1. TB Host-Directed Therapy (HDT) Candidate Agents
December 2015 – Tuberculosis Clinical Research Branch, DAIDS, NIAID

2. TB HDT Rationale
TB drug resistance continues to develop and HDT will be effective despite resistance and may help to prevent increases
Few new anti-TB drugs are in or entering clinical evaluation and some have issues with safety and significant PKIs interactions
MANY candidate HDT drugs await clinical evaluation for rapid adaptation for TB – re-purposing avoids agent development costs
If only a small % of potential new classes of HDT drugs are useful, the number of available TB agents will be greatly expanded
HDT agents for TB may also have therapeutic benefit for other infections and also may serve as vaccine “potentiators”

3. 1. Improving TB-induced immune defects
Rationale for Specific, Small Molecule Adjunctive Immunomodulators in TB Rx
1. Improving TB-induced immune defects
– Particularly macrophages/other innate cells/autophagy
2. *Decreasing inflammatory tissue pathology/damage*
AND protected Mtb sanctuaries
Less inflammation, necrosis, caseation, granulomas, lessened inhibitory molecules
Improved immune cell access/function
Improved anti-TB drug delivery to bacilli
“NRP” TB may reactivate to be killed more quickly by TB drugs
3. Overall Effect  Improved TB clearance occurs in models

4. NIAID RFA - HDT for TB: New approaches
Objective: Identify and characterize adjunctive host-directed therapy agents to improve treatment outcomes
Agents with evidence of therapeutic benefit in animal models
and have FDA approval or be in Phase II or III trials
Mechanism: Phased UH2 / UH3 (5 yrs)
UH2 – Pre-clinically identify/optimize specific agent and dosing regimen to advance to POC clinical trial and trial preparation
UH3 - Phase II POC Trial performance

5. Four NIAID UH2/UH3 TB HDT Awards
Autophagy Inducers (Deretic)
Statins and other lipid-metabolism modulators (Karakousis)
Imatinib (Kalman)
Anakinra (Flynn) interleukin-1 receptor antagonist (IL-1Ra)
[Metformin grant application under review]

6. Simvastatin increases the in vivo activity of the first-line tuberculosis regimen J Antimicrob Chemother 2014; 69: 2453–2457
Relative to the standard oral regimen of R/H/Z, addition of 25 mg/kg simvastatin in a BALB/c mouse model reduced lung cfu by an additional 1 log10 at Day 28 (P=0.01) and by a further 1.25 log10 at Day 56 (P=0.01)

7. Collectively, these data indicate that MET is a promising
In Mtb-infected mice, adjunctive metformin (MET) reduces the intracellular growth of DS a DR Mtb in an AMPK–dependent manner
MET increased production of mitochondrial reactive oxygen species and facilitates phagosome-lysosome fusion
MET reduced inflammation and lung pathology, and enhanced
specific immune responses and efficacy of conventional TB drugs
Collectively, these data indicate that MET is a promising
candidate host-adjunctive therapy for improving TB treatment

8. Tyrosine Kinase Inhibition
Imatinib – abl and c-kit targets
Myelomonocytic targets –
Improved autophagy and phagosomal acidification
Mobilization of myeloid progenitor cells
Decreases the number and function of immune suppressor cells in some malignancies
Studies in NHP (Rhesus macaques) TB and TB-SIV models with MDR Rx combinations are ongoing and appear promising

9. Planned BMGF Sponsored 5-arm Phase II Trial
PDE-4 inhibitor - CC-11050
Auranofin (complexed gold)
Everolimus (mTOR inhibitor)
Vitamin D
Standard DS TB regimen

10. Next Generation HDT?
First-generation HDT candidates were chosen as “anti-inflammatory agents”, or empirically, e.g., steroids, anti-TNF, statins, imatinib, metformin
Next generation HDT agents will be TARGETED agents chosen to reverse specific abnormalities caused by Mtb in
Cell regulatory pathways of infected/immune reactive cells
- “Precision Medicine”
Immune effector functions of immune reactive cells
- “Immuno-oncology”
For BOTH innate and adaptive TREATMENT AND VACCINE Responses

11. Cell regulatory pathways - Precision Medicine Advances for TB
“Precision Medicine” Therapeutics
Targeted HDTs have revolutionized cancer treatment by reversing effects of “molecular drivers” subverting core cell regulatory pathways
Some of the same cell pathways exploited by cancers are also disrupted by Mtb to enhance survival/proliferation, e.g., altered cholesterol metabolism AND to impair reactive immune cell function by disruption of cell metabolism
Many precision medicine agents now approved or in clinical trials can be re-purposed for TB and other infections

