via multiple pathways, but a preferred pathway in speed has been determined, with three IFs coupling to 30s bases and coordinately modulating the speed of tRNA coupling. IF-2 plays a central role in the overall guide path, ensuring correct selection of fMet-tRNAfMet. Initiation Factor (IF) 2 controls the fidelity of translation initiation during 30s IC assembly by selectively increasing the binding rate of the 50s ribosomal subunit to the 30s Initiation Complex (IC) carrying N-formyl-methionyl-tRNA (fMet-tRNAfMet). The research finds that the domain III of IF2 plays a key allosteric role in the activation of IF2, which indicates that the domain can be used as a target for developing novel antibiotics.

Example 4

The CapG bacterial capsular polysaccharide is a major component of biofilms that protect bacterial cells from environmental stresses. They also play an important role in pathogenicity. As shown in FIG. 5, a BITC molecule exists in the T (threonine) at position 8, the T at position 12 or the R (arginine) at position 13 of the CapG protein. The pathogenicity of staphylococcus aureus depends on successful adaptation of the microorganism to the host and the coordinated expression of virulence factors. Staphylococcus aureus is usually surrounded by a thin capsule, of which the type 5 (CP 5) and type 8 (CP 8) Capsular Polysaccharides (CPs) are the most common clinical isolates. Capsular polysaccharide forms a thick layer of carbohydrate on the cell surface, rendering it anti-phagocytosis and helping staphylococcus aureus persist in the blood of infected hosts. Enzymes that synthesize bacterial capsular polysaccharides are attractive antibacterial targets.

Example 5

Cyclic-di-AMPcyclic-di-AMP (C-di-AMP) is a bacterial signal nucleotide synthesized by a variety of human pathogens. This broad and specific bacterial product is recognized by infected host cells to trigger the innate immune response. As can be seen from FIG. 6, K (lysine) at position 337 in the Cyclic-di-AMP protein has a BITC molecule. The definite function of the secondary messenger molecule C-di-GMP in controlling gene expression has been established in various bacterial species. There is now strong evidence that cyclic dinucleotides can control biofilm formation and pathogenic gene expression in a variety of bacteria including important human pathogens such as pseudomonas aeruginosa. It has also been shown that the signal molecule is widely present in bacteria, but is not found in higher eukaryotes, and can be used as a danger signal in eukaryotic cells, facilitating the study of immunomodulatory and immunostimulatory properties of C-di-GMP. The clear identification of C-di-AMP in the cytoplasm of S.aureus and the ability to modulate its levels provides the possibility to identify target proteins or other compounds by which such cyclic dinucleotides exert their functions and modulate cellular processes. Detection of C-di-AMP in the host cytoplasm primarily leads to induction of type I interferons via the STING-cGAS signaling pivot, but is also associated with activation of the NF-kB pathway. In their long-term interactions, the host and pathogen co-evolve to control the activation of C-di-AMP by innate immunity. In bacterial terms, the amount of intracellular released C-di-AMP allows for the manipulation of host responses to exacerbate infection by avoiding immune recognition or conversely overloading through the STING-cGAS pathway.

