Combating a Master Manipulator: Staphylococcus aureus

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Combating a Master Manipulator: Staphylococcus aureus Immunomodulatory Molecules as Targets for Combinatorial Drug Discovery Kadalipura P Rakesh, Manukumar H. Marichannegowda, Shobhith Srivastava, Xing Chen, Sihui Long, Chimatahalli S KARTHIK, Putswamappa Mallu, and Hua-Li Qin ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00088 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on October 30, 2018

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ACS Combinatorial Science

Combating a Master Manipulator: Staphylococcus aureus Immunomodulatory Molecules as Targets for Combinatorial Drug Discovery Kadalipura P. Rakesh1, Manukumar H. Marichannegowda2*, Shobhith Srivastava3, XingChen1, Sihui Long4, Chimatahalli S. Karthik2, Putswamappa Mallu2 and Hua-Li Qin1** 1Department

of Pharmaceutical Engineering, School of Chemistry, Chemical Engineering and

Life Science, Wuhan University of Technology, 205 Luoshi Road, Wuhan, 430073, PR, China 2Department

of Chemistry, Sri Jayachamarajendra College of Engineering, Mysuru-570006,

Karnataka, India 3Department

of Pharmacology and Therapeutics, King George's Medical University, Chowk,

Lucknow, 226003, India 4Key

Laboratory for Green Chemical Process of Ministry of Education, Hubei Key Laboratory of

Novel Reactor and Green Chemical Technology, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan, Hubei, China Corresponding authors *E-mail: [email protected] Fax: +91-8884354447, **E-mail: [email protected] Fax: +86 27 87749300. Fax: +86 27 87749300. Abstract Staphylococcus aureus is a bacterial pathogen that can cause significant disease burden and mortality by counteracting host defences through producing virulence factors to survive the immune responses evoked by infection. This emerging drug-resistant pathogen has led to a decline in the efficacy of traditional antimicrobial therapy. To combat these threats, precision antimicrobial therapeutics have been created to target key virulence determinants of specific

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pathogens. Here we review the benefits of, progresses in, and roadblocks to the development of precision antimicrobial therapeutics using combinatorial chemistry. Introduction Staphylococcus aureus(S. aureus) is a gram-positive, nosocomial normal human microflora that causes common skin infections which result in 11-14 million outpatient visits and nearly 500,000 hospitalizations each year in the United States. S. aureus is responsible for 76% of all skin and soft tissue infections in community and healthcare settings. It is often difficult to treat S. aureus because of its resistance to widespread antibiotics, such as in the case of methicillin-resistant S. aureus (MRSA).1 This provides an example of the many ways bacterial pathogens use to resist and escape the defensive strategies of the host immune system (Fig. 1).

Figure 1: A schematic network of how S. aureus counteracts the innate immune defence system. S. aureus colonizes the skin by neutralizing the acidic environment of human sweat via the production of ammonia (NH4+) from L-arginine catabolism. Greater colonization releases 2 ACS Paragon Plus Environment

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phenol-soluble modulins (PSMs) to recruit neutrophils, mast cells, and proteases to combat host defences. S. aureus produces many surface-anchored and secretary virulence factors, including peptides contributing to inhibition of, or escape from, the innate immune response.2 For example, the bacteria secrete proteases to destroy specific components of the host immune system, and also secrete proteins to bind precursors of host proteases that would otherwise attack the bacteria. Such host protease modulators include Coagulase (Coa) and von Willebrand factor-binding protein (vWbp), staphylokinase (SAK), α-toxins such as prothrombin, plasminogen, and A disintegrin and metalloprotease 1 (ADAM1) activation, inhibitors such as extracellular adherence protein (Eap), superantigen-like proteins 1 (SSL1) and 5 (SSL5), S. aureus collagen adhesin (Cna), Staphylococcal complement inhibitor (SCIN), extracellular fibrinogen binding protein (Efb), serine aspartate glycosyltransferases A (SdgA) and B (SdgB), CP/LP C3 proconvertase, metalloproteases, C1 complex formation, AP C3 convertase, and human neutrophil-derived

cathepsin

inhibitors.3

G

In

addition,

S. aureus adhesion during colonization and infection is also robust enough to counteract several host defence programs such as complement activation. Neutrophils are among the most important components of the immune defence mechanisms at local infection sites. They respond to chemotactic stimuli, which are identified by G protein-coupled receptors (GPCRs) such as formyl peptide receptor 1 (FPR1). The FPR1 recognizes formylated bacterial peptides, which are produced by bacterial protein biosynthesis using

formylated

methionine

and

are

characteristic

of

bacterial

infection.

