Python Cathelicidin CATHPb1 Protects against Multidrug-Resistant

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Python Cathelicidin CATHPb1 Protects against Multidrug-Resistant Staphylococcal Infections by Antimicrobial-Immunomodulatory Duality Shasha Cai, Xue Qiao, Lan Feng, Nannan Shi, Hui Wang, Huaixin Yang, Zhilai Guo, Mengke Wang, Yan Chen, Yipeng Wang, and Haining Yu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00036 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 22, 2018

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Journal of Medicinal Chemistry

Python

Cathelicidin

Multidrug-Resistant

CATHPb1

Protects

Staphylococcal

Infections

against by

Antimicrobial-Immunomodulatory Duality

Shasha Cai1¶, Xue Qiao1¶, Lan Feng1, Nannan Shi1, Hui Wang1, Huaixin Yang1, Zhilai Guo2, Mengke Wang1, Yan Chen2, Yipeng Wang2*, Haining Yu1*

1

School of Life Science and Biotechnology, Dalian University of Technology, Dalian,

Liaoning 116024, China;

2

Department of Pharmaceutical Sciences, College of

Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215123, China

1

ACS Paragon Plus Environment

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ABSTRACT

Multidrug-resistant Staphylococcus aureus, including MRSA (Methicillin-Resistant) and VRSA (Vancomycin-resistant), causes serious healthcare-associated infections, even sepsis and death. Here, we identified six novel cathelicidins (CATHPb1-6) from Python bivittatu, and CATHPb1 displayed the best in vitro pharmacological and toxicological profile. We further show that CATHPb1 exhibited evident protection in mice MRSA/VRSA infection models, either given 24h before or 4h after infection. The protection was all effective through different administration routes, but was blocked by in vivo depletion of monocyte/macrophages or neutrophils. CATHPb1 can rapidly and massively modulate macrophages/monocytes and neutrophils trafficking to the infection site, and potentiate their bactericidal functions. Meanwhile, CATHPb1 remarkably augmented neutrophil-mediated bacteria killing by facilitating neutrophil extracellular traps (NETs) formation and preventing its degradation. Acting through MAPKs and NF-κB pathways, CATHPb1 selectively enhanced the levels of chemokines while reducing the production of pro-inflammatory cytokines without undesirable toxicities. The much improved serum half-life and stabilities confer CATHPb1 an excellent prospect to become a novel therapeutic agent against multidrug-resistant staphylococcal infections.

2


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1. INTRODUCTION Infection treatment has become a worldwide challenge because of the rapid evolution of bacterial resistance. In the last decade, more and more multidrug-resistant pathogens,

such

as

methicillin-resistant

Staphylococcus

aureus

(MRSA),

vancomycin-resistant S. aureus (VRSA), vancomycin-resistant Enterococcus (VRE)

1-3

,

and the so-called “superbugs”, dubbed NDM-1 (New Delhi metallo-beta-lactamase 1-producing Enterobacteriaceae) 4, have emerged due to the heavy use of traditional antibiotics, and pose a catastrophic threat to people all over the world 3. This situation has hastened an aggressive search for novel approaches of anti-infective therapy, or novel antimicrobial agents with special mechanism that hardly incurs bacteria resistance. Among novel approaches currently under research, the immunomodulatory therapy is an attractive one as it is an excellent supplement to direct antibiotic therapies and potentially has broader applications, and meanwhile few concerns of drug resistance 5. Innate immunity is the first line of host defense system against pathogens 6. It can be triggered by the binding of molecular signatures (also known as pathogen-associated molecular patterns, PAMPs) from microbial cells, such as proteins, lipids and nucleic acids, to pattern recognition receptors (PRRs), including toll-like receptors (TLRs) and NOD-like receptors (NLRs) 6. PRRs also sense host PAMPs that are symbolic of disease and cellular damage. Within minutes to hours after binding, a broad, relatively nonspecific, innate immune response is activated both at the cellular and the molecular level

6,

7

. Numerous cells and molecules, including mucosal epithelial cells, 3



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tissue-resident and blood circulating phagocytic cells (neutrophils, monocytes and macrophages), cytokines, chemokines, complement system components and host defense proteins and peptides are incorporated in a complex and extremely orchestrated response to PAMPs

7, 8

, which effectively prevent or clear infection. However, inappropriate

activation of innate immunity may lead to excessive production of pro-inflammatory cytokines, and increase the risk of harmful inflammation, some respiratory infections, or even severe sepsis and septic shock 9. Recently, a range of innate immune modulators have been reported, including host-defense peptides (HDPs), agonists of TLRs and NLRs, and other natural bacterial ligands

