Structure-guided Design in the Discovery of Antimicrobials

Structure-Guided Design in the Discovery of Antimicrobials

Colin FishwickColin Fishwick

Professor of Medicinal ChemistryProfessor of Medicinal ChemistryUniversity of Leeds, School of ChemistryUniversity of Leeds, School of Chemistry

The threat of antibiotic resistance

“A post-antibiotic era – in which common infections and minor injuries can kill – far from being an apocalyptic fantasy, is instead a very real possibility for the 21st century” – WHO 2015

Urgent need for new antibacterial agents Urgent need for new antibacterial agents effective against bacteria resistant to multiple effective against bacteria resistant to multiple antibioticsantibiotics DUE TODUE TO::Overuse of antibioticsOveruse of antibioticsLack of new antibacterial agentsLack of new antibacterial agents SOLUTION:SOLUTION:Find new molecular targets and/or novel Find new molecular targets and/or novel inhibitors in order to combat resistanceinhibitors in order to combat resistance

KJ Simmons, I Chopra, CWG Fishwick. Structure-based discovery of antibacterial drugs Nature Rev Microbiol, 2010, 8, 501-510

In silico assisted inhibitor discovery

docking /virtual high-throughput

screening (vHTS)

X-ray structure ormodel of enzyme

SPROUT de novomolecular design

inhibitorstructures

actual compounds

enzyme assays

compoundlibraries

laboratorysynthesis

structural refinement

Ligand-basedvHTS

Example 1: Topoisomerases

• DNA is compacted by supercoiling.

• Must be relaxed to allow replication and transcription.

• Topoisomerases control supercoiling.

• Gyrase and Topo IV are Type II (ATP dependant) topoisomerases and share 40% sequence identity, though their functions are distinct and independently essential to bacterial cell survival.

• They are (i) Highly conserved; (ii) Essential; (iii) lacking a close mammalian homologue.

• Inhibiting both at same time- ‘dual targeting’ potentially slows onset of resistance

DNA Gyrase and Topoisomerase IVInhibitor binding sites

Known inhibitors, but none in clinical use

Novobiocin

ATP binding site

DNA binding siteFluoroquinolone antibiotics widely used

Moxifloxacin

OO

OH

O

OO

OHO NH2

ONH

O

HO

N

OO

HO

N

F

H

H

OMeNH

• In-house crystallographic data showing a triazolopyridine inhibitor bound to the E. faecalis GyrB ATP binding site was used as a platform in the design of novel inhibitors

SPROUT was used in a bid to mimic these interactions via a novel scaffold

N

N

N

N N

O

ONH

N

H HO

OH

OH

HN

HO

NH2H2N

HN

N55

D82

R85

Bioorg. Med. Chem. Lett., 2009, 19, 894-899

Confidential

Primary Scaffold Screening

Compound GyrB IC50 (µM)a MIC (µg / mL)

SA EF SP HI

Triazolopyridine 0.041 8 4 8 -

2 158.3 >64 >64 >64 >64

3 6.0 >64 >64 32 >64

4 >754 >64 >64 >64 >64

5 >716 >64 >64 >64 >64

6 >530 >64 >64 >64 >64

7 >513 >64 >64 >64 >64

8 74 >64 >64 >64 >64

9 1.8 >64 >64 >64 >64

10 1.4 >64 >64 >64 >64

a E. coli GyrB ATPase assay; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes ATCC 51339; HI, H. influenzae ATCC 51907

• ca. 5-10 compounds for each scaffold, most active shown.

Ian Yule, Sarah NarramoreIan Yule, Sarah Narramore

SAR

Compound 3

N

NH

O

NH

NH

ON

N

N

NN

NH

NH

O

NHO

Exploring ‘Compound 3’ de novo library

• Further examination of the model identified a key location into which compounds could be extended• A substantial route optimization effort was required to access structural variants of 3 which could further probe the pocket. A 35 compound library was prepared.