S. aureus counteracts this component of immune attack by producing FPR1, a chemotaxisinhibiting protein (CHIP), to block formylated peptide recognition.In addition, S. aureus secretes a highly specific inhibitor of FPR1-related formyl peptide receptor 2 (FPR2/ALX; initially 3 ACS Paragon Plus Environment


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denoted formyl-peptide receptor-like 1, FPRL1) of human neutrophils,the FPR2/ALX-inhibitory protein (FLIPr) and the reason for secretion of FLIPr by S. aureus has remained mysterious.4 Even though the physical pathways of infection have been established, it is still not understood how the skin commensal S. aureus virulence factors cause S. aureus to become virulent.

Two

quorum

sensing

(QS)

molecules,

LuxS

and

Agr

are

known

in

S. aureus.5 The master virulence (Agr-accessory gene regulatory) program is a two-component system responding to bacterial density and is involved in controlling the expression of phenolsoluble modulins (PSMs) (Fig. 1).Among the seven PSMs, PSMα is highly cytotoxic to immune cells including keratinocytes, but which secretary factor is responsible, and the mechanism by which S. aureus counteracts keratinocytes remain unknown.6 To prevent serious infections, neutrophils function by releasing various neutrophil serine proteases (NSPs) such as neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CG) upon NSP activation from stored azurophilic granules. These NSPs cleave secreted virulence factors, receptors and chemokines to regulate immune response to the pathogen.7, 8 S. aureus produces serine protease-like (spl) virulence factors as well, via the spl operon on the vSaβ pathogenicity island, which carries six serine protease genes (splA to splF). Staphopain A (ScpA) and staphopain B (SspB) are cysteine and serine proteases and are able to combat host immune by attacking the N-terminal domains of CXCR2 and CD31, respectively.3 These secreted molecules inhibit the neutrophil serine proteases involved in ending the journey of S. aureus in the host and also extracellular adherence protein (Eap) homologs EapH1 and EapH2 act as unique class of noncovalent inhibitors, in the nanomolar range as manipulators of the immune system and innate immunity8 (Fig. 1). Apart from these interactions, pathogen takes an advantage to become resistant to antibiotic effects. In this regard, it is necessary to understand


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the multi-factorial counteracting mechanism by this pathogen and also, novel designing strategies for developing drugs to specific targets are needed.7 The urgency of antibiotic discovery, and its role in combating the rise of antimicrobial resistance, has emerged as a global challenge in modern medicine, as we aim to prevent the ultimate death of people infected by even common bacteria. Many antibiotics are developed for clinical use targeting bacterial cell-wall synthesis, DNA replication, Folic acid metabolism, membrane structure, and protein synthesis to avoid resistance.9 However, MRSA currently is resistant to standard-of-care (SOC)drugs and their clinical utility in the future is drastically diminished by the accessible targets in MRSA.10Many factors cause MRSA to become resistant through horizontal transfer of genes, such as acquiring drugresistant targets (biofilm forming genes, β-lactamase genes, and ion channel genes) by bypass mechanisms, and mutations (Fig. 2). Four key mechanisms of antibiotic-resistance developed by MRSA include efflux pump, alteration in drug target, enzymatic hydrolysis of the active drugs and changing the membrane permeability to antibiotics.11,12



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Figure 2: The different drug targets in S. aureus and clinical trials for discovering promising future antibiotic candidates. As a consequence, over the last few decades many pharmaceutical companies have started screening early-stage molecules against new targets and have invested a huge amount of money to such programs but gained little success in treating multi-drug resistant pathogens.13,14 Thus, the huge investments did not lead to profitable products and accordingly, many companies have exited this therapeutic area (Fig. 2). Due to huge global burden of S. aureus and its epidemic nature currently only a few antibiotic treatments are left such as Clindamycin15,16, Doxycycline17, Linezolid15,18,19,Trimethoprim-sulfamethoxazole (TMP-SMX)20,21, Daptomycin15, 22-24,

Teicoplanin25,26, and Vancomycin15 etc.

Combinatorial strategy: Discovery and development of new antibiotics From the early 1960s through the 1970s, many antibiotics were developed using natural sources and chemicals via a semi-synthetic approach. As a consequence of the over/misuse of broad-spectrum antibiotics, drug-resistant bacteria emerged within a short period of time. With advancements in molecular biology, bacterial genome analysis, and targeted-drug discovery by combinatorial chemistry approaches, a number of promising lead compounds were identified and optimized as potential targets. According to FDA, due to bacterial resistance the approved drugs are losing their efficacy to resistant bacterial strains, and to overcome this problem in treating infectious bacteria, new drug designing approaches are needed for future treatment.27 But, after decade-long efforts, expected results did not become a reality, for several reasons, including: i) the inability of the lead compounds to cross the bacterial cell wall, and ii) the narrow spectrum of the biocidal lead compounds did not meet required criteria for further development. The pharmaceutical companies primarily focused on developing broad-spectrum antibiotics due to


