The structure-activity relationships study of natural products on antibacterial activity and biofilm eradication activityAntibiotic resistance is one of the greatest threats to human health. Chronic infections and recurring bacterial infections are caused by the increasing resistance to antibiotic. The formation of biofilm is one of the reasons that cause tolerance to antibiotics. In this review, natural products like halogenated phenazine and antimicrobial peptides (anthranilic acid) are investigated. The structure-activity relationships studies are performed to measure the effect of different positions or substitutions on antibacterial and biofilm eradication activity. The modification of clinically used statin drugs are mentioned as a feasible solution to solve antibiotic resistant problem. More research should be taken on natural products as they destroy biofilms and prevent bacteria from becoming resistant.BackgroundBacteria tends to float freely as single cells. When these free-floating microorganisms are located on the surface, the microbial community is formed. Biofilm is the surface that is attached to this community (1). For example, dental caries is a typical biofilm-induced disease. The acidogenic Streptococcus sp. are the main caries-associated pathogens. The pathogens that adhere to the surface of the teeth can form dental plaque biofilm firstly (2). Then, the bacteria embedded in the biofilm produce acidic microenvironment. It leads to the inflammation around teeth, which may gradually develop into dental caries. Dental caries affects 3.5 million people globally and costs over 120 billion dollars in the USA alone (3).Antibiotics are used to inhibit the growth of microorganisms, while the overuse of antibiotics causes the increase in antibiotic resistance. Since the microbes are protected by the biofilm shield, they have become extremely resistant to antibiotics or other antimicrobial agents. In contrast, the free-floating microbes are easily killed by antibiotics or antimicrobial agents (1). Therefore, the development of new antibacterial drugs should focus not only on planktonic bacteria, but also on the potential damage to biofilms. In the United States alone, 17 million new biofilm infections occur every year, leading to 550,000 fatalities per year (1). Finding effective antibacterial therapies is becoming increasingly important. Antibacterial therapyVarious Gram-positive and Gram-negative bacteria are resistant to existent antibiotic drugs. However, the research and development of new antibiotics require a lot of money and time. Developing modification from the existing drug is a feasible solution since drugs in clinical use have existing data on pharmacokinetics and pharmacodynamics (4).Statins are commonly prescribed medicines, taken daily by almost 200 million people worldwide for primary and secondary prevention of cardiovascular diseases (5). But statins also have inherent antibacterial properties. The seven statin drugs (atorvastatin (ATV), fluvastatin (FLV), lovastatin (LVS), pitavastatin (PTV), pravastatin (PRV), rosuvastatin (RSV), and simvastatin (SMV)) that have been approved for clinical use were examined against E. coli and S. marcescens. Based on the minimum inhibitory concentration (MIC) results and structure activity relationship (SAR) studies to determine which statin is shown the highest antibacterial activity. Among seven tested statins, none of them showed significant antibacterial activity against selected Gram-negative bacteria. While simvastatin and pitavastatin-lactone show a good antibacterial effect against S.aureus infections. By comparing the chemical structure of statins, it is suggested that the lactone ring and methyl group may affect the antibacterial activity.Simvastatin with a hydrophobic ring gives antibacterial activity at MIC of 64.0 µM. Pitavastatin gives obvious antibacterial properties at MIC of 128.0 µM. However, lovastatin with lactone structure did not show any antibacterial effect. The only difference in chemical structure between simvastatin and lovastatin is the number of methyl groups in the ester side chain. It is suggested that the presence of dimethyl may determine whether the drug has an antibacterial effect. The wall teichoic acids are produced from peptidoglycan layers of many Gram-positive bacteria and contain alanine residues (6). The bacteria attached to the environment via the non-polar interactions between a methyl group and the alanine residue by Van der Waals force. The cyclopropyl ring in PTV-lactone structure is able to bind with an alanine residue by hydrophobic interactions. Thus, the decrease of number of alanine residues results in the reduction of biofilm formation (7). Meanwhile, comparing simvastatin-OH acid and pitavastatin, the compound with lipophilic lactone ring have a better antibacterial effect.Statin with antibacterial activity against S. aureus                SMV                     PTV-lactoneStatin with no significant antibacterial activity against   S. aureus                SMV-OH                    PTVStatin with no antibacterial activity against S. aureus        LVSTable 