Therapeutic targeting of vimentin by ALD-R491 impacts multiple pathogenic processes to attenuate acute and chronic colitis in mice.

BACKGROUND: Vimentin, an intermediate filament protein, crucially contributes to the pathogenesis of inflammatory bowel disease (IBD) by interacting with genetic risk factors, facilitating pathogen infection, and modulating both innate and adaptive immune responses. This study aimed to demonstrate preclinical proof-of-concept for targeting vimentin therapeutically in IBD across diverse etiologies. METHODS: The small molecule compound ALD-R491 was assessed for vimentin binding using microscale thermophoresis, off-target effects via Eurofins screening, and therapeutic effects in mice with dextran sulfate sodium (DSS)-induced acute colitis and in IL-10 KO with spontaneous colitis. Parameters measured included body weight, survival, disease activity, colon length, and histology. The study analyzed intestinal proinflammatory cytokines, Th17/Treg cells, and epithelial barrier molecules, along with gut microbiota profiling. RESULTS: ALD-R491 specifically bound vimentin with a dissociation constant (KD) of 328 ± 12.66 nM and no off-target effects. In the DSS model, orally administered ALD-R491 exhibited dose-dependent therapeutic effects, superior to 5-ASA and Tofacitinib. In the IL-10 KO model, ALD-R491 significantly delayed colitis onset and progression, with near-zero disease activity index scores over a 15-week treatment. ALD-R491 consistently showed in both models a reduced proinflammatory cytokine expression, including TNF-α, IL-1ß, IL-6, IL-17, IL-22, a rebalanced Th17/Treg axis by reducing RORγt while enhancing FoxP3 expression, and an improved epithelial barrier integrity by increasing intestinal expressions of Mucin-2, ZO-1 and Claudin5. The intestinal dysbiosis was restored with enriched presence of probiotics. CONCLUSIONS: Targeting vimentin exhibits significant therapeutic effects on various facets of IBD pathogenesis, representing a compelling approach for the development of highly effective treatments in IBD.

ALD-R491 regulates vimentin filament stability and solubility, cell contractile force, cell migration speed and directionality.

Metastasizing cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumors in the clinic. However, the role of vimentin in cell motility, and if the assembly of non-filamentous variants of vimentin into filaments regulates cell migration remains unclear. We observed that the vimentin-targeting drug ALD-R491 increased the stability of vimentin filaments, by reducing filament assembly and/or disassembly. ALD-R491-treatment also resulted in more bundled and disorganized filaments and an increased pool of non-filamentous vimentin. This was accompanied by a reduction in size of cell-matrix adhesions and increased cellular contractile forces. Moreover, during cell migration, cells showed erratic formation of lamellipodia at the cell periphery, loss of coordinated cell movement, reduced cell migration speed, directionality and an elongated cell shape with long thin extensions at the rear that often detached. Taken together, these results indicate that the stability of vimentin filaments and the soluble pool of vimentin regulate the speed and directionality of cell migration and the capacity of cells to migrate in a mechanically cohesive manner. These observations suggest that the stability of vimentin filaments governs the adhesive, physical and migratory properties of cells, and expands our understanding of vimentin functions in health and disease, including cancer metastasis.

A Vimentin-Targeting Oral Compound with Host-Directed Antiviral and Anti-Inflammatory Actions Addresses Multiple Features of COVID-19 and Related Diseases.

Damage in COVID-19 results from both the SARS-CoV-2 virus and its triggered overactive host immune responses. Therapeutic agents that focus solely on reducing viral load or hyperinflammation fail to provide satisfying outcomes in all cases. Although viral and cellular factors have been extensively profiled to identify potential anti-COVID-19 targets, new drugs with significant efficacy remain to be developed. Here, we report the potent preclinical efficacy of ALD-R491, a vimentin-targeting small molecule compound, in treating COVID-19 through its host-directed antiviral and anti-inflammatory actions. We found that by altering the physical properties of vimentin filaments, ALD-491 affected general cellular processes as well as specific cellular functions relevant to SARS-CoV-2 infection. Specifically, ALD-R491 reduced endocytosis, endosomal trafficking, and exosomal release, thus impeding the entry and egress of the virus; increased the microcidal capacity of macrophages, thus facilitating the pathogen clearance; and enhanced the activity of regulatory T cells, therefore suppressing the overactive immune responses. In cultured cells, ALD-R491 potently inhibited the SARS-CoV-2 spike protein and human ACE2-mediated pseudoviral infection. In aged mice with ongoing, productive SARS-CoV-2 infection, ALD-R491 reduced disease symptoms as well as lung damage. In rats, ALD-R491 also reduced bleomycin-induced lung injury and fibrosis. Our results indicate a unique mechanism and significant therapeutic potential for ALD-R491 against COVID-19. We anticipate that ALD-R491, an oral, fast-acting, and non-cytotoxic agent targeting the cellular protein with multipart actions, will be convenient, safe, and broadly effective, regardless of viral mutations, for patients with early- or late-stage disease, post-COVID-19 complications, and other related diseases. IMPORTANCE With the Delta variant currently fueling a resurgence of new infections in the fully vaccinated population, developing an effective therapeutic drug is especially critical and urgent in fighting COVID-19. In contrast to the many efforts to repurpose existing drugs or address only one aspect of COVID-19, we are developing a novel agent with first-in-class mechanisms of action that address both the viral infection and the overactive immune system in the pathogenesis of the disease. Unlike virus-directed therapeutics that may lose efficacy due to viral mutations, and immunosuppressants that require ideal timing to be effective, this agent, with its unique host-directed antiviral and anti-inflammatory actions, can work against all variants of the virus, be effective during all stages of the disease, and even resolve post-disease damage and complications. Further development of the compound will provide an important tool in the fight against COVID-19 and its complications, as well as future outbreaks of new viruses.