6, 10

. These innate defense regulators (IDRs) are either natural peptides most

abundantly present in skin keratinocytes, mucosal epithelial cells, circulating neutrophils and myeloid bone marrow cells, or synthetic antimicrobial and immunomodulatory peptides derived from the natural HDPs 9. HDPs are characterized by its small size (10-50 amino acids), with an overall positive charge and a substantial proportion of hydrophobic residues 11. They have long been touted for their potential to fill the current void in novel antibiotic discovery. However, development of HDPs is currently facing obstacles under the pre-clinical phase, because of the relatively low anti-infection activity and high detrimental side-effects, including cytotoxicities and mast cell degranulations 12

9,

. Among the IDRs, cathelicidin, a gene-encoded endogenous peptide that serves as a

critical immune modulator catches more and more attentions owing to its rapid and potent antimicrobial activity without significant toxicity 4


10, 13

. To date, three

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cathelicidin-derived peptides have been advanced into phase 3 clinical-efficacy trials 11. We here identified six novel natural occurring cathelicidins (CATHPb1-6) from Python bivittatu. All mature peptides were synthesized, purified and screened antimicrobial

activity,

and

CATHPb1

exhibited

the

best

dual

in

vitro

antimicrobial-immunoregulatory activities with great stabilities. In current study, we further demonstrated that CATHPb1 has protective effect in a broad range of in vivo infection models, especially MRSA and VRSA mice model, by local and systemic administration. CATHPb1 was not only directly antimicrobial and anti-biofilm in vitro, but also immunomodulatory in vivo. Subsequently, we explored the mechanisms underlying the CATHPb1’s duality both at the cellular and molecular level, and investigated the signaling pathways whereby CATHPb1 triggered a series of immunomodulatory responses. The altered immune cell trafficking and effector levels in animal models further proved that these immunomodulatory mechanisms are operative in vivo. Finally, the toxicity and stability of CATHPb1 were evaluated for its potential in the systemic application. Given that CATHPb1 could not cause the drug resistance due to the special anti-infection mechanism, CATHPb1 could be a promising agent applicable in the situation that widely-used antibiotics are less effective, such as vancomycin and methicillin.

2. RESULTS 2.1 Identification and characterization of CATHPbs. 5



Six novel cathelicidins (GenBank Accession Numbers for CATHPb1-6: MF185714; KY937973; MG737674; MG737675; KY937975; KY937974) were acquired by tblastn from NCBI (National Center for Biotechnology Information) followed by 3’-RACE PCR screening the boa cDNA library (Figure S1). Sequence alignments showed that CATHPbs all share same gene structure: a conserved cathelin domain with four invariantly spaced cysteines flanked by an N-terminal signal peptide and a cationic C-terminal mature antimicrobial peptide (Figure S2). Six CATHPbs in Table S1 were then synthesized, purified (Figure S3) and screened for their antimicrobial and cytotoxic activity. As shown in Table S2, CATHPb1, 2, 4 possess broad-spectrum antimicrobial activity with CATHPb1 having the best MIC from 4.69 to 37.5 µg/mL, whilst CATHPb3, 5, 6 did not show detectable activity up to 100 µg/mL. This may be explained by their helical content and positive charge (Table S1) that mainly contribute to the antimicrobial capacity of short cationic amphiphilic peptides. Furthermore, the immunoregulatory effect of CATHPbs were evaluated in mouse peritoneal macrophages (MPMs) with or without LPS. All CATHPbs did not induce the production of inflammatory cytokines, while CATHPb1 and 2 showed chemotactic effect by increasing MCP-1 production (Figure S4 and S5). LPS-induced TNF-α, IL-1β, IL-6, and MCP-1 secreted from MPMs were remarkably and dose-dependently blocked by CATHPb1, whilst CATHPb2 and 4 exhibited weaker inhibitory effect, and CATHPb6 only suppressed cytokine production at the highest dose. CATHPb3 and 5 did not show immunoregulatory effect in vitro. Although CATHPb1 and 4 both directly antimicrobial 6


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and regulated the host innate immune response, CATHPb1 exhibited negligible cytotoxicity towards mammalian cells, much lower than CATHPb4 (Table S3), and therefore was selected for template for further study.

2.2 Effects of CATHPb1 in infection models. Extrapolating from our previous discovery that CATHPb1 had the best in vitro antimicrobial and immunomodulatory properties without cytotoxicities, we further examined its efficacy in mice models of aggressive bacterial infection with S. aureus, a major cause of nosocomial infections, especially the multidrug-resistant S. aureus (MRSA/VRSA). CATHPb1 (10 mg/kg) was administered through different routes after the mouse was intraperitoneally (i.p.) challenged with MRSA/VRSA for 4 hours. Substantial protection was observed when CATHPb1 (10 mg/kg) was given either locally by the i.p. injection or at distant sites, by intramuscular (i.m.), subcutaneous (s.c.) or intravenous (i.v.) injection (Figure 1A), indicating that peptide application can be effective by both local and systemic administration. CATHPb1 significantly decreased the bacterial counts in peritoneal lavage (P

Python Cathelicidin CATHPb1 Protects against Multidrug-Resistant

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