Ismail Fliss and Riadh Hammami

Current trends in antimicrobial agent research:chemo- and bioinformatics approaches

Antimic 2017, Québec, 21-23 juin

A Multidisciplinary Team

Amina SghairiFrançois Bédard

Riadh Hammami Sylvie Rebuffat Éric Biron Muriel Subirade

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Chemical additives and salts: very controversial barriers

Prohibition of antibiotics as a growth promotor

Very few new antibioticsResistance phenomenon

MAPs

Sustainable alternative

Natural

Safe (resistance)

Why the urgent need for antimicrobial peptides (AMP)?

AMPs: Widely distributed compounds through Nature

o Key components of innate, non-adaptive immune defense systems of multicellular organisms.

o Set of cationic and amphiphilic peptides, 20 to 50 amino acids, broad spectrum of antibacterial and antifungal activity.

o Great structural diversityo Mechanism of action: permeabilization of the cell membrane by a

detergent-like effect or by pore formation.

Plant AMPs

Microbial AMPs

Milk AMPs

Fish AMPS

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AMPs: Ubiquitous active compounds

o Small cysteine-rich AMPs

o Bacteriocins (nisin)

o Latoferrin, lactoperoxidase

o Cecropin family

• Narrow spectrum

• MIC in nM

• Non-toxic

• Thermal stability

• Mode of action (multiple)

• Food grade (GRAS)

• Non-ribosomal synthesis

• Large spectrum

• MIC in μM

• Toxic

Past

Antibiotics Vs AMPs

Future

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Enzymeribsome

Antibiotics

A

C

D E

Bβ-lactams, vancomycin

Fluoroquinolones, rifamycins

Daptomycin, lipopetides

macrolides

tetracyclines

aminoglycosides

trimethoprim,

sulfonamides

nisin, nukacin ISK-1

pediocin, nisin A, dysgalacticin

colicin, microcin B17

microcin J25

Protein synthesis

Replication and

transcription

Cell wall

synthesis

Perturbation de

membrane

composition

Perturbation of

membrane efflux

pediocin PA-1

Septum formation

benzamides,

N-heterocycles,

phenols

garvicin A,

lactoccin 972

Mode of action

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PEPTIDES

ANTIMICROBIENS

Extraction/purificationChimio-and bioinformatics

Recombinant organisms

• Identification

• Characterization

• Extraction

• Purification

• Development of databases

• Sequence analysis

• Development of new analogues

• Modeling and molecular dynamics

• Molecular interactions (in silico)

• Synthesis

• Identification de genetic

• Cloning

• Expression

• Production (microcin J25)

1 2

3

Bacterials Plants

Animals

Strategies for the production of AMPs

• Random Methods• Time consuming and

expensive• Variables (tests used)• Low yields

• Rapid• Inexpensive• Good correlation with

biological activity• High yields

Bioinformatics analysis tools and methods

o Bioinformatics: Concept using mathematics, statistics, computer science, technology and molecular biology

o Bioinformatic methods: include data collection, information management, sequence analysis, molecular interactions and advanced systems simulations.

oDatabases

oGenomics tools

oBioinformatics tools

oPrograms

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Contain genomic, proteomic and functionalinformation

Classification of bioinformatics tools and methods

o Thousands of peptides have been identified

o Very few having known biological activities, structures or sequences

o Several information absent or dispersed in the scientific literature (not available to potential users).

o The majority of the sequences are listed in UniProtKB / Swiss-Prot: universal and non-specific database.

o Urgent need for:

o Collect, filter and analyze these information

o Store in more specialized resources to improve efficiency of use

Why databases for AMPs?

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Database contents and tools• Literature

•Microbiology

•Physicochemical

•Taxonomy

Query

•BLAST

• FASTA

• SSEARCHHomology Search

•ClustalW

•MUSCLE

•T-Coffee

Multiple Sequence Alignment

•Various Physicochemical parameters

•Wheel representation

•Hydropathy

Physicochemical profile

•2D: NPS@

•3D: Modeller

Structure Prediction

• Sequences

• Structures

•Database dumpData Recovery

A database is a set of structured largecollection of data organized especiallyfor rapid search and retrieval by acomputer.

Summary of databases dealing with AMPs

Bactibase: web-accessible database for bacteriocins

o Created in 2007o 123 bacteriocins produced by both Gram-

positive and Gram-negative bacteria.

o Information is very easy to extract and allows rapid prediction of relationships structure/function and target organisms

o The BACTIBASE database is freely available at http://bactibase.pfba-lab.org

o 47742 visitors since January 2016

Gram-positive bacteriocins : a new structure-based classification 10 20 30 40 50 60 70

Group Genus Bacteriocin .........|.........|.........|.........|.........|.........|.........|....