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financial incentives. This critical fiscal goal in drug discovery leads to a quest for ‘blockbuster drugs’ i.e., drugs generating more than $1 billion of revenue yearly. In this regard, new combinatorial approaches are needed to design promising and efficient drug-like candidates, which are used for clinical trials in future to treat bacterial infections. The majority of aminoglycoside antibiotics are isolated from soil bacteria streptomyces and acetinomycetes species. After penicillin, streptomycin [1] is the second antibiotic which was employed in clinical trials whereas it was the first drug in the class of aminoglycoside isolated in 1944 by Waksman.28,29 In 1949, Waksman identified Neomycin [14] but due to its poor bio availability, cell penetration and toxicity, it is only used in topical applications. Kanamycin [3] was identified in 1957 and it was widely used until the discovery of Gentamicin [2]. Gentamicin was isolated in 1963 from Micromonospora and it was a clinically successful candidate. After Gentamicin success, researchers were interested to search for similar antibiotics and identified some of them such as Tobramycin [5], Dibekacin [6], Amikacin [7], Arbekacin [8], Isepamicin [9], Gentamicin B [10], Gentamicin C1 [11], Gentamicin C2 [12], Gentamicin C1A [13]. Aminoglycoside antibiotics of 2-deoxy streptamine derivatives attached to 4,5,6-substitutents of amino sugars are powerful broad-spectrum antibiotics which target a rRNA helix of the mRNAtRNA decoding centre of the bacterial 30S ribosomal subunit,30-32 thus affecting the accuracy of bacterial translation and causing inhibition of cell growth. These antibiotics specifically bind to the functional RNA-A decoding site (Fig. 3, Table 1).33-36 Due to increased bacterial resistance to aminoglycosides, a new approach leads to semi-synthetic glycosides in which Amikacin [7] is the first in class clinically tested.



H 2N OH N OH

OH O HO HO

NHO H 3C OHC

HO O O N OH

HO

NH2

H 3C HN H 3C

NH2

H N CH32

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O

CH3

H 2N HO

OH H 2N

NH2

O NH2

2

1

R R4

R2 HN

R1

3

O R5

2-Deoxy streptamine NH2

O HO O

HO

R6 NH R10 O R8

HN R

7

R9

Figure 3: Representative powerful antibiotics derived from aminoglycosides against S. aureus. Table 1: The derivatives of the 2-deoxy streptomine used for the synthesis of antibiotics Antibiotics

R1

R2

R3

R4

R5

R6

R7

R8

R9

R10

3.

Kanamycin A

H

H

OH

OH

OH

H

H

H

OH

CH2OH

4.

Kanamycin B

H

H

OH

OH

NH2

H

H

H

OH

CH2OH

5.

Tobramycin

H

H

OH

H

NH2

H

H

H

OH

CH2OH

6.

Dibekacin

H

H

H

H

NH2

H

H

H

OH

CH2OH

7.

Amikacin

H

H

OH

OH

OH

COCHOH

H

H

OH

CH2OH

H

H

OH

CH2OH

H

H

OH

CH2OH

(CH2)2NH2 8.

Arbekacin

H

H

H

H

NH2

COCHOH (CH2)2NH2

9.

Isepamicin

H

H

OH

OH

OH

COCHOH CH2NH2

10. Gentamicin B

H

H

OH

OH

OH

H

CH3

OH

CH3

H

11. Gentamicin C1

CH3

CH3

H

H

NH2

H

CH3

OH

CH3

H 8

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12. Gentamicin C2 13. Gentamicin C1A

CH3

H

H

H

NH2

H

CH3

OH

CH3

H

H

H

H

H

NH2

H

CH3

OH

CH3

H

After streptomycin use against Mycobacterium tuberculosis, and penicillin as a second compound employed for the therapy, subsequent compounds are also developed, such as neomycin [14] used for tropical application due to poor bioavailability and paromomycin [15] not used systematically due to poor potentency.37

HO HO HO

R O NH2 O O O HO

H 2N

HO

NH2 NH2

OH

OH

O

NH2 14 = Neomycin B, R = NH2 15 = Paromomycin, R = OH

The amino and hydroxyl groups of aminoglycoside antibiotics contribute to the binding site in RNA. To better understand the mechanism, tobramycin was deoxygenated in a combinatorial approach 4ʹ-deoxy, 2ʹ-deoxy and 4ʹʹ-deoxy tobramycins [16-19] showed potent nature against S. aureus (Fig. 4, Table 2).37

3

R H 2N

O HO

R

2

OH

HO

R4

O H 2N

O

R1 NH2

NH2

Figure 4: The combinatorial approaches for tobramycin derivitization. Table 2: The derivatives of Tobramycin antibiotic from a deoxy generation approach.