Example 6

The main end product of the anaerobic sugar metabolism of the Ldh Staphylococcus aureus is lactic acid, which can inhibit TCA cycle, increase lactic acid level can cause up-regulation of butanediol pathway, and maintain redox balance under nitrification stress, which plays a key role in bacterial virulence and biofilm formation. From FIG. 7, it is understood that R (arginine) at position 45 of the Ldh protein has a BITC molecule. The pathogenesis of staphylococcus aureus involves multiple metabolic pathways, the pathogen having a complete TCA cycle, playing an important role in the growth and colonization of bacteria. At high glucose concentrations, the Pentose Phosphate (PP) pathway is up-regulated and the tricarboxylic acid (TCA) cycle is inhibited; in addition, oxidation of pyruvate and cytochrome content are reduced when Staphylococcus aureus is grown under high sugar conditions. The reduced activity of TCA cycle enzymes is likely due to their inhibition of biosynthesis; this phenomenon is known as glucose effect or carbon catabolite repression. Lactate dehydrogenases can be divided into two classes according to the specificity of d-or l-lactate. Class I includes L-iron lactate cytochrome c oxidoreductase (L-LCR) and NAD-dependent L-lactate dehydrogenase, while class II includes three moieties. In mammals, lactate is not only a regulator of cellular redox homeostasis, but also a shuttle of l-lactate metabolism in the mitochondria of human hepatocellular carcinoma G2 cells. In Pyricularia oryzae, d-lactic acid, a major metabolite that crosses the mitochondrial membrane gap, is oxidized to pyruvate by d-lactate dehydrogenase 1 (MoDLD 1) with the formation of FADH2, which fuels the respiratory chain of fungi.

Example 7

Pyruvate kinase Pyk catalyzes the irreversible conversion of phosphoenolpyruvate, and thus controls the level of pyruvate in the organism. Therefore, high pyruvate formation under anaerobic conditions does not contribute to energy production, but favors up-regulation of biosynthetic pathways involved in biofilm formation, one of the key pathogenic factors. From FIG. 8, it is clear that one BITC molecule is present in R (arginine) at position 264 or C (cysteine) at position 266 of the Pyk protein. S. aureus primarily harvests energy from glucose catabolism through glycolysis and the krebs cycle. The final product of glycolytic pyruvate enters the TCA cycle and regulates the energy levels associated with the pathogenicity of the organism. Pyruvate kinase (Pyk) belongs to a group of transferases coupled with the free energy of PEP hydrolysis, using K + and Mg2+ as cofactors, producing ATP and pyruvate.

Example 8

EzrA from figure 9 it is known that there is a BITC molecule for S (serine) at position 256, T (threonine) at position 257 or S at position 260 of the EzrA protein. Bacterial cell division is a highly regulated process in which cells undergo a series of temporally and spatially controlled events, producing two identical daughter cells. In almost all bacteria, cell division is initiated by the polymerization of a tubulin-like protein, ftsZ, to form a ring structure at the site of future division. This Z-loop serves as a scaffold to recruit other proteins involved in cell division, leading to the assembly of a multi-protein complex. EzrA is an intact membrane protein in the schizont complex, conserved in gram-positive bacteria with low GC content. It is well documented that the primary role of EzrA in s.aureus is to maintain proper FtsZ assembly kinetics in the metaphase of the cell, helping to coordinate cell growth and cell division, rather than preventing the Z-loop from assembling in an inappropriate position. One hypothesis that EzrA functions in cells in the metaphase of bacillus subtilis is that, as cytokine levels increase, ezrA promotes Z-loop remodeling by accelerating Z-loop disassembly. Thus, the absence of EzrA results in a stable Z-loop that may be present for a longer time in the bacterial cell cycle. In staphylococcus aureus, the septum is the only site of cell wall synthesis. Thus, it is conceivable that in the absence of EzrA, there is more time to synthesize the cell wall at the membrane, and therefore additional cell wall material may be incorporated into the membrane. A septum containing more cell wall material may result in larger hemispheres within the daughter cells, resulting in larger daughter cells. These cells may further increase in size due to cell wall synthesis scattered around the EzrA mutant cells. These mechanisms may explain the larger cell size observed in the s.aureus EzrA mutant.