12. Precision Medicine and TB - Approaches
What research is needed?
Identification of therapeutic targets - Precise determination of the immune cell regulatory pathways disrupted by Mtb to cause immunopathogenesis
Screening - A wide variety of clinically available precision medicine agents for effect on TB infection in improved in vitro “granuloma model” systems
Animal models (as appropriate)  POC clinical Trials

13. Molecular Targets of Precision Medicine Agents of Relevance for TB HDT
Evidence for PG role or benefit
NOT YET EXPLORED for PG or benefit
AMPK
P13K-AKT-mTOR
MAPKs – JNK, ERK
Protein kinases: abl, c-kit, VEGF, EGFR, JAK/STAT
Mevalonate pathway
Autophagy inducers
Rho/ROCK
Ras
Cathelicidin/AMPs –
HDACs as inducers
Wnt/beta-catenin
MMPs
PARPS
Sirtuins – STACs/inhibitors
Hedgehog
Notch
HIF-1α
Other kinases – SIK, FAK-Src, S6…
Inflammasomes
DPP-4
ERS/UPR reduction
Angiotensin II receptors
Bcl-2
GSK-3
Many more

14. Reversing TB immunosuppressive Mechanisms
1) Immune checkpoint modifications (Directly on Mtb-reactive cells)
Immune cell co-receptor checkpoints
Prevent inhibitory T-cell co-receptor/ligand signaling
– AGENTS - PD-1, CTLA-4, LAG-3, A2aR blockers (OX40* is stimulatory)
Immunometabolic checkpoint modifications
Regulate immune cell differentiation and function by pathway controlled cellular metabolism, e.g., to enhance Th1/TH17 and memory CD8+ T cell responses
- AGENTS - AMPK, sirtuin1/HIF-1α, mTOR inhibition*  VACCINE potentiation
* Used in combination for vaccine potentiation - animals
2) Block Suppressor cell induction (Indirect effect on Mtb-reactive cells)
Tregs, MDSCs, M2 polarized macrophages induced by Mtb and BCG vaccination
- AGENTS – IDOi, ATRA, TKIs, PDE-5i, bisphosphonates, others

15. Leveraging the exciting advances in precision medicine for the development of innovative next-generation HDTs may lead to entirely new paradigms for treatment and prevention of tuberculosis and other infectious diseases.

17. BACK-UPS

18. Targeting Immunometabolic Cell Regulatory Pathways to improve Vaccine Responses
AMPK Stimulation
Regulates CD4 T cell responses to infection by control of a glucose-sensitive metabolic checkpoint for Th1 and Th17 development
Controls metabolic transition of active/glycolytic effector CD8 T lymphocytes to quiescent T cells to support CD8 T-cell memory development
Sirtuin1/HIF1a signaling
Sirtuin1 inhibition enhances HIF1a activity in DCs to improve Th1 T cell differentiation and to decrease Tregs
HIF1a activity stabilizing agents improve Th1 responses by inhibiting HIF-PHs (Hypoxia- Inducible Factor prolyl hydroxylases – PHDs)
mTORC inhibition – including with BCG vaccination
Improves memory CD8+ T cell generation in animal models – see “Beyond Adjuvants: Immunomodulation Strategies to enhance T-cell immunity” Vaccine 33S (2015) B21-28.

19. Anti-inflammatory PRO-inflammatory And Cell Death SIRT
PAMP or DAMP
OxLDL
RTK
PRR
β-AR SR
Adenylate
Cyclase
PI3K
ROS
cAMP
ATP
ERK
AKT
PTEN
Oxidative
Stress
JNK ER
STRESS
OxLDL
mTOR
PKA
PDE
Autophagy
Foamy Macrophage M2
DAMPs
PARP
AMPK
NF-κB
Anti-inflammatory
PRO-inflammatory
And Cell Death
SIRT

20. Metformin
Decreases glycolation (pyridoxamine and buformin may be better) reacts with dicarbonyls
Decreased TNF and other inflammatory CKs by inhibiting ERK/EGR-1 pathway and suppressing scavenger receptors (CD36 and SR-A)
AMPK activation
Inhibits mTOR – enhances autophagy in TB infected macrophages mediated by PPAR-gamma
Inhibits PARP activation and activates Bcl-6
Decreases effects of RAGE signaling on some cells and inhibits HMGB1 release
Decreases NF-kB expression/Suppresses ROS formation

21. Statins HIGHLY PLEIOTROPHIC EFFECTS AGE/RAGE effects
Decrease MPO-dependent AGE generation
Control AGE-mediated histone modification
Displaces RAGE from membrane
Decrease RAGE expression by inhibition of Rac-1
Decreases LOX-1 effects
Enhance Efflux pumps for lipids
Matrix metalloproteinase down-regulation
And Others - Will all of these be sufficiently additive to significantly impact TB-DM treatment outcomes?
Hitone mods

22. Metabolic pathways in T cell fate and function Immunometabolism: linking metabolism and immune function Trends in Immunology April 2012, Vol. 33, No. 4