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Compounds/derivatives

R1

R2

R3

R4

5

Tobramycin

OH

OH

OH

OH

16

4’- deoxy-tobramycin

H

OH

OH

OH

17

2’- deoxy-tobramycin

OH

H

OH

OH

18

4”- deoxy-tobramycin

OH

OH

H

OH

19

6- deoxy-tobramycin

OH

OH

OH

H

S. aureus becomes resistant to the aminoglycosides due to decrease in drug uptake, accumulation and expression of the bacterial enzymes to modify the antibiotic to inactivate it and many other reasons (Fig. 2).38,39 There is a need for understanding how to design a drug to escape the modifying enzymes which are involved in the bacterial resistance. Some of the bacterial enzymes modify the aminoglycosides by catalysing the covalent modification at specific amino and hydroxyl functions, which leads to modifying the drug and the energy-dependent phase II [EDP-II] accelerated drug uptake. The enzymes modifying aminoglycosides include Nacetyltransferases (AAC), which use acetyl-coenzyme A as donor and affect amino functions, and O-nucleotidyltransferases (ANT) and O-phosphotransferases (APH), which both use ATP as donor and affecthydroxyl functions. The functions affected in typical aminoglycosides (kanamycin and gentamicin derivatives) are on positions3, 2ʹ, and 6ʹ for AAC, positions 4ʹ and 2ʹʹ for ANT; andpositions 3ʹ and 2ʹʹ for APH (Fig. 5).40


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AAC(2') - G, T, N, Dbk

APH(3') -I = K, GmB -II = K, GmB, (A) -III = K,A,I, GmB -IV = K, A, I -VI = K, GmB, A, I -VII = K, (A)

OH HO H2N 3' ANT(2') -I G, T, Dbk, S, K

O OH H 2N

2' OH OH H 2N 6' O 6 OH NH2 4 O 1

3

ANT(4') - I = T, A, I, K, Dbk -II = T, A, I, K

NH2

APH(2'')+AAC(6') G,T,N,A,I.K,Dbk

AAC(6') -I = T, N, A, K, Dbk, S, (I) -II = G, T, N, K, Dbk, S -III = T, N, A, I, K -IV = G, T, N, A, K AAC(3) -I = G, S -II = G, T, N, Dbk, S -III = G, T, K, Dbk, S -IV = G, T, N, Dbk, S -VI = G, T, N, S, (T, K)

Figure 5: The resistance causing aminoglycoside-modifying enzymes acting at kannamycin B (this aminoglycoside is susceptible to drug modifying enzymes).40 To design a successful desired active compound against aminoglycoside-modifying enzymes, we need to combined approaches of known conventional pharmacology, structuralactivity-relationship (SAR), medicinal chemistry to explore the powerful candidates against a broad range of infections causing bacteria, and novel approaches to be applied in the same line of antibiotics are currently working. To understand the exchange of chemical moieties to escape the resistance causing enzymes, 2-deoxy-streptomycine was chosen to explain the combinatorial approaches to design a novel target molecule (Fig. 6).38



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Fluorination in position 3' Removal of 3' hydroxy group Enzyme inactivation Protein kinase inhibitors

Hydroxymethalation in position I Fluorination in position 5 and 2'' Inversing the chirality in position 5

APH(3')

AAC(2') OH HO

O

H2N 3'' OH H 2N

2' OH OH H 2N 6' O 6 OH NH2 4 O 1

3

ANT(2'') APH(2'') Amination in position 2'' Acylation in position 2'' Fluorination in position 5 and 2''

NH2

ANT(4') Fluorination in position 4'

AAC(3) Fluorination in position 5 Hydroxymethalation in position I Inversing the chirality in position 5 Alkylation in position I

Alkylation in position I Aminoacylation in position I Inversing the chirality in position I

Figure 6: Chemical modifications performed on kanamycin B by specific aminoglycosidemodifying enzymes to cause resistance (check figure 5 for enzyme acting on each position). Hanessian et al.41 showed the class of 4,5-disubstituted 2-deoxystreptamine aminoglycosides, including butirosin, paromomycin, and neomycin share a common binding site, mode-of action, and powerful biocidal properties. But, they were never used as anti-infective due to susceptibility to multiple enzymes which modify their structure. The most prominently used class for clinical purposes belongs to 4,6-disubstituted 2-deoxystreptamine,including gentamicin, tobramycin, amikacin, isepamicin, and arbekacin. Due to continuous use, S. aureus becomes resistant to the above drugs due to emergence of modifying enzymes, prevalently 4ʹ-Onucleotidyltransferase,

ANT(4ʹ)-I,

3ʹ/5ʹʹ-O-phosphotransferase,

APH(3ʹ/5ʹʹ)-III,

and

the

bifunctional 2ʹʹ-O-phosphotransferase and 6ʹ-Nacetyltransferase, APH(2ʹʹ)/AAC(6ʹ), whose respective target preferences are shown in Figure 7.42-45


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APH(2')/AAC(6') H 2N 1

APH(2'')/AAC(6')

4

B HO

X

O no ring

6'

NH2

HO

O

5

NH2 4'

OH

ANT(4')-I

H2N 3' OH

C O O

O A

APH(3'/5'')-II

OH

5''