Example 9

ABC transporters, ATP-binding proteins, can find ATP-binding cassette (ABC) transporters in bacteria, archaea and eukaryotes, as it is one of the largest super families. Their functional roles are very diverse, ranging from the introduction of essential nutrients into bacterial cells to the conferring of multidrug resistance on cancer cells. As can be seen from FIG. 10, a BITC molecule exists in K (lysine) at position 15 or T (threonine) at position 16 of the ABC transporter protein. ABC transporters are divided into two classes: in bacteria, ABC transporters may export substrates, including drugs and antibiotics, or mediate the absorption of essential nutrients; however, most ABC transporters found in fungi and parasites function almost exclusively as exporters, mediating substrate transfer from ATP-rich cytosol out of cells or into intracellular organelles. It is noteworthy that certain ABC transporters found in bacteria, pathogenic fungi, and parasites are associated with resistance to antimicrobial drugs. ABC transporters are of great clinical significance in mammals. For the ABC input class, inefficient ATP hydrolysis may occur without substrate, although at a slower rate than when the substrate binds. For multispecific transporters (including the multidrug transporter ABCB 1), it is not clear whether there is only one binding site or multiple sites.

Example 10

Aminoglycoside 6'-N-Acetyltransferase aminoglycosides are bactericidal antibiotics, and it can be seen from FIG. 11 that Y (tyrosine) at position 135 or S (serine) at position 138 of the Aminoglycoside 6' -N-Acetyltransferase protein has a BITC molecule. Treatment of serious infections caused by multidrug resistant (MDR) bacteria is becoming more complex and prohibitively expensive. There is therefore an urgent need to develop new therapies against these pathogens, not only to design new antibiotics, but also to find adjuvants that, in combination with existing drugs, circumvent resistance. The latter strategy extends the useful life of the antibiotics already in use, but these are becoming ineffective due to the spread of the drug resistance characteristics. Aminoglycosides can interfere with translational fidelity, produce incorrect primary sequence proteins, and thus cause a variety of toxic physiological effects, and ultimately cell death. These antibiotics play an important role in the treatment of life-threatening infections caused by gram-negative bacteria and in combination with other antibiotics in the treatment of infections caused by gram-positive bacteria. Although bacteria have developed various mechanisms to combat aminoglycosides, enzyme inactivation is most prevalent in the clinical setting.

Example 11

Udk has been reported to determine the genetic relationship between T7 bacteriophage and its host E.coli, identifying four host genes, among which the gene Udk encodes uridine/cytidine kinase, an enzyme that converts uridine and cytidine to their respective ribonucleoside monophosphates, UMP, and CMP. As can be seen from FIG. 12, K (lysine) at position 102 of Udk protein has a BITC molecule. The most effective phosphate donors for this reaction are GTP and dGTP. The udk gene is not essential for E.coli and is part of the pyrimidine ribonucleotide salvage pathway. Overexpression of udk, an E.coli gene encoding uridine/cytidine kinase, limited the growth of T7 phage.

Example 12

AurSabat et al cloned and sequenced the structural gene (aur) encoding the aureolysin (aurey), and in addition, they demonstrated that the aur gene appears in two allelic forms (type I and type II) and is strongly conserved among human S.aureus isolates, suggesting that this protease may have important housekeeping functions. From fig. 13, it can be seen that a BITC molecule is present in S (serine) at position 131 of the Aur protein. However, it is not clear whether all isolates from various livestock species retain the aur gene with both allelic forms. Furthermore, the enzymatic properties of the metalloproteases of type I and type II isolates are unknown.

Example 13

HslU from FIG. 14, it can be seen that a BITC molecule exists in the 91 st T (threonine) or 95 th Y (tyrosine) of HslU protein. ATP-dependent proteases are an important cellular machinery that plays an important role in regulating protein turnover and clearing damaged proteins. They utilize the chemical energy generated by ATP hydrolysis, convert it into mechanical force to unfold the protein substrate, and transfer it to the proteolytic chamber for degradation. These ATP-dependent protease cells separate the proteolytic active site from the cytosol, thereby preventing uncontrolled entry of cytosolic proteins into the active site. HslVU is one of ATP-dependent bi-component proteases in bacteria, and consists of HslV protease and HslU ATPase. HslV is a homologue of the 20S proteasome β subunit. It forms a barrel dodecameric IC complex by stacking hexameric rings of two identical HslV subunits, each containing an N-terminal Thr (Thr 1) proteolytically active site. A hexameric HslU atpase belonging to the AAA + atpase family binds to one or both ends of the HslV dodecamer to form an hsvu complex. In the hsvu complex, the central pores of HslU and HslV are aligned such that HslU transfers the substrate polypeptide chain to the inner proteolytic compartment of HslV via the pore channel.