Optimal region for structural branching; Phosphate channel

C-4 structural Optimisation

R1 R2 GyrB IC50 (µM)* MIC (µg / mL)

SA EF SP

2-thiazolyl NMe-pip 33.2 >64 >64 >64

3-tolyl NMe-pip 18.0 >64 >64 >64

2-thiazolyl 3-pyridyl 0.56 >64 64 64

3-chlorophenyl 1-imidazolyl 0.55 >64 32 64

3-chlorophenyl 1-pyrazolyl 2.96 8 1 4

3-pyridyl NHCH2(3-py) 0.66 >64 8 8

3-tolyl NHC6H11 2.12 >64 >64 >64

2-thiazolyl 3-toluidyl 0.098 >64 >64 >64

3-tolyl 3-toluidyl 0.099 1 0.5 32

3-chlorophenyl H 1.6 16 16 16

* E. coli GyrB ATPase assay; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes ATCC 51339

N

NH

O

NH

NH

OR1

R2

Initial structural optimisation at C4


Possible role of intramolecular H-bonding?


N-heterocycles at C-4

R cLogPIC50s (µM) MICs (µg / mL)

GyrB ParE SA EC

0.79 49 105 64 >128

1.56 0.26 0.68 16 >128

-0.02 5.2 1.0 64 >128

0.11 8.9 1.2 >128 >128

1.22 59 5.3 64 >128

NN

NN

Br

NN

N

N

NN

N

N

NH

O

NH

NH

O

RN


R1 R2 GyrB IC50 (µM)* MIC (µg / mL)

SA SA + HS EF SP

Ph NHPh 0.040 1 nd 0.25 2

3-tolyl NHPh 0.140 1 nd 0.125 2

3-Cl-Phe NHPh 0.150 0.5 nd 0.125 4

3-pyridyl NHPh 0.170 1 4 0.125 1

6-mor-3-py NHPh 0.111 2 16 0.25 1

6-acetam-3-py NHPh 0.039 2 8 0.125 8

6-triaz-3-py NHPh 0.153 >16 >16 0.25 >16

phenyl NH-3-Py 0.430 1 2 2 0.25

3-pyridyl NH-3-Py 0.093 8 nd 0.5 1

phenyl OPh 4.90 >16 >16 >16 >16

* E. coli GyrB ATPase assay; SA, S. aureus ATCC 29213; EF, E. faecalis ATCC 29212; SP, S. pyogenes ATCC 51339; SA + HS, S. aureus + 50% horse serum

• Core structural element of R2 = NHAr was retained and amide varied leading to a series of potent antibacterial compounds with low levels of serum binding

N

NH

O

NH

NH

OR1

R2

R1 =

R1 =

R1 =

N

NO

N

AcHN

N

N

N

N

Amide optimisation


• Bacteria have developed β-lactamase enzymes which cleave the amide bond in the β-lactam antibiotics to render the drug molecules ineffective.

• This method of antibiotic resistance is particularly prevalent in Gram-negative bacteria.

• There are four different classes of enzymes: A, B, C and D.

• Class B are made up of the metallo-β-lactamases whereas classes A, C and D are the serine-β-lactamases.

Example 2: -lactamases

• Most important class of β-lactamase enzymes as they can hydrolyse all β-lactams except for the monobactam aztreonam.

• 3 different subclasses: B1, B2 and B3.

• B1 has two zinc ions in the active site but can be active in the mono or di-zinc forms.

• B2 is only active in the mono-zinc form whereas the B3 subclass is active in the di-zinc form.

• A number of structurally related variants of MβLs, including IMP-1, VIM-2 and NDM-1 which all belong to the B1 subclass.

Metallo--lactamases

Example 2: -lactamases

Boronates to mimic tetrahedral intermediate?

Bind to MBL

planar tetrahedral

Mechanism-based inhibition

In vitro screening reveals broad spectrum activity

Brem et al, Nature Commun., 2016

In vitro screening reveals broad spectrum activity


2

Structural elucidation


Conclusions

In silico fragment-based molecular design can be In silico fragment-based molecular design can be used to rapidly produce novel enzyme inhibitors.used to rapidly produce novel enzyme inhibitors.

The initially-designed molecules can act as The initially-designed molecules can act as templates for further rounds of enhancement via templates for further rounds of enhancement via synthesis and de novo design. synthesis and de novo design.