G 1

a

Ac Actargadine ---------------SSGWVCTLTIECGT-----V---------ICAC-------------------------- -1

Bs Mersacidin ---------CTFTLPGGGGVCTLTSEC-----------------IC---------------------------- -1

La Lacticin 3147A1 --CSTNTFSLSDYWGNNGAWCTLTHEC-M-----A---------WCK--------------------------- 0

b

Lac Plantaricin Wa ---KCKWWNISCDLGNNGHVCTLSHEC-Q-----V---------SCN--------------------------- +2

La Bacteriocin J46 -------------KGGSGVIHTISHEVIYNSWNFVF--------TCCS-------------------------- +2

La Lacticin 481 -------------KGGSGVIHTISHECNMNSWQFVF--------TCCS-------------------------- +2

Sta Nukacin ISK1 -------------KKKSGVIPTVSHDCHMNSFQFVF--------TCCS-------------------------- +4

Ko Variacin ---------------GSGVIPTISHECHMNSFQFVF--------TCCS-------------------------- +1

St Streptococcin AFF22 --------------GKNGVFKTISHECHLNTWAFLA--------TCCS-------------------------- +3

St Streptococcin AM49 --------------GKNGVFKTISHECHLNTWAFLA--------TCCS-------------------------- +3

Ru Ruminococcin A --------------G-NGVLKTISHECNMNTWQFLF--------TCC--------------------------- +1

St Mutacin H29B ------------NRWWQGVVPTVSYECRMNSWQHVF--------F----------------------------- +2

St Mutacin 2 ------------NRWWQGVVPTVSYECRMNSWQHVF--------TCC--------------------------- +2

G 2

Stm Ancovenin ---------------------CV-QSCS--FGPLTW--------SCD-GNTK---------------------- 0

Stm Duramycin C ---------------------CA-NSCS--YGPLTW--------SCD-GNTK---------------------- 0

Stm Cinnamycin ---------------------CR-QSCS--FGPFTF--------VCD-GNTK---------------------- 0

G 3

En Bacteriocin 31 ------------ATYYGNGLYCNKQKCWVDWNKASREIG----KIIVNG-NVQHGPWAP--R------------ +2

Leu Leucocin C -------------KNYGNGVHCTKKGCSVDWGYAWTNIANNSVMNGLTG---GNAGWHN--------------- +4

Li Listeriocin 743A -------------KSYGNGVQCNKKKCWVDWGSAISTIGNNSAANWATG---GAAGW----KS----------- +4

Lac Sakacin P -------------KYYGNGVHCGKHSCTVDWGTAIGNIGNNAAANWATG---GNAGW-N--K------------ +4

En Mundticin -------------KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATG---GAAGW-S--K------------ +4

En Mundticin KS -------------KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATG---GAAGW----KS----------- +4

Lac Prebacteriocin 423 -------------KYYGNGVTCGKHSCSVNWGQAFSCSV----SHLAN-F--GHG------KC----------- +6

Leu Leucocin A -------------KYYGNGVHCTKSGCSVNWGEAFSAGV----HRLANG---GNGFW----------------- +4

Leu Leucocin BTa11a -------------KYYGNGVHCTKSGCSVNWGEAFSAGV----HRLANG---GNGFW----------------- +4

Leu Mesentericin Y105 -------------KYYGNGVHCTKSGCSVNWGEAASAGI----HRLANG---GNGFW----------------- +4

Lac Plantaricin C19 -------------KYYGNGLSCSKKGCTVNWGQAFSCGV----NRVATA---GHGK------------------ +6

Lac Bavaricin MN ------------TKYYGNGVYCNSKKCWVDWGQAAGGIG----QTVVXGW--LGGAIPG--K------------ +3

Car Divercin V41 ------------TKYYGNGVYCNSKKCWVDWGQASGCIG----QTVVGGW--LGGAIPG--KC----------- +3

Bs Coagulin A -------------KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATG---GHQGTH---KC----------- +6

Car Divergicin M35 ------------TKYYGNGVYCNSKKCWVDWGTAQGCI------DVVIG--QLGGGIPGKGKC----------- +3

En Enterocin A --------TTHSGKYYGNGVYCTKNKCTVDWAKATTCIA----GMSIGGF--LGGAIPG--KC----------- +3

En Enterocin CRL35 -------------KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAANLATG---GAAGW----KS----------- +4