3'' D HO 4'' OH

APH(3')- III

NH2

Neomycin B

NH2 HO APH(2'')/AAC(6')

Steric discrimination between enzymes and ribosomes

H N 1 O HO

X

O no ring

HO

4

D 3''

O

5

C O O

6'

NH2 B

5''

X

O A

APH(2')/AAC(6')

NH2

X

4'

ANT(4')-I

H 2N

3'

X

OH

X

APH(3'/5'')-II

APH(3')- III

NH2

4''



NH2

APH(2'')/AAC(6')

X

HO

Page 14 of 39

Steric discrimination between enzymes and ribosomes

H N 1

O HO 2'' H 2N O C O HO

6'

NH2 4

B

O A

APH(2')/AAC(6')

X NH2

OH 4' H2N 3' OH

O OH

ANT(4')-I APH(3')- III

OH Amikacin APH(2')/AAC(6')

Me

Me H Me NH2 H 2N 1 6' NH 4 OH O B 2'' O HN O A 4' X C OH 3' O H 2N X Me OH

ANT(4')-I APH(3')- III

Gentamicin complex (C1 shown)

Figure 7: Representative members of the 2-deoxystreptamine aminoglycoside classes characterized by 4,6-disubstitution (amikacin and gentamicin C1)or 4,5-disubstitution (neomycin B).

Arrows

indicate

positions

targeted

by

modifying

enzymes

prevalent

in

S. aureus (black, complete resistance; gray, medium resistance; white and crossed, evaded). Based on a report from Hanessian et al.41, a semisynthetic 4,6-disubstituted 2deoxystreptamine candidate, suchas arbekacin, was found to overcome the action of a subset of the

aforementioned

enzymes

used

by

S.

aureus

against

active

components

of

aminoglycosides.46In order to overcome the drug resistance strains of MRSA, they synthesized derivatives of arbekacin, and among them 5,4″-diepi-arbekacin (TS2037) emerged as the most potent anti-bacterial agent against S. aureus having MIC value of 0.25 µg/mL compared to 14 ACS Paragon Plus Environment

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arbekacin’s 0.25 µg/mL. Even though, many anti-MRSA drugs are in the clinical investigations in which aminoglycoside TS2037 is the one promising lead candidate as antimicrobial and further interest in aminoglycoside research may find a way into further evaluation.47 (A) TSO237

(B) Arbekacin

OH OH 6'' O 4'' H 2N

2'' OH O HN HO O

H 2N

2' OH OH 6' O NH2 O

H 2N

1 OH

OH 6'' HO O 4'' H2N 2''OH HO

3 NH2

O HN O

HO 1

2' OH OH 6' O NH2 O

H 2N

3 NH2

H 2N

Figure 8: Chemical structures of TS2037 (A) and arbekacin (B). Many types of compounds48 are investigated in the clinical research pipeline as candidates against drug resistant MRSA such as A. New aminoglycosides: Plazomicin (Phase 3 currently underway: NCT01970371) against multi-drug resistant (MDR) Enterobacteriaceae; B. β-lactamases, cephalosporinase, and carbapenemase inhibitors in combination with old and new -lactams such as 1) Avibactam; 2) Ceftolozane; 3) Vaborbactam (RPX7009); 4) Relebactam; Siderophore-containing β-lactams, 5) S649266; C. New cephalosporins and -lactams that have activity against -lactamase and carbapenemase-producing bacteria. The compound S649266 (Phase 3 development: CREDIBLE-NCT02714595) showed β-lactamase inhibition but it is not used in combination with β-lactamases inhibitors; it is highly active against Gram-negative bacteria. D. New pleuromutilins: Lefamulin (BC-3781) is a semi-synthetic compound effective against MRSA by inhibiting the bacterial protein machinery (Phase 3 development: CABPNCT02813694); E. New tetracycline: omadacycline is a tetracycline derived in a subclass called aminomethylcycline for the treatment of acute bacterial skin and skin structure infection (ABSSSI) (Phase 3 development enrolled: NCT02531438); F. New macrolides: solithromycin 15 ACS Paragon Plus Environment


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phase 2/3 pivotal trial (NCT02605122) in pediatric patients (2 months to 17 years of age) with CABP has been initiated; G. New fluoroquinolones and DNA gyrase inhibitors: Delafloxacin (completed two Phase 3 trials known as “PROCEED” in which the comparator was vancomycin + aztreonam: NCT02679573) against ABSSSI; H. New oxazolidinones: Cadazolid (Phase 3: IMPACT:

NCT01987895),

MRX-1(Phase

2:

NCT02269319),

and

Sutezolid

(Phase

2:NCT01225640); I. New fatty acid biosynthesis inhibitors: Debio1452 is a type of Fab1 inhibitor, active against all resistant staphylococcal strains by inhibiting Enoyl-acyl carrier protein reductase (Phase 2:NCT02426918) and CG400549 another inhibitor (Phase 2:NCT01593761) for the treatment of ABSSSI caused by MRSA; J. New folate biosynthesis inhibitors: Iclaprim is a type of diaminopyrimidine and next-generation inhibitor of dihydrofolate reductase (DHFR) (Phase 3 development: NCT02607618) against ABSSSI of MRSA and MSSA showing low propensity to develop resistance in bacteria and K. Defensin: mimetic peptidesBrilacidin (Phase 2 studies: NCT02052388) against ABSSSI of MRSA including non-dividing bacterial cells by acting on cell membrane and without altering the cell membrane of mammalian cells causing cytotoxicity. Only a few of these candidates receive approval at the end of phase 3 trials. All the above candidates are modified versions of known compounds that have previously shown antibacterial actions so far.9 Efforts to identify novel lead candidates via combinatorics for chemical biology investigations and drug discovery programs are underway. Through one such approach, Nicolaou et al.49 designed a combinatorial library of 65 new benzopyran derived cyanostilbenes (MIC 21>22 (MIC 3, 12, 13 µg mL-1 respectively) (Fig. 9). Secondly, the free phenolic


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moiety at the terminal aromatic ring confers its potency compared to original >1000 members in stilbene library containing other substituents such as ethers, esters, halogens, nitro groups, sulfonates, and heterocycles, which are inactive at the original concentrations (MIC 99.0%) and its noncytotoxic nature confers a high therapeutic value in attenuating bacterial infections. These further calls for more novel molecules to be synthesized against MRSA in combinatorial approaches for future antibiotic drug discovery. A) L-Ile2

H N

O N H H

D-N-Me-Phe1

L-Ile11

D-Gln4 H 2N H N O HO

L-Ile5

O

O N H

H H N

O

O

N H H

H N O

H

O O

O

N H H O OH D-Thr8

H N

O

N H HN O L-Alag

H N

NH

NH L-Allo-End10

D-All--Ile5 33


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B) a

Cl

AllocHN

b

O

OH T A

c AllocHN

O

NHFmoc d O I e T A

AllocHN

O I T A

NHTrt

f g O

O

tBu

L NH2 O I I S T A COOH

tBu Boc MeN Phe I S R I

Boc MeN Phe I S R I

h

O I I S T A

L NHTrt

tBu

tBu i j HN

NH2

HN H N

O N H

H N O HO

O

O

O N H

H N O

O N H

H N O

O N H OH

O H N

N H HN O

O

34

Figure 11: The highly potent natural teixobactin 33 (A) and (B) synthesis of D-arg4-leu10teixobactin (34) starting from a 2-chlorotritylchloride resina. a(a) Fmoc-Ala-OH (4 equiv.) and DIPEA (8 equiv.) in DCM, 3 h. (b) Piperidine (20%) in DMF followed by 3 equiv. of AllocHND-Thr-OH

and 3 equiv. of HATU with 6 equiv. of DIPEA, 1.5 h. (c) Fmoc-Ile-OH (10 equiv.),

DIC (10 equiv.), and 5 mol % DMAP in DCM, 2 h, followed by a capping with Ac2O/DIPEA 10% in DMF and 20% piperidine in DMF. (d) Fmoc-Leu-OH (4 equiv.) and HATU (4 equiv.) with DIPEA (8 equiv.) in DMF, 1 h, followed by 20% piperidine in DMF. (e) Trt-Cl (10 equiv.) and 15% Et3N in DCM, 1 h. (f) [Pd(PPh3)4]0 (0.2 equiv.) and PhSiH3 (24 equiv.) in dry DCM, 1 × 20 min and 1 × 45 min. (g) Fmoc/Boc−AA(PG)-OH (4 equiv., AA = amino acid, PG = protecting group), DIC/Oxyma (4 equiv., MW, 10 min), followed by 20% piperidine in DMF (3



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min and 10 min). (h) TFA/TIS/DCM = 2:5:93, 1 h. (i) HATU (1 equiv.) with DIPEA (10 equiv.) in DMF, 30 min. (j) TFA/TIS/H2O = 95:2.5:2.5, 1 h. Working towards discovering new drugs-mechanisms The Pew Charitable Trust reported nearly all the antibiotics approved from the last three decades are modified forms of the already known classes of robust compounds against resistant bacteria. Currently, the special mechanism of action against resistant microbial candidates is understood for only a few antibacterial candidates (Fig. 12). In the following, three important candidates are discussed to highlight their upcoming promise against drug-resistant diseases. Analog 35 (ACHN-975, a Phase 1 candidate from Achaogen Inc.) is a new class of antibacterial candidate (Fig. 12A). It inhibits LpxC- an enzyme involved in lipid biosynthesis in Gram-negative (Enterobacteriaceae-Pseudomonas aeruginosa),and is inactive against Grampositive bacteria.54 Analog 36 (Brilacidin, PMX-30063, a Phase 2 candidate from Cellceutix Corp.) is a nonpeptide chemical mimic (Fig. 12B), and an investigational new drug (IND) representing a new class of synthetic (arylamidefoldamer) mimetic of host defence protein, which is the first line of defence against microbial infection in many species, including ‘superbug’ MRSA. Brilacidin showed less cytotoxicity to mammalian cells through selectively targeting the bacteria by direct disruption of the membrane to cause its death. Due to its unique mechanism and novel action of mimicking host innate immune response (first time proven to be successful in fighting against infections evolved over millions of years), this new drug is less likely to become bacteria resistant.55-58


