In Vivo Imaging of the Activity of Host Defense Peptide Mimetics in - - PowerPoint PPT Presentation

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In Vivo Imaging of the Activity of Host Defense Peptide Mimetics in a Mouse Model of Invasive Candidiasis Gill Diamond 1, *, Lisa K. Ryan 1 , Rezwana Parveen 1 , Amy G. Hise 2 , Katie B. Freeman 3 , and Richard W. Scott 3 1 Department of Oral

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In Vivo Imaging of the Activity of Host Defense Peptide Mimetics in a Mouse Model of Invasive Candidiasis

Gill Diamond1,*, Lisa K. Ryan1, Rezwana Parveen1, Amy G. Hise2, Katie B. Freeman3, and Richard W. Scott3

1 Department of Oral Biology, University of Florida, Gainesville FL USA; 2 Department of Pathology, Case Western Reserve University, Cleveland OH USA; 3 Fox Chase Chemical Diversity Center, Doylestown, PA USA

* Corresponding author: gdiamond@dental.ufl.edu

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Graphical Abstract

In Vivo Imaging of the Activity of Host Defense Peptide Mimetics in a Mouse Model of Invasive Candidiasis

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mBD1-/- mBD1-/-

+RFP AMP Mimetic Optimize mimetic structure In vivo imaging

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Systemic fungal infections are increasingly common, especially in immune compromised patients. Even with newly developed drugs, there remain issues of limited spectrum, side effects, and the development of resistance. Host defense peptides (HDPs) have been examined recently for their utility as therapeutic antifungals, especially due to the low levels of resistance that develop. Unfortunately, the peptides exhibit poor pharmacologic properties in vivo. We have demonstrated the potent activity of nonpeptidic compounds that mimic HDPs in both structure and function against clinical strains of Candida albicans associated with oral and invasive candidiasis in mouse models. However, to test numerous compounds in vivo requires large numbers of mice, with multiple time points, and requires immunosuppression of the mice using cyclophosphamide, which can influence pharmacological parameters. We have identified a strain of mouse that develops invasive candidiasis without the need for immunosuppressive

  • drugs. When we infect these mice with a strain of C. albicans that constitutively

expresses Red Fluorescent Protein, we can quantify the infection in real time by in vivo imaging. We can further observe the reduction in fluorescence in infected mice after treatment with an HDP mimetic. Together our results demonstrate a novel in vivo method for screening new antifungal drugs. Keywords: Antimicrobial peptide; antifungal; Candida; peptidomimetics

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Introduction- Antimicrobial peptides

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  • Short, generally cationic, broad-spectrum

antimicrobial proteins

  • Found at mucosal surfaces

– Skin secretions in fish and amphibians – Oral cavity, trachea, small intestine, female reproductive tract in mammals

  • Found in myeloid cells

– Neutrophils, alveolar macrophages

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Types of AMPs

  • Linear

– Amphipathic a-helical

  • Cysteine-rich

– b-sheet

  • Peptides with specific amino acids

– Rich in His, Pro or Trp

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Linear peptides

  • Magainin (frog skin)
  • Pleurocidin (fish skin)
  • Cecropin (insect)
  • Protegrin (pig

leukocytes)

  • LL-37 (human cells)

Form cationic amphipathic a-helices

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Cysteine-rich peptides

  • a-defensins

– PMNs, small intestine

  • b-defensins

– Epithelial cells, some blood cells

  • q-defensins

– Rhesus monkey PMNs

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Natural Roles of AMPs

  • Antimicrobial defense of surfaces

– magainins on amphibian skin – b-defensins on mammalian epithelium

  • Oxygen-independent antimicrobial activity of

phagocytic cells

– a-defensins in PMNs

  • Chemotactic agents for innate immune defense cells

– b-defensins, LL-37

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Antimicrobial Peptides as Therapeutics

  • Naturally occurring
  • Broad-spectrum

antimicrobials

  • Little resistance
  • Low antigenicity
  • Protease sensitive
  • Expensive to

produce and purify

  • Often are

inactivated by

  • ther proteins

GOOD BAD

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Peptide Mimetics

  • Structurally similar to

active portion of AMP

– Cationic, amphipathic

  • Protease resistant
  • Inexpensive to

produce

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Activity of an AMP mimetic against oral pathogens

Beckloff et al., Antimicrobial Agents Chemother., 51:4125, 2007

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Hypothesis

  • Antimicrobial peptide mimetics could be

useful therapeutic agents to treat fungal infections

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Activity of a peptide mimetic against Candida spp. in vitro

Species MIC, µg/ml

  • C. albicans

4-8

  • C. tropicalis

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  • C. parapsilosis

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  • C. glabrata

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  • C. dubliniensis

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  • C. krusei

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Screening for New Compounds

Co Compound An Anti-C.