1: The antibacterial activity of simvastatin (SMV), pitavastatin (PTV)-lactone, simvastatin (SMV) hydroxy, pitavastatin (PTV) and lovastatin (LVS).Antimicrobial peptides (AMPs) Structural modification of existing drugs can only slow the development of bacterial resistance and does not reduce the resistance of bacteria to antibiotics. The protein and peptides that are isolated from plants and animals contain antimicrobial properties. This would be a better source of new therapeutic agents (8). Antimicrobial peptides (AMPs) are considered to be the potential antimicrobial agent. They are small proteins secreted by the innate immune system with high effectivity for inhibiting microorganism growth. However, AMPs are susceptible to proteolytic degradation and lose activity due to binding with serum proteins. The incorporation of unnatural amino acids may increase the bioavailability. AMPs are composed of hydrophobic and hydrophilic amino acids which mimic the properties of natural peptides like cationic charged lysine and arginine (9). The electrostatic interaction with microorganisms was mediated by positively charged amino acids on AMPs. The secondary structure of the AMPs was formed during this process which facilitated the antimicrobial effect. Lipoteichoic acid (LTA) is the major component of the membrane of Gram-positive bacteria. AMPs are able to bind with LTA, resulting in membrane disruption by pore formation. Due to the fact that the bacterial membranes contain a greater amount of anionic lipids, the peptide with the hydrophobic group can easily get partitioned into the lipid bilayer and cause bacteria died (10).The anthranilic acid scaffold was found to be peptidomimetic derivative. The different electron withdrawing groups (such as F, Cl, Br) or donating group (OCH3) substitutions were investigated to determine electronic influence on biological activity. Primary amine, tertiary amine, quaternary ammonium salts, and guanidine are represented in the side chain which would affect antimicrobial activities (Figure 2).Figure 2: Chemical structure of anthranilic acid and series of side chain.Both electrons withdrawing and donating groups resulted in improvement in minimum inhibitory concentration (MIC) value. Fluorine and bromine are the most active substitution. Bromo-substituted compound disrupts 83% of S. aureus biofilms at 64µM. While fluoro-substituted showed 92% disruption of S. aureus biofilms at 64µM. Among the four series, guanidinium compound which was designed to mimic the arginine residue showed the highest antibacterial activity with MIC value of 15.6 µM against S. aureus. As the membrane of mammalian cells is mainly composed of zwitterionic phospholipids including phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, which present net neutral charge. Therefore the guanidino cationic group would be able to bind with the membrane via electrostatic interaction (11). When guanidinium interacted with membrane, the bidentate or multiple hydrogen bonds were formed, facilitating the penetration of peptide through membrane. The drug resistance was induced during traditional chemotherapeutics treatment. The peptide is a new antibiotic that can replace traditional treatment, it can destroy biofilms and prevent bacteria from becoming resistant to AMP. Naphthylthiazole compoundsThe guanidine substitution increases the antibacterial effect on the anthranilic acid scaffold. It is suggested that guanidine may enhance the antibacterial activity as a potential functional group. Therefore, it needs to be proved whether the guanidine group as a substituent can enhance the antibacterial activity of other chemical skeletons, it is difficult to find a new chemical scaffold. Phenylthiazole as the existing chemical scaffold has the advantages of rapid sterilization and antibiofilm activity (12). A total of thirty-two naphthylthiazole derivatives were synthesized in the experiment, among which the compound with ethylenediamine and methylguanidine side chains show a good antibacterial effect against methicillin-resistant Staphylococcus aureus (MRSA).Naphthylthiazole compoundR1.MIC=64?g/mL2.MIC=16?g/mL3. MIC=64?g/mLTable 2: Naphthylthiazole compounds with different side chain and the MIC value of each derivative.Among thirty-two naphthylthiazole derivatives, all compounds with lipophilic side chain present poor antibacterial property. As can be seen in Table 2, when the side chain is replaced by a guanidine group, the compound is detected to reveal most promising anti-MRSA activity and disrupt biofilm by 76% at MIC of 16 ?g/mL. This means that guanidine is one of the most effective derivative compared to other substituents. The mechanism of action of guanidine functionalised compound is inhibiting the synthesis of bacterial cell wall (13). While the guanidinyl analog (number 1) and the dimethylguanidinyl derivative (number 3) were four-times less active than methylguanidine.Halogenated phenazinesBiofilms were considered to be medically relevant in mid-20th century, while the majority of conventional antibiotic were discovered pre-1970 (14). This could be the main reason why clinically