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O O NH OH Me Me NH2

A)

HN

HO 35

CF3 NH

B)

O

O

HN NH2 H 2N

N

N

O N H

O N H

N

N H

NH2 N

NH2

HN

CF3 36

C)

The lack of strong basic group confers to DLX weak acid character

O

O

F

OH

N

N Cl

HO

F

N

H 2N Increase the stability of the heterocycle

F

37

Figure 12: New antibiotics in drug-discovery and clinical trials with new mechanism of actions. A. ACHN-975, B. Brilacidin, and C. Delafloxacin. Analog 37 (Delafloxacin (DLX) (Fig. 12C)) is a recent (July 2017) US Food Drug Administration (FDA) approved drug with the trade name BAXDELA. It possesses excellent broad spectrum activity against clinically relevant bacteria including MRSA and MSSA, which cause ABSSSI in skin. DLX operates in a biological system by changing its potent character in response to pH, immune defence, pathogenic counteracting mechanisms, and circulating environments. The C7 basic group in DLX acts as a zwitterion and is distributed inside of the phagolysosome (pH 5-5.5). The subsequent deprotonation of DLX helps retain bacteria in host.59 DLX has been used as a fourth-generation fluoroquinolone (FQ) and as dual DNA gyrase 23 ACS Paragon Plus Environment


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(topoisomerase II) and topoisomerase IV inhibitor to shut down the growth of S. aureus by deactivating the prime functions of cell growth by interfering with cell DNA replication, transcription, repair, and recombination.60DLX also has significant activity in acidic media compared to other quinolones against S. aureus which has resistance to quinolones, in the future drug discovery programs have been concentrating to develop drug-like candidates to overcome resistance problem associated with diseases. Is S. aureus 100S ribosome response important for future therapeutic discovery against the sleeping factor of S. aureus? Hibernating factors are the perfect targets! The bacterial 30S and 50S subunit ribosomes are extensively studied cellular components involved in translating a universal code into proteins in the 70S complex. The 100S ribosome is ubiquitously found in all bacterial phyla (Fig. 2). A common feature of all biological process is conservation of energy, because protein synthesis accounts >50% of energy cost. This study reveals that the lack of any 100S ribosome makes cells prone to suicide due to rapid degradation of ribosomes. The hibernation-promoting factor (HPF) is involved in homodimerization of 100S ribosome, which is a translationally silent complex. The 100S ribosome is of temporal abundance in different phyla, however, the regulation and disassembly of 100S ribosome is unknown.61 In Escherichia coli (E. coli) 100S dimers were found in a stationary phase when nutrient was scarce, but in S. aureus, the 100S dimer was found throughout all phases including, nutrient rich ones. In addition, studies have suggested that the formation of 100S dimer during the logarithmic growth phase of S. aureus is responsible for the shutdown of the translational efficacy of a few genes and loss of HPF, demonstrating massive ribosome degradation entering


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the

stationary

phase,

which

correlates

with

the

rate

of

cell

death.

In

E. coli three ribosome-silencing factors (YfiA- inactivates the 70S and recycles to translation, RMF- ribosome modulation factor, and HPF- dimerization of 100S into inactive 100S complex) are related to binding ribosome subunit and blocking the binding of messenger RNA (mRNA), anticodon region of A-, P-, and E-transfer RNA (tRNA). However, the absence of RMF or YfiA in S. aureus hpf gene codes another form of HPFSA, which is twice the size of HPFEC with dual functions as N-terminal HPFSA to block the mRNA and as anti-codon tRNA binding site. These interesting highlights demonstrate that understanding of the distinct 100S structure of the S. aureus compared to E. coli complex may be a potential and novel species-specific therapeutic target and the best strategy to unlock the pathogenesis.61,62 Matzovet al.61 reveal that,even though the 100S complex in gammaproteo-bacteria and gram-positive bacteria serves the same functions, their dimerization and structural orientation are different. The unique interaction of HPF with 100S and the hampering of the complex formation by targeting their unique signature between twin ribosome emphasize the distinctive Grampositive specific anti-bacterial treatment (Fig. 2). In the future, strategies to overcome and counteract the S. aureus mechanisms during pathogenic conditions are warranted. Precision in target drug discovery Development of precision drugs to wipe out bacterial bugs during pathogenic state in the body protected by its well-developed counteracting mechanisms by overcoming drug-resistance is an upcoming new target based approach in drug-discovery programs. The commonly used antibiotics today were discovered during the “Golden Age” of antibiotics and they are the first and most successful class of drug candidates which can treat a broad spectrum of targeted infections. These types of antibiotics have lost their patent protection and have been used as