Example 15

As shown in FIG. 15, T (threonine) at position 40, R (arginine) at position 41, or K (lysine) at position 44 of ThrE protein present a BITC molecule.

Example 16

As shown in FIG. 16, K (lysine) at position 105 of PhiSLT ORF401-like protein exists as a BITC molecule.

Example 17

As shown in FIG. 17, R (arginine) at position 55 of Phi PV83 orf 19-like protein presents a BITC molecule.

Example 18

As shown in fig. 18, a BITC molecule is present in S (serine) at position 77, S at position 78, or K (lysine) at position 79 of the yci protein.

Example 19

As shown in FIG. 19, there is a BITC molecule in T (threonine) at position 487, S (serine) at position 488, or K (lysine) at position 492 of the Aldehydehydrogene B protein.

Example 20

As shown in FIG. 20, a BITC molecule is present in the R (arginine) at position 170 or the R at position 171 of the LysR substrate binding domain protein.

Example 21

As shown in FIG. 21, R (arginine) at position 39 of the agcS _2 protein presents a BITC molecule.

Example 22

As shown in FIG. 22, K (lysine) at position 149, R (arginine) at position 152, or T (threonine) at position 154 of the DiHydrolipoamide acetyltransferase component protein present a BITC molecule.

Example 23

As shown in fig. 23, Y (tyrosine) at position 149 or T (threonine) at position 148 of the Exotoxin protein presents a BITC molecule.

Example 24

As shown in fig. 24, there is a BITC molecule for K (lysine) at position 305, T (threonine) at position 306, K at position 308 or K at position 309 of the bshC protein.

Example 25

As shown in FIG. 25, K (lysine) at position 78 of Ribosol protein L7Ae protein has a BITC molecule.

Example 26

As shown in FIG. 26, a BITC molecule is present in the 149 th S (serine) or 151 th S (serine) of the hemN protein.

Example 27

As shown in fig. 27, K (lysine) at position 81, K at position 82 or K at position 83 of infC protein has a BITC molecule.

Example 28

As shown in FIG. 28, Y (tyrosine) at position 490 of menE _1 protein has a BITC molecule.

Example 29

As shown in FIG. 29, there is a BITC molecule in the C (cysteine) at position 93 of the Toxin, beta-grapsp domain protein.

Example 30

As shown in FIG. 30, Y (tyrosine) at position 180 of CobB protein has a BITC molecule.

Example 31

As shown in FIG. 31, R (arginine) at position 17 or S (serine) at position 20 of nadE protein exist as a BITC molecule.

Example 32

As shown in FIG. 32, Y (tyrosine) at position 392 of dapE protein presents a BITC molecule.

Example 33

As shown in fig. 33, K (lysine) at position 245 or S (serine) at position 236 of the cbiX protein presents a BITC molecule.

Example 34

As shown in fig. 34, there is one BITC molecule present at K (lysine) at position 278 or K at position 282 of the ysdC protein.

Example 35

As shown in FIG. 35, T (threonine) at position 515, Y (tyrosine) at position 519, or K (lysine) at position 520 of the gltB _1 protein have a BITC molecule.

Example 36

As shown in FIG. 36, S (serine) at position 22 of srmB protein presents a BITC molecule.

Example 37

As shown in FIG. 37, T (threonine) at position 2 of the argF protein presents a BITC molecule.

Example 38

As shown in FIG. 38, K (lysine) at position 132 or C (cysteine) at position 133 of the N-acetyltransferase domain-stabilizing protein has a BITC molecule.