Cellular activity difficult to predict- once Cellular activity difficult to predict- once established can be improved via chemistryestablished can be improved via chemistry

Acknowledgements

Ian YuleIan YuleSarah NarramoreSarah NarramoreRicky CainRicky CainRachel JohnsonRachel JohnsonProf. Peter JohnsonProf. Peter JohnsonDr. Alex O’NeillDr. Alex O’Neill

Prof. Chris SchofieldProf. Chris SchofieldDr. Lloyd CzaplewskiDr. Lloyd CzaplewskiProf. Tony MaxwellProf. Tony MaxwellDr. Clare StevensonDr. Clare StevensonDr. Jim SpencerDr. Jim SpencerDr. Jurgen BremDr. Jurgen Brem

EPSRC, BBSRC, Medical Research Council, Medicines for Malaria Venture, EPSRC, BBSRC, Medical Research Council, Medicines for Malaria Venture, Biota Europe Ltd (UK), Wellcome Trust, NIHBiota Europe Ltd (UK), Wellcome Trust, NIH

R = NH-PNB

R = NH2

R = NHCONHEt

R = OH

R = thiazol-2-yl amino

R = OHR = Cl

R = imidazol-1-yl (34 %)

R = 4-me-piperazin-1-yl

R = imidazol-1-ylR = 4-me-piperazin-1-yl

R = imidazol-1-yl

R = 4-me-piperazin-1-yl (80 %)

m-toluidineHCl

EtOH51%

N

EtO2CHN

Cl N

EtO2CHN

R N

HN

NH

O

R

NH

O

N

EtO2CR

R N

EtO2CR

Cl N

EtO2CR

NH

NH

O

N

R

NH

NH

ONH

OAr

N

HO2CR

NH

NH

O

p-MeOCH2NH2 PhMe, ,62%

62 %

NaOH (aq),

90 %

TFA, Et3SiH98 %

EtNCO, 43 %

POCl3, 79 %

ArNH2, HOBTEDC.MeI, DMF, rt, 35 %

ImH, NaH, DMF or

NMe-pip, TEA

Pd(OAc)2, xantphosN-ethylurea, KOtBu,

79-92 %

NaOH (aq),

56 -97 %

ArNH2, HOBT, EDC.MeI, DMF, rt,

5-51 %

routes based upon dichloropyidine


• Proving a link between target (enzyme) activity and antibacterial activity is important in early evaluation of a novel antibacterial compound

• Promiscuous cytotoxic activity (e.g. membrane damaging) observed alongside coincidental enzyme activity can be mis-leading

Wild-typeGyrB

Wild-typeParE

dualinhibitor

Wild-typeBacterium

antibacterialactivity

Mode of action studies via mutagenesis



Wild-typeGyrB

Wild-typeParE

dualinhibitor

Wild-typeBacterium


X X




Wild-typeGyrB

Wild-typeParE

dualinhibitor

Wild-typeBacterium


X X

X X


R1 R2MIC (µg / mL)

SA T173N / T167N

(GyrB / ParE)SA SA T173N (GyrB) SP SP A53S (ParE)

Ph NHPh 1 2 2 16 >64

3-Cl-Phe NHPh 0.5 1 4 32 >64

3-pyridyl NHPh 1 4 1 2 >64

SA, S. aureus (ATCC 29213); SA T173N, S. aureus GyrB mutant (ATCC 080519); SA T173N / T167N, S. aureus GyrB / ParE mutant (ATCC 090108); SP, S. pyogenes (ATCC 51339); SP A53S, S. pyogenes ParE mutant (ATCC 071201).

N

NH

O

NH

NH

OR1

R2

Point mutation in GyrB ATPase site


R1 R2MIC (µg / mL)

SA T173N / T167N


Ph NHPh 1 2 2 16 >64

3-Cl-Phe NHPh 0.5 1 4 32 >64



N

NH

O

NH

NH

OR1

R2

Point mutation in ParE ATPase site


R1 R2MIC (µg / mL)

SA T173N / T167N


Ph NHPh 1 2 2 16 >64

3-Cl-Phe NHPh 0.5 1 4 32 >64



N

NH

O

NH

NH

OR1

R2

Dual mutation in GyrB and ParE sites


R1 R2MIC (µg / mL)

SA T173N / T167N


Ph NHPh 1 2 2 16 >64

3-Cl-Phe NHPh 0.5 1 4 32 >64



N

NH

O

NH

NH

OR1

R2

Dual mutation in GyrB and ParE sites


Yule, Narramore, Yule, Narramore, et alet al (2014), (2014), European Journal of Medicinal ChemistryEuropean Journal of Medicinal Chemistry , 86, 31 - 38 , 86, 31 - 38

Structure-guided Design in the Discovery of Antimicrobials

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