23. T-cell Activation Immune Checkpoints
Checkpoints for development of effective cellular immunity
T cell Co-receptors
Blocking PD-1 and CTLA-4 checkpoints with antibodies improves antigen- specific T cell functions – reverses senescence
The next generation of immune checkpoint blockade for clinical evaluation will include oral A2aR antagonists. Some are in trials (e.g., oral istradefylline) to increase immune responses to tumors (by T-cells, macrophages, NK cells)
TB connection - Journal of Infectious Diseases 2014;210:824–33
“… a role for the purinergic pathway in the host response to M. tuberculosis. Dampening inflammation through signaling via the adenosine A2A receptor may limit tissue damage but may also favor bacterial immune escape.”

24. Myeloid-derived Suppressor Cells (MDSCs)
MDSCs are immunosuppressive lesion-infiltrating cells
Tumor-associated MDSCs are being targeted in cancer vaccine and other immunotherapies to improve host immune responses
Large numbers of MDSCs develop during active TB in humans and BCG infection recruits MDSCs
In a mouse model, BCG-induced MDSCs blocked T cell proliferation and dampened Ag-specific priming in lymph nodes
Interventions - MDSC suppression or conversion to improve cancer immunoRx by
Sirtuin1 inhibition directs a switch to a M1 lineage by glycolytic activation of cells through enhanced HIF1a function
All-trans retinoic acid, PDE-5 and c-KIT inhibition (sunitilib), bisphosphonate…

25. Innovative New Research Tools for Use at Single Cell Level
High through-put assays to define effects of molecular drivers of disease on individual cell regulation and to identify intervention targets
Epigenomic assays – defining the regulome
ChIP (Chromatin Immunoprecipitation)-based assays (acetyl, methyl, TF binding, etc.)
Chromatin accessibility assays (ATAC-seq, FAIRE-seq, …)
DNA methylation sequencing – bisulfite methylation sequencing (BMS)
Advanced proteomics, metabolomics…
Combined for simultaneous use – interactomes
DNA + RNAseq, CHiP + DNAseq, ChIP + BMS, Chip+proteomics, etc.
Sensitive detection of histone-altering gene polymorphisms (acetylation ChiP-DNAseq to find haQTL SNPs) – on a genome-wide basis using cohorts < 100, MAF ’s < 1%
Results:
Correlating changes in specific cell’s signaling (and influence of genetics, epigenetics)
gene expression (transcription/translation/post-translational modifications)
effects on cell metabolism and defense functions (phenotype)

26. “Signaling Network Modeling” for Outcomes Single cell methods are key to this modeling
With a sufficient signaling database for a specific cell type – develop mathematical modeling for effects of signaling changes on cellular outcomes
-- gene expression  cell metabolism/function
Adaptive – modify model as new data becomes available
In silico study of network dynamics to predict “network nodes” of most vulnerability to intervention
Modeling can predict effect of targeted drug interventions and if combination therapy would be significantly more effective
After drug intervention – include dosing effects to determine minimal effective dose for a drug
 The future of therapeutic development for most diseases

27. Immune Protection vs. Inflammatory Damage
Immune reaction regulation will need to change over the time course of an evolving immune reaction to pathogens
Initially -
Enhance robust and usually highly inflammatory reaction to first control and then kill and begin to remove the pathogen -- or react to vaccine
HDT’s best use may be to reverse pathogen immune disruptive effects
Later - ? Exactly when
Inflammatory down-regulation must INCREASE as the immune process evolves in order to limit tissue damage and implement tissue repair
NOW- HDT role may be to help modulate inflammation/damage – also to improve access and function of immune cells and antibiotics
 Timing/context of use of different HDTs may be crucial

28. Key Cellular Regulatory Signaling Pathways
Core pathways
PARP family
mTOR
Sirtuin family
AMPK
Other key pathways – with known links to TB pathogenesis
Cellular Kinase networks (and Phosphatases)
MAPK and cascades
Kinases – TKs – (e.g., abl, c-kit, VEGF, JAK/STAT) and MANY more classes
Small molecule GTPases
Wnt/beta-catenin family
Hedgehog, Notch1/SOCS3….
Implicated in BCG-induced suppression of immune responses

29. * US FDA-approved or available OTC # Approved in other countries with stringent regulatory authorities Bold: Preclinical testing against MTB being performed

30. HDT CAVEATS May not work Could cause harm
PK/PD issues - delivery to site of action in active form with sufficient exposure
Extrapolation from different disease models/states
In vitro and animal model (rodent) data not translating
Actions depend on “tissue/cellular context”
Complexity of regulation/signaling – counter-reactions
ETC.
Could cause harm
Worsen TB disease course
Increase lung damage
Impact on HIV co-infection