Example 39

As shown in FIG. 39, a BITC molecule exists in K (lysine) at position 57, T (threonine) at position 59, or S (serine) at position 60 of the DNA-3-methyaddine glycosylase protein.

Example 40

As shown in fig. 40, K (lysine) at position 711, T (threonine) at position 713, or K at position 714 of the mfd protein has a BITC molecule.

Example 41

As shown in FIG. 41, K (lysine) at position 362, K at position 366 or K at position 364 of the Lipoprotein protein has a BITC molecule.

Example 42

As shown in fig. 42, R (arginine) at position 372 of gyrA _1 protein has a BITC molecule.

Example 43

As shown in FIG. 43, a BITC molecule exists in the T (threonine) at position 8 or the K (lysine) at position 6 of the NADH dehydrogenase protein.

Example 44

As shown in FIG. 44, the format dehydrogenation, alpha subbunit protein, has a BITC molecule at K (lysine) at position 803, S (serine) at position 808 or Y (tyrosine) at position 812.

Example 45

As shown in FIG. 45, T (threonine) at 257 th position of the LysR family translational regulator protein has a BITC molecule.

Example 46

As shown in FIG. 46, T (threonine) at position 568 of the Putaive membrane protein has a BITC molecule.

Example 47

As shown in fig. 47, there is a BITC molecule at Y (tyrosine) at position 225 or K (lysine) at position 228 or Y at position 231 or S (serine) at position 232 or K at position 233 of the entC protein.

Example 48

As shown in FIG. 48, R (arginine) at position 47 of aroC _3 protein presents a BITC molecule.

Example 49

As shown in FIG. 49, K (lysine) at position 942 or T (threonine) at position 945 of the uvrA _3 protein present a BITC molecule.

Example 50

As shown in FIG. 50, T (threonine) at position 112, S (serine) at position 118, Y (tyrosine) at position 119, or K (lysine) at position 121 of glcB _2 protein have a BITC molecule.

Example 51

As shown in FIG. 51, T (threonine) at position 298 or Y (tyrosine) at position 300 of irtA protein exists as a BITC molecule.

Example 52

As shown in FIG. 52, a BITC molecule is present in R (arginine) at position 951 or K (lysine) at position 950 of Superfamily I DNA/RNAhelicase protein.

Example 53

As shown in FIG. 53, K (lysine) at position 962 or Y (tyrosine) at position 963 of Collagen adhesin protein has a BITC molecule.

One or more of the 53 benzyl isothiocyanate targeted binding proteins can be used for preparing a BITC targeted antibacterial agent or a BITC targeted antitumor drug; the multivalent targeting fusion protein containing the benzyl isothiocyanate targeting binding protein can also be used for preparing a BITC targeting antibacterial agent or a BITC targeting antitumor drug; the nucleic acid for coding the 53 benzyl isothiocyanate targeted binding proteins and/or the nucleic acid for coding the multivalent targeted binding protein can also be used for preparing a BITC targeted antibacterial agent or a BITC targeted antitumor drug.

The invention discloses multi-target synergistic action by adopting bioinformatics analysis, proteomics identification and the like to promote the sterilization effect of BITC on staphylococcus aureus, comprehensively analyzes the covalent binding proteins of 53 benzyl isothiocyanate in staphylococcus aureus, totally relates to 99 action modification sites, takes one or more of the covalent binding proteins of the 53 benzyl isothiocyanate as a target, develops a targeting staphylococcus aureus antibacterial agent by bioinformatics structure simulation and molecular docking aiming at the corresponding binding target, can greatly improve the sterilization effect of the antibacterial agent, reduces the generation of drug-resistant bacteria and treats clinical or food-borne staphylococcus aureus pollution.