used antibiotics like penicillin and vancomycin have no significant effect on biofilms. These clinically used antibiotics are mainly the product of microbial warfare which indicates that microbial compounds may have biofilm eradication activities.Phenazines were considered to have antibiotic metabolites through the microbial interaction between Pseudomonas aeruginosa and Staphylococcus aureus in the lungs of Cystic Fibrosis (CF) patients (14). The S. aureus lung infection often occurs in the young CF patients. As these CF patients age, Pseudomonas aeruginosa coinfects the lung and eradicates the established S. aureus biofilms through phenazine antibiotics to compete for CF patient’s lung (15). Among various phenazine analogues, halogenated phenazine (HP) are considered to have potent antibacterial activities and biofilms disruption activities. Based on the structure activity relationship (SAR) studies, the 6-, 7- and 9-position of HP scaffold would affect antibacterial activities (14).The minimum inhibitory concentration (MIC) value is measured to generate antibacterial profiles of HPs. The compounds 61 and 63 (Figure 1) report most potent antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) with MIC value less than 0.1?M, while compounds 57 and 58 are completely inactive against MRSA (MIC>100?M). In the biofilm eradication assays, 7-substituted HPs 63 analogue is the most potent biofilm eradicator against MARS (minimum biofilm eradication concentration or EMBC values=2.35?M). The compound 61 shows good biofilm eradication activities with EMBC=4.69?M.The substitution on 6 and 7 positions of HP improve the activities, while the substitution on 9-position of HP shows poor activities against bacteria. Phenazine antibiotics are a class of redox-active metabolites produced by Pseudomonas and Streptomyces bacteria. Initially, it was suggested that the phenazine heterocycle ring could generate superoxide anions which killed various bacteria and fungi (15). Several active HPs were assayed in MIC experiment against MRSA through binding with tiron (a superoxide quenching agent). However, the result indicated that the antibacterial activities were not suppressed by the addition of tiron. Thus, HPs do not produce antibacterial effect through a redox-active mechanism.              Figure 1: Halogenated phenazine (HP) and its analogues.In fact, halogenated phenazine analogues are able to bind copper (?) and iron (?) to produce antibacterial activities (16). The function groups on positions 6, 7 and 9 of the HP scaffold would affect the binding ability between metal cations and HP scaffold. The 6-position of HP (compound 64) is the distal position relative to the metal binding site of the HP scaffold. It would not affect HP-metal binding event. In addition, the bromine substitution on the 6 position enhances bacterial membrane permeability compared to other substitution like ether (16). When the 9-position of compounds 57 and 58 have both been occupied, they are proved to be inactive against MRSA. It is suspected that the steric bulk of 9-position impedes binding between metal and HP scaffold. In contrast, the potent antibacterial compound 61could chelated copper (?) and iron (?) effectively (4-6). Thus, it could be concluded that 6 and 7 position of HP would enhance the antibacterial activities. In contrast, the 9 position could reduce activities.  Conclusion Severe infections, longer hospital stays and increased mortality are caused by the increase of antimicrobial resistance. That is the biofilms protect bacteria from being detected by antibiotic agent and the clinically used antibiotics have no significant effect on disrupting biofilms. Meanwhile, it would cost much time and money to develop a new antibiotic. Few pharmaceutical companies continue discovering new antibiotics and the development of new antibiotics is far less than the mutation of bacteria. Thus, the modification of existing drugs that have antibacterial properties like statins may be one of the solutions. Because statins have existing data on pharmacokinetics and pharmacodynamics which could save time. There is a need to discover how to reduce the resistance of bacteria to antibiotics. Natural products like halogenated phenazines and antimicrobial peptides are the good starting point. Halogenated phenazines produce antibacterial and biofilm eradication activity through chelating metal (?) cations. Antimicrobial peptides (AMPs) are the small protein that secreted by the innate immune system which would be the new therapeutic agents. As the potential functional group, guanidine is added to both anthranilamides and naphthylthiazole lead compounds. The result indicates that guanidine has the probability to enhance the antibacterial activity of the compound.   References (1)  Nizalapur S, Kimyon O, Yee E, Ho K, Berry T, Manefield M, et al. Amphipathic guanidine-embedded glyoxamide-based peptidomimetics as novel antibacterial agents and biofilm disruptors. Org Biomol Chem; 2017 Feb 10; 15(9):2033-51(2)  Chen L, Jia LL, Zhang Q, Zhou XR, Liu ZQ, Wang FW, et al. 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