TB DRUG DISCOVERY BIOASSAY RESOURCES
Scott Franzblau
Director, Institute for Tuberculosis ResearchProfessor, Department of Pharmaceutical Sciences

Multiple UIC units support TB drug discoveryChallenges and Solutions in Early-Stage TB Drug Discovery
Challenge
Virulence of M. tuberculosis (Mtb) +lack of avirulent surrogate species withsimilar drug susceptibility

Solution
Use virulent but drug-sensitive Mtb
Powered Air Purifying Respirators (PAPR)
Biosafety Level 3 lab

Slow growth of Mtb, 3 weeks for colony
formation

Metabolic surrogates of viability:• Resazurin reduction (Microplate Alamar BlueAssay; MABA)• Luciferase reporter genes• Intracellular ATP

Early ID of treatment shortening potential

Determine killing of non-growing culture by Low
Oxygen Recovery Assay (LORA)

Mouse models take 1.5 – 2.5 months

qPCR reduces time by 3 weeks

1st & 2nd Generation Bacterial Luciferase Reporters
First Generation
Plasmid-borne
Requires substrate (n-decanal)End-point assay

Second Generation
Integrated into chromosome
Codes for both enzyme and substrate
Enables Kinetic Assays

Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, et al. (2010) Optimisation ofBioluminescent Reporters for Use with Mycobacteria. PLOS ONE 5(5): e10777.

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Phenotypic-based screening: hit ID
HTSvs Mtb luxABCDE
96 or 384 well100K samples/month>85-90%luminescence

MIC

Cytotoxicity IC50

vs Mtb H37Rv
Microplate Alamar Blue Assay (MABA)

vs Vero cells
Resazurin reduction

SI = IC50/MIC >10

In vitro profilingPhenotypic-based Screening: In vitro Hit Profiling
Property

Assay

Potential to shorten treatment

MIC vs non-replicating Mtb culture (LORA)

Killing effect (vs. growth inhibition only)

Minimum bactericidal concentration (MBC)

Persistent suppression of growth following
compound clearance

Post Antibiotic Effect (PAE)

Ability to kill bacteria within hostmacrophage

Intramacrophage activity (EC90)

Synergy or antagonism in combination withestablished or experimental TB drugs

diaMOND to determine FICs

Frequency of mutation to resistance andtarget identification

Selection of resistant mutants and WGS

Non-Replicating Mtb luxABCDE: Low Oxygen Recovery Assay (LORA)
R99HOHO12OO

OR6HO N

11Low oxygen-adapted M. tuberculosis
carrying luxABCDE63OO

2 x 104

OOO
OH

28h “recovery”
in air

CFU in 24well plates

luminescence

10 days under <0.16% oxygen
No replication!
Cho S, Lee HS, Franzblau S. Microplate Alamar Blue Assay (MABA) and Low Oxygen Recovery Assay (LORA) for
Mycobacterium tuberculosis. Methods Mol Biol. 2015;1285:281-92. PubMed PMID: 25779323.

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MBC by CFU vs Bioluminescence Against Replicating M. tuberculosis luxABCDE
2 x 105 RLU
CompoundR u f o m y c in ( s t a t ic )10 6( M IC 0 .0 2 u g /m L )C o n c ( u g /m L )7/14 days

14 days, 37 ºC

Log RLU

10 5

10 .2 5

10 40 .0 6 2 5
1030 .0 1 5 6 2 50 .0 0 3 9 0 6

10 2B a c t c o n tr
10 1
0Plating on 7H11
agar plate

510152025

T im e (d a y s )Luminescence is read~daily

E c u m ic in ( c id a l) M IC ( 0 . 0 7 u g / m L )10 64 weeks
MBCMBC, Time-killinformation

Log RLU

10C o n c (u g /m L )1052 .5
10 4
100 .6 2 50 .1 5 6 2 530 .0 3 9 0 6 310 2
10B a c t c o n tr

10Total time required

42 days

51015T im e (d a y s )14 days
82025

Post Antibiotic Effect Can Shed Light On Target Vulnerability

1010 6R if a m p ic in

510 4
10

1 0 0 X M IC

31 0 X M IC
1 X M IC

10 2

L o g (R L U )L o g (R L U )10 6

B act C ont

10 102468101210I s o n i a z id

510 4
101 0 0 X M IC

31 0 X M IC
1 X M IC

10 2B act C ont

10 1014268101214

T im e ( d a y s )T im e ( d a y s )Bacterial control

2h

10 610 6

E c u m i c in

10 5
10 4
1 0 0 X M IC
10 3

1 0 X M IC

10 2

1 X M ICB act C ont

10 10246810

T im e ( d a y s )1214

L o g (R L U )PAETreatedL o g (R L U )Cell density by RLU

4

R u f o m y c in

10 5
10 4
1 0 0 X M IC

10 3

1 0 X M IC

10 2

1 X M ICB act C ont

10 102468101214T im e ( d a y s )Time9Activity Against Macrophage-Internalized Bioluminescent Mtb
5x104 Macrophages
seeded to 96 WP

Infect with
H37Rv_LuxABCDE
5x105

Treat withamikacinovernight

Wash with PBS
Add test compounds

Incubate for
6-7 days

Bioluminescence

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Phenotypic based screening: spectrum of activity
Property

MIC vs

Cross-resistance to existing TB drugs

H37Rv-isogenic strains mono-resistant to INH,rifampin, ethambutol, kanamycin, bedaquiline,moxifloxacin, etc.

Effective against clinical isolates fromdifferent geographical regions

6 global clade representatives

Broad or narrow spectrum antimycobacterial activity

M. abscessus, M. avium, M. ulcerans, M. bovis,etc.

Broad or narrow spectrum antibacterial
activity

ESKAPE panel of 4 Gram - and 2 Gram +bacteria

Efficacy in Acute and Chronic Mouse Infection Models of TB
Aerosol infection of ~100 female BALB/c mice with low dose of M. tuberculosis Erdman
Acute infection model
Chronic infection model
Tx begins Day 10Tx begins Day 3138
2.6 Log CFU reduction1) Mice sacrificed 3 days after final dose2) Lung homogenates serially diluted & plated3) Colonies counted after 3 weeks incubation

qPCR w PMA dye to reducetime to data by 3 weeks

CT36

100 mpk
1.7 Log CFU reduction

3450 mpk

32

0.58 Log CFU reduction

25 mpk30vehicle

284567Log 10 CFU TBA-208 IP

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Target profile of TB drug lead
Assay
MIC
Vero cytotoxicity IC50
Vero IC50/MIC
LORA/MABA
MBC/MIC
EC90 vs intramacrophage Mtb
MIC vs H37Rv/drugR strains & global clades
MIC vs G+, G-/MIC vs Mtb
MIC with serum or albumin
Combinations (diaMOND)
Mouse infection model log10 lung CFU reduction

Ideal<0.1 uM>100 uM>1000<5<5< 1 uM
<2x>50<4Synergistic
3Acceptable
5 uM>20 uM>50
Any
Any<20 uM<8x>10<20Not antagonistic1