  • C. albicans

GD GDH2346 (µg µg/ml) Cy Cyto toto toxicity ty An Anti-ba bacte teria ial; l; co commen ensals MIC (µ (µg/ml) MT MTD (m (mg/kg) EC EC50 (µM) IC IC50 MI MIC NI NIH3T3 He HepG2 OK OKF6/ TE TERT

St Streptoc

  • coc
  • ccus

sa salivarius Ac Actinomyces vi viscosus

PM PMX70004 4.88 4-8 52 31 68 16 32 10 PM PMX519 4.93 4-8 439 >1000 >1000 >64 >64 17 PM PMX1408 4.24 4 311 453 466 16 4 < 2.5 PM PMX1502 1.44 4 436 885 766 >64 >64 20 PM PMX1570 1.09 2 108 310 371 8 4 10 PM PMX1576 1.03 2 149 288 502 8 4 5 PM PMX1591 2.2 2 461 904 ND 32 8 ND PM PMX1625 2.08 2 523 723 718 64 16 15

Ryan et al., Antimicrob. Agents Chemother. 58:3820, 2014

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No Development of Resistance

20 40 60 80 100 120 140 2 4 6 8 10 12 14 16 18 20 Fold increase in MIC Passage mPE PMX30016 Fluconazole PMX10149 PMX519

Hua et al., Mol. Oral Microbiol. 25: 418, 2010

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Peptide mimetics are active in a mouse model of oral candidiasis

1 2 3 4 5 6 7 water 519 1502 1570 nystatin cfu/tongue X105

Ryan et al., Antimicrob. Agents Chemother. 58:3820, 2014

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Lead compound, PMX1502 (also called C4)

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Activity in a model of invasive candidiasis-kidney burden

1 2 3 4 5 6 Infected control – 2h Infected control - 24 h Fluconazole - 10 mg/kg C4 - 14.5 mg/kg C4 - 29.1 mg/kg C4 - 2 X 14.5 mg/kg C4- 21.8 + 14.5 mg/kg Log10 CFU

Menzel et al., Sci. Rep. 7:4353, 2017

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Disseminated Candidiasis Model; Survival Study

100% survival in 10 and 15 mg/kg C4 groups, no overt toxicity 40% survival in the fluconazole group Menzel et al., Sci. Rep. 7:4353, 2017

10 20 30 40 50 60 70 80 90 100 5 10 15 Percent survival Days post infection Control Fluconazole C4 5mg/kg C4 10mg/kg C4 15 mg/kg

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Screening of new compounds in vivo

Chowdhury et al, J. Fungi, 4:30, 2018

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Results and discussion

  • 1. AMP mimetics can be designed and screened to obtain highly

active antifungal drugs that act in vivo to treat oral and invasive fungal infections.

  • 2. Screening large numbers of mimetics in vivo requires large

numbers of mice, especially for dose response and time course studies.

  • 3. We wished to develop a mouse model of invasive candidiasis

that would require fewer mice, to allow for more efficient screening of AMP mimetic activity against fungal pathogens in vivo.

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  • 1. Development of a non-immunosuppressed mouse model of

invasive candidiasis a) Used C57Bl/6 mice deficient in mouse b-defensin 1 (mBD1-/-) b) Injected 5x105cfu C. albicans IV into tail vein c) Quantified viable cfu in kidneys

22 100000 200000 300000 400000 500000 600000 Day 1 Day 3 Day 7 cfu Day post infection mBD-1 KO WT

mBD1-/- mice are a good strain to test antifungal drugs without the need for immunosuppressive pre-treatment

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  • 2. Develop a fluorescent strain of C. albicans

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  • C. albicans GDH2346-RFP

Can readily visualize and quantify fluorescence of Candida in the Xenogen in vivo imaging system (IVIS)

  • C. albicans GDH2346
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  • 3. Infect with C. albicans-RFP (5x104cfu). Inject AMP mimetic (C6) 2

hours post infection. Image mice daily

Quantify region of interest

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  • 4. Quantify fluorescence daily by IVIS

0.00E+00 5.00E+06 1.00E+07 1.50E+07 2.00E+07 2.50E+07 3.00E+07 2 4 6 8 10 12 14 Radiance Day post infection no drug C6, 20mg/kg

Compound C6 inhibits growth of C. albicans over time

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  • 5. Dose response (day 10 post infection)

No infection 0mg/kg 5 10 20

Compound C6 inhibits infection in a dose-dependent manner

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Conclusions

  • 1. We have developed a novel, non-lethal infection model for

invasive candidiasis in mice.

  • 2. We have used a fluorescent strain of C. albicans to allow
  • bservation and quantification of infection in real time.
  • 3. We have shown that antimicrobial peptide mimetics can be

tested in this model to assist in more efficient and rapid screening of novel antifungal agents.

  • 4. This will hopefully lead to more efficient in vivo screening
  • f antifungal drugs, with the use of less mice.

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Acknowledgments

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Diamond Lab Case Western Reserve U Mobaswar H. Chowdhury, Ph.D. Amy G. Hise, MD Lorenzo Menzel, Ph.D. William Ruddick, M.S. Rezwana Parveen, B.S. David Brice, Ph.D. Lisa K. Ryan, Ph.D. (UF Medicine) PolyMedix/FCCDC Rick Scott, Ph.D. Katie Freeman, Ph.D.