# Preclinical and Translational Assessment of Small Molecule IRF5 Inhibitors in Lupus- Relevant Systems

## INTRODUCTION

### Transcription factor with essential role in immune regulation

- IRF5 is a transcription factor downstream of Toll-like receptors 7, 8, and 9
- Primarily expressed in Dendritic cells, B cells, Monocytes, and Macrophages
- Critical role in autoantibody production, type I interferon, and proinflammatory cytokines

## Preclinical validation

### IRF5-deficient and heterozygous mice exhibited strong disease protection in several models of SLE with reductions in type I interferon levels and autoantibodies

## Interferon Gene Signature
## IRF5 Gene Signature 125

### Genetic validation

- Genetic polymorphisms in IRF5 are associated with increased risk of SLE, Sjogren’s syndrome, and other autoimmune diseases

### IRF5: Master Transcriptional Regulator Impacting Three Clinically-Validated Pathways in Autoimmunity

#### Provides Complete Cytokines

**Figure 1**. Smart Allostery Platform Enables Discovery of Potent and Selective IRF5 Inhibitors

Single-Digit Nanomolar Potency Across Multiple Cell Types and Whole Blood

| Assay      | IC50(nM) |
|------------|----------|
| Binding    | 4        |
| THP-1     | 6        |
| Monocytes  | 3        |
| B cells    | 4        |
| Neutrophils| 3        |
| PBMCs      | 5        |
| Whole Blood | 3       |

### TLR Agonist HOT-A (nM) Concentration (nM)

Compounds demonstrated exceptional selectivity over IRF3, TLR2/ NFkB pathway, and a relevant panel of >400 kinases

**(A)** Complete inhibition of TLR agonist-induced TNF production in human monocytes, B cells, PBMCs, and whole blood by the IRF5 inhibitor HOT-A.
**(B)** Average IC50 values are shown for HOT compounds across multiple assays.

**Figure 2.** IRF5 Inhibition Blocks Cytokine Production and Plasmablast Differentiation Induced by Anti-Sm /RNP Immune Complexes in B Cells

**(A and B)** Human B cells were pretreated with IRF5 inhibitors or vehicle before being cultured in the presence of soluble CD40L (sCD40L) and IL-21 and stimulated with Anti-Sm/RNP immune complexes. **(A)** Dose-dependent inhibition of cytokine production was observed after overnight incubation.

**Figure 3.** Potent Inhibition of Cytokines in SLE PBMCs with TLR Stimulation

**(A)** SLE PBMCs were pretreated with a titration of HOT-A before stimulation with a TLR agonist. HOT-A blocked the production of various cytokines in a dose-dependent manner.

**Figure 4.** IRF5 Inhibition Demonstrates Excellent Potency And Outperforms Relevant Inhibitors

**(A)** Whole blood was stimulated with a TLR agonist and TNF was measured after overnight incubation.
**(B)** Human B cells were stimulated with Anti-Sm/RNP immune complexes (RNA IC) and TNF was measured after overnight incubation.

**Figure 5.** Oral Dosing of HOT-A Blocks Cytokine and mRNA Responses Driven by TLR Stimulation in a Dose-Dependent Manner

**(A-D)** Humanized NOG-EXL mice were dosed orally with HOT-A before challenging the mice with a TLR agonist. Doses are listed in mg/kg (mpk).

**Figure 6.** Oral Dosing in Cyno Provides Complete Inhibition of the IRF5 Pathway for 24h at Lowest Dose

### Single PO Dose in NHP

**(A)** PK profile of HOT-A following oral dosing in Cynomolgus monkeys. Dotted lines indicate IC50/IC90 values based on Cyno whole blood potency.

## CONCLUSIONS

- Potent and selective inhibition of a previously undruggable transcription factor with a traditional small molecule
- Complete inhibition of B cell cytokine production and plasmablast differentiation induced by immune complexes
- Outperforms relevant inhibitors in activated SLE PBMCs
- Excellent in vivo potency and inhibition of IFN and IRF5 gene signatures; 24h complete inhibition observed in Cyno at lowest tested dose
- Potential to significantly impact a broad range of autoimmune diseases