Pe Pediocin PA1 -------------KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATG---GHQGNH---KC----------- +6

Car Pisciocin V1a -------------KYYGNGVSCNKNGCTVDWSKAIGIIGNNAAANLTTG---GAAGWN---KG----------- +3

Car Piscicolin 126 -------------KYYGNGVSCNKNGCTVDWSKAIGIIGNNAAANLTTG---GAAGWN---KG----------- +3

Lac Bavaricin A -------------KYYGNGVHXGKHSXTVDWGTAIGNIGNNAAANXATG---XNAGG----------------- +3

En Enterocin SEK4 ------------ATYYGNGVYCNKQKCWVDWSRARSEIIDRGVKAYVNGFTKVLGGIGG--R------------ +5

La Lactococcin MMFII -------------TSYGNGVHCNKSKCWIDVSELETYKA----GTVSN--PK--DILW--------------- +1

En Enterocin P -----------ATRSYGNGVYCNNSKCWVNWGEAKENIA----GIVISGW---ASGLAGMGH------------ +2

Car Carnobacteriocin BM1 ------------AISYGNGVYCNKEKCWVNKAENKQAIT----GIVIGGW---ASSLAGMGH------------ +3

Car Pisciocin V1b ------------AISYGNGVYCNKEKCWVNKAENKQAIT----GIVIGGW---ASSLAGMGH------------ +3

Lac Curvacin A ------------ARSYGNGVYCNNKKCWVNRGEATQSII----GGMISGW---ASGLAGM-------------- +3

Lac Sakacin A ------------ARSYGNGVYCNNKKCWVNRGEATQSII----GGMISGW---ASGLAGM-------------- +3

Car Carnobacteriocin B2 -------------VNYGNGVSCSKTKCSVNWGQAFQERYTAGINSFVSG---VASGAGSIGRRP---------- +4

G 4

a Sta Epidermin ---------------IASKFICTP-GCAK-TGSFN---------SYCC-------------------------- +2

Sta Gallidermin ---------------IASKFLCTP-GCAK-TGSFN---------SYCC-------------------------- +2

St Mutacin 1140 ---------------FKSWSLCTP-GCAR-TGSFN---------SYCC-------------------------- +2

St Mutacin BNy266 ---------------FKSWSFCTP-GCAK-TGSFN---------SYCC-------------------------- +2

St Streptin ----------------GSRYLCTPGSCWK-LVCFTTT--------VK--------------------------- +3

b La Nisin A ---------------ITSISLCTP-GCK--TGALMGCNM----KTATCHCSIHVSK------------------ +2

La Nisin Z ---------------ITSISLCTP-GCK--TGALMGCNM----KTATCNCSIHVSK------------------ +4

Bs Subtilin ---------------WKSESLCTP-GCV--TGALQTCFL----QTLTCNC--KISK------------------ +2

G5 Sta Epicidin 280 --------------SLGPAIKATRQVCPKATRFVTV--SCK-KSDCQ--------------------------- +5

Sta Pep 5 --------------------KT-----LKATRLFTV--SCKGKNGCK--------------------------- +8

G 6

Bs Cytolysin ----------------GDVHAQTTWPCATVGVSVAL---CP-TTKCTSQC------------------------ +1

La Lacticin 3147A2 --------------TTPATPAISILSAYISTNTCPT-------TKCTRAC------------------------ +2

Lac Plantaricin Wbeta ----------------SGIPCTIG---AAVAASIAV---CP-TTKCSKRCGKRKK------------------- +7

G7

Lac Acidocin J1132 beta --------------GNPKVAHCASQI----GRST------A-WGAVSGA------------------------- +3

Lac Plantaricin 1.25 beta ---KKKKKKVACTWGNAATAAASGAVXGILGGPTGALAGAI-WGVSQCASNNLHGMH----------------- +8

G8

Car Carnobacteriocin A ---DQMSDGVNYGKGSSLSKGGAKCGLGIVGGLATIPSGPLGWLAGAAGVINSCMK------------------ +2

En Enterocin B ENDHRMPNELN--RPNNLSKGGAKCGAAIAGGLFGIPKGPLAWAAGLANVYSKCN------------------- +4

G 9

Lac Gassericin T --------RNNWAANIGGVGGATVAGWALGNAVCGPACGFVGAHYVPIAWAGVTAATGGFGKIRK--------- +5

Lac Lacticin FlafA --------RNNWQTNVGGAVGSAMIGATVGGTICGPACAVAGAHYLPILWTAVTAATGGFGKIRK--------- +5

Lac Plantaricin S alfa --------RNKLAYNMGHYAG--------KATIFG-----------LAAWALLA-------------------- +4