Page 26 of 39

generic, low-priced drugs. This has led to their overuse, misuse, and the evolution of antibiotic resistance. In turn, this has drawn the attention of infectious diseases specialists.13The resulting global burden has spurred large pharmaceutical companies to re-direct resources to develop drugs to overcome the problem of resistance in medicines. To combat real threats, increasing effort is being focussed to develop precision antimicrobial therapeutics that target key virulence determinants of specific pathogens such as S. aureus63 while leaving the remainder of the host microbiota undisturbed. Due to the nonspecificity of current antimicrobials, ‘superbug-S. aureus’ can take advantage of present antibiotics and is predicted to kill more than 10 million each year by 2050.64 Also, World Bank suggests by 2050 the consequences of elevated antibiotic-resistant infections could cost the world economy by an estimated $100 trillion.65 To address this emerging crisis, current research focuses on elucidating the destructive property of broad-spectrum antibiotic treatment on the beneficial host microbiota. These new types of therapeutics, hitherto called ‘precision antimicrobials’, include both new anti-virulence compounds to inhibit bacterial pathogenesis and persistence, and bacteriostatic/bactericidal to minimize bacterial pathogens. Very recently, a commensal strain Staphylococcus lugdunensis selectively removes and inhibits the metabolism of S. aureus by producing a natural compound called ‘lugdunin’ (a cyclic peptide), but its precise mechanism of action remains unknown.66 Like this, many promising inhibitors are uncovered having an attenuated role against S. aureus pathogenesis through lipoteichoic acid synthesis, RnpA-mediated RNA degradation, sortase activity, and iron-sulfur cluster assembly. These examples shed light into the microbial world of small molecule-based treatment approaches.67-70 Conclusion


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The emergence of drug-resistant pathogen MRSA through counteracting the host immune system and declining traditional antibiotic therapy by misuse, causes a rise in the resistance to broad-spectrum antibiotics. To combat this threat, the challenges lie in the development of precision candidates such as texiobactin and vancomycin that target key virulence determinants of specific pathogens while leaving other microbiota undisturbed. These candidates face a traditional hurdle in development processes such as bioavailability, toxicity, and manufacturing scalability. In traditional antibiotics, the scope is broad by targeting core bacterial process common in all strains in a group to avoid cross-reactivity with host cellular process. In contrast, the precision antimicrobials designed to target specific bacteria occur only in a defined subset of pathogens, without affecting either the host or beneficial bacteria within the microbiota. Developing therapies that target the host reservoir of pathogens, rather than simply the site of infection, may help reduce disease burden and/or prevent recurrence. To which, the “precision” or “ultra-narrow” candidates are tailor-made as a result of understanding the various stages of pathogen lifestyle. The design of therapeutics that disrupt the critical pathways in virulent pathogens such as MRSA is warranted, and represents the ‘future’ to overcome the resistance problem. Acknowledgment The authors declare no conflict of interest. We are grateful to the Wuhan Applied Fundamental Research Program of Wuhan Science and Technology Bureau (Grant NO. 2017060201010216) and Wuhan University of Technology for financial support. Author contributions



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The technical content was designed and the manuscript was written by MHM, suggestions were given by HLQ and PM; and corrections were edited by KPR, SS, SL, CSK and references were checked by XC. Transparency declaration The authors declare no conflict of interest.

References and Notes [1]

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[2]

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[3]

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[4]

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[5]

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Graphical abstract Combating a Master Manipulator: Staphylococcus aureus Immunomodulatory Molecules as Targets for Combinatorial Drug Discovery Kadalipura P. Rakesh1, Manukumar H. Marichannegowda2*, Shobhith Srivastava3, XingChen1, Sihui Long4, Chimatahalli S. Karthik2, Putswamappa Mallu2 and Hua-Li Qin1** 1

Department of Pharmaceutical Engineering, School of Chemistry, Chemical Engineering

and Life Science, Wuhan University of Technology, 205 Luoshi Road, Wuhan, 430073, PR, China 2

Department of Chemistry, Sri Jayachamarajendra College of Engineering, Mysuru-570006,

Karnataka, India 3

Department of Pharmacology and Therapeutics, King George's Medical University, Chowk,

Lucknow, 226003, India 4

Key Laboratory for Green Chemical Process of Ministry of Education, Hubei Key

Laboratory of Novel Reactor and Green Chemical Technology, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan, Hubei, China

ACS Paragon Plus Environment

Combating a Master Manipulator: Staphylococcus aureus

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