<xnotran> (Helicobacter pylori), (Staphylococcus argenteus), (Pseudomonas aeruginosa), (Streptococcus hemolyticus), (Listeria monocytogenes), (Escherichia coli), (Cronobacter spp), (Yersinia enterocolitica), (Bacillus cereus), (Streptococcus pneumoniae), (Klebsiella pneumoniae), (Morganella spp), (Providencia spp) (Neisseria gonorrhoeae), (Salmonella enterica), (Salmonella enterica serovar Typhi), (Acinetobacter baumannii), (Enterococcus faecalis), (Enterococcus faecium), (neisseria meningitidis), (Haemophilus influenzae), (Vibrio cholerae), (Clostridioidesdifficile), (Neisseria gonorrhoeae) , BLAST 53 , . </xnotran>

Aiming at common cancer cells such as lung cancer cells, liver cancer cells, prostate cancer cells, esophageal cancer cells, leukemia cells and the like, aiming at one or more of 53 target proteins, searching corresponding homologous proteins in the cells such as the lung cancer cells, the liver cancer cells, the prostate cancer cells, the esophageal cancer cells, the leukemia cells and the like through BLAST, and aiming at the homologous proteins and BITC binding sites, performing targeted drug screening and targeted drug design to develop specific anti-cancer drugs aiming at one or more targets of a certain cancer cell.

Claims (10)

1. <xnotran> BITC , , / , CshA , cshB , prolipoprotein diacylglyceryl transferase , translation initiation factor IF-2 , capG , cyclic-di-AMP , ldh , pyk , ezrA , ABC transporter , aminoglycoside 6' -N-Acetyltra-nsferase , udk , aur , hslU , thrE , phiSLT ORF401-like , phi PV83 orf 19-like , yycI , aldehyde dehydrogenase B , lysR substrate binding domain , agcS _2 , dihydrolipoamide acetyltransferase component , exotoxin , bshC , ribosomal protein L7Ae , hemN , infC , menE _1 , toxin, beta-grasp domain , cobB , nadE , dapE , cbiX , ysdC , gltB _1 , srmB , argF , N-acetyltransferase domain-containing , DNA-3-methyladenine glycosylase , mfd , lipoprotein , gyrA _1 , NADH dehydrogenase , formate dehydrogenase, alpha subunit , lysR family transcriptional regulator , putative membrane , entC , aroC _3 , uvrA _3 , glcB _2 , irtA , superfamily IDNA/RNA helicase Collagen adhesin . </xnotran>

2. The BITC targeted antibacterial agent of claim 1, wherein the targeted binding protein is a protein targeted to bind with benzyl isothiocyanate in Staphylococcus aureus.

3.A BITC targeted antibacterial agent prepared using a protein homologous to the benzyl isothiocyanate targeted binding protein of claim 1.

4. A nucleic acid encoding the benzyl isothiocyanate targeted binding protein and/or multivalent targeted fusion protein of claim 1 or 2.

5. A nucleic acid encoding a homologous protein of the benzyl isothiocyanate targeted binding protein of claim 3.

6. Use of a nucleic acid according to claim 4 and/or claim 5 in the preparation of a BITC-targeted antibacterial agent or a BITC-targeted anticancer drug.

7.A recombinant host cell comprising one or more nucleic acids encoding a benzyl isothiocyanate target binding protein and/or a multivalent target fusion protein of claim 4 operably linked to a promoter.

8.A recombinant host cell comprising one or more nucleic acids encoding the homologous proteins to the benzyl isothiocyanate target binding protein of claim 5 operably linked to a promoter.

9. Use of a recombinant host cell according to claim 7 and/or claim 8 in the preparation of a BITC-targeted antibacterial agent or a BITC-targeted anticancer drug.

10.A BITC targeted anticancer drug prepared using the benzyl isothiocyanate targeted binding protein and/or the multivalent targeted fusion protein of the benzyl isothiocyanate targeted binding protein according to any one of claims 1 to 3, or the homologous protein according to claim 3, or the nucleic acid according to claim 4, or the recombinant host cell according to claim 7, or the recombinant host cell according to claim 8.

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