G 1

0 Lac Acidocin LF221B ---NKWGNAVIGAATGATRGVSWCRGFG-PWGMTACALG----GAAIGGYLGYKSN------------------ +6

Lac Lacticin FlafX ---NRWGDTVLSAASGAGTGIKACKSFG-PWGMAICGVG----GAAIGGYFGYTHN------------------ +3

Lac LactobinA ---NRWTNAYSAALGCAVPGVKYGKKLGGVWGAVIGGVG----GAAVCGLAGYVRKG----------------- +6

G 1

1 Lac Lactocin 705 -------------GMSGYIQGIPDFLKGYLHGISAAN-------KHKKGRL----------------------- +6

Car Divergicin 750 -------------KGILGKLGVVQAGVDFVSGVWAG---------IKQSAKDHPNA------------------ +3

G 1 2 Bs Sublancin 168 --------------GLGKAQCAALWLQCASGGTIGCGGG----AVACQNYRQFCR------------------- +3

Lac PlnJ ---------------GAWKNFWSSLRKGFYDGEAGR--------AIRR-------------------------- +4

The classification of bacteriocins from G(+) is complicated by their heterogeneity and increasing numberClassification schemes have had to continuously evolve

MilkAmp: dairy derived AMPshttp://milkampdb.org/home.php

o Created in 2012o 371 entries: 9 hydrolysates,

299 AMPs, 23 peptides predicted as antimicrobial and 40 non-active peptides.

o Freely available at http://milkampdb.org/,

o Should be useful to develop biologically active peptides for both pharmaceutical and food sectors

PhytAmp: Plant derived AMPshttp://phytamp.hammamilab.org/main.php

o 24 janv. 2012271 plant AMPs

o Expected to grow quickly with the rapid development of genomic and proteomic

o https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2686510/

Genomics based bioinformatics tools

Computational tools dedicated to: .o Development of whole-genome sequencing and expression

profiling technologies;

o Identify homologs of known genes (comparing new sequences with sequences of biochemically characterized active peptides);

o Identify genes that encode AMPs

o Large target-screening approaches and provide new insight into antimicrobial mechanisms of action (MOAs)

Structure-based drug design methods: QSAR methods

o One of the most broadly used chemoinformatics approacheso Quantitative models that correlate the variation in biological activity with the

variation in molecular structureo Applied to closely related sequence variantso QSARs /artificial neural networks (ANNs): accelerate the discovery

and design of AMPs and applied to a larger peptides with high sequence diversity. o Successfully used for as antiviral/anti-HIV inhibitory peptideso Various commercial programs for QSAR: MOE, ACD labs and Tsar

Artificial neural networks

o Artificial neural networks: mathematical modeling algorithms

o Reliable and efficient way for in silico identification of novel AMPs

o Successfully applied for prediction of the antifungal activities

o ANN analysis can be performed with Tsar or Neural Network Toolbox

Structure-based drug design methods: Fuzzy logic modeling

o Fuzzy logic modeling : analysis of AMPs to distinguish active from inactive peptides with impressive accuracy.

o Fuzzy calculation can be performed using Gait-CAD

Molecular simulations and dynamics

o Demonstrate the inhibition activity and elucidate the mode of action of AMPs ( effect on bacterial membranes, formation of transmembrane ion-permeable pores and detergent-like effect)

o Use of live bacterial strains (availability and handling)

o Current area of research involves simulating bacterial membrane and AMP interactions with phospholipid bilayers

Membrane externe:Bicouche de phospholipides

Peptidoglycane

Membrane interne:Bicouche de phospholipides Membrane interne:

Bicouche de phospholipides

Couche épaisse de peptidoglycane

Acide lipoteichoique(LTA)

Acide teichoique(TA)

Molecular simulations and dynamics

Listeria monocytogenesEscherichia coli

Molecular simulations and dynamics

https://www.youtube.com/watch?v=c2utqcDdX_w

Concluding remarks

o Urgent need for new antimicrobials

o Experimental approaches

o The recent development in genomics and bioinformatics tools combined to existing structural and functional data offer a new opportunity to develop novel peptide candidates as antimicrobial molecules.

o Bioinformatics approaches: Rapid, reproducible, low cost, safe, high yields

o Amina Sghairi

o Mohamed Kacem Ben Fradj

o Jeannette Ben Hamida

o Eric Biron

oMuriel Subirade

o Sylvie Rebuffat

Acknowledgments