Repurposing of Pan-ErbB Inhibitors to Protect from Coronaviral Infection, Inflammation, and Lung Injury

Background: There is an urgent need for more effective approaches to prevent the acute and long-term lung complications associated with COVID-19 and ideally also provide readiness for future outbreaks with coronaviruses. For the past decade, our laboratory has been studying the role of cellular kinases in intracellular trafficking of RNA viruses and as targets for broad-spectrum antivirals. We have provided a proof of concept for the potential feasibility of the host-targeted broad-spectrum antiviral approach by demonstrating that the inhibition of two cellular kinases, AAK1 and GAK, by novel or approved anticancer drugs, sunitinib and erlotinib, protects mice from dengue and Ebola viruses. Since the therapeutic index of this drug combination is narrower for SARS-CoV-2 infection, in this proposal, we focus on repurposing independent kinase inhibitors targeting different pathways that emerged from our recent work. Rationale: To address the gap in treatment of pandemic coronaviruses, we screened 4,413 compounds for agents that rescue Vero E6 cells from SARS-CoV-2-induced lethality. Among the promising hits were several compounds with potent activity against members of the epidermal growth factor receptor (ErbB1-4) family of kinases, including lapatinib, an approved oral anticancer drug with a favorable safety profile, ibrutinib, an approved BTK inhibitor with potent anti-ErbB activity, and several investigational drugs (saptinib, afatinib, tesevatinib). These pan-ErbB inhibitors suppress replication of SARS-CoV-2 and unrelated viruses in human cells with a high barrier to resistance. ErbB4, but not lapatinib's cancer targets ErbB1 and ErbB2, is required for SARS-CoV-2 entry and alphavirus infection and is a molecular target mediating lapatinib's antiviral effect. In human adult lung organoids (ALOs), lapatinib protects from SARS-CoV-2-induced activation of pathways implicated in non-infectious acute lung injury and fibrosis downstream of ErbBs (p38-MAPK, MEK/ERK, and AKT/mTOR), pro-inflammatory cytokine production, and epithelial barrier injury. This project's goals are to define the roles of ErbBs in SARS-CoV-2 infection, inflammation, acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and lung fibrosis and the antiviral as well as direct tissue- protective potential of existing pan-ErbB inhibitors as attractive repurposing candidates. Hypotheses: Pan-ErbB inhibition is a safe strategy for treating COVID-19 and future coronaviral infections, which not only suppresses viral infection via ErbB4, but also reduces inflammation, ALI, and fibrosis by inhibiting ErbB1/2-mediated signaling. Specific Aims: Our main objective is to advance the repurposing of lapatinib, an attractive, approved, safe candidate drug, and back-up approved and investigational ErbB-inhibitors, for combating COVID-19 and providing readiness for future coronavirus outbreaks. Our specific aims are: Aim 1: Determine the in vitro therapeutic potential of pan-ErbB inhibitors as an anti-pan-coronavirus strategy. Aim 2: Characterize the ex vivo and in vivo effects of pan-ErbB inhibitors on viral replication, inflammation, acute and chronic tissue injury in unique human ALOs and characterized rodent models of SARS-CoV-2. Aim 3: Probe the roles of ErbBs in SARS-CoV-2 infection, inflammation, acute and chronic lung injury and decipher the mechanism of antiviral and tissue protection of pan-ErbB inhibitors ex vivo and in vivo. Study Design: In Aim 1, we will study the effect of lapatinib, ibrutinib, sapitinib, tesevatinib, and afatinib on cell viability and replication of SARS-CoV-2 isolates, including recently identified variants of concern, of interest or of high consequence (VoI/C/HC) in human cell lines. Their effect in combination drug treatment will also be assessed. To establish an expanded broad-spectrum potential, their effect against replication of SARS-CoV and MERS-CoV will also be studied. Lastly, their barrier to resistance will be defined in SARS-CoV-2 infected cells. In Aim 2, we will study ErbB inhibitors (alone or in promising combinations [Aim 1]) for their effect on viral replication, cytokine production, epithelial barrier integrity, and lung fibrosis in human ALOs from six donors. Two prioritized ErbB inhibitors will also be tested for tolerability, and efficacy (morbidity, mortality, viral load, inflammation, injury) in K18-hACE2 mouse and hamster SARS-CoV-2 models. The in vivo barrier to resistance will also be defined. In Aim 3, we will use genetic, dominant interfering and pharmacological approaches in human ALOs and/or rodent models to mechanistically probe the roles of ErbBs in the SARS-CoV-2 life cycle and disease pathogenesis. We will map the signals downstream of ErbBs and probe the roles of Angiotensin II and viral spike binding to the NRP-1 co-receptor candidate in ErbBs activation in SARS-CoV-2 infection. We will also validate ErbBs as antiviral targets and molecular targets mediating the antiviral and tissue protective effect of these drugs. Lastly, we will monitor target engagement and identify a pharmacodynamic (PD) biomarker for in vivo target modulation. Our multidisciplinary team and unique resources-including Co-Investigators Drs. Das (University of California, San Diego) and Martinez (Texas Biomedical Research Institute), collaborators Drs. Mochly-Rosen and Alvira (Stanford University), Soker (Wake Forest University), and industry advisors (Stanford SPARK) with expertise in virology, kinase biochemistry, organoids, lung biology and injury, animal models, PK/pharmacodynamics (PD), regulatory, and drug development-is ideally suited for this project. Short-Term Impact: Basic science: Our work will characterize unexplored virus-host interactions critical for SARS-CoV-2 infection and pathogenesis of ALI and lung fibrosis with implications to other coronaviruses. Technological: This project will advance a human lung organoid model that uniquely captures the diversity of proximal airway and distal alveolar cell types, is propagable, cost-effective, and personalized. Once furthered, this resource can be broadly applied to study the pathogenesis of respiratory conditions and may find potential use for phase 0 clinical trials. It will also advance a mouse model to monitor in vivo dynamic of CoV-2 infection. Translational: This study may deliver an approved, oral small molecule candidate designed to not only suppress coronaviral infection but also protect from inflammation and lung injury. Based on the excellent selectivity index in ALOs, and existing PK data, lapatinib's approved dose for cancer should sustain concentrations at levels >5-10 than the EC50 for antiviral and -inflammatory effect in serum, and >50-fold higher in lungs. Lapatinib's safety profile as monotherapy over weeks-months is favorable, with the most common side effect being diarrhea. Notably, most side effects in the package insert are based only on combination with toxic antimetabolites, as used in cancer, and not on monotherapy. Since it does not target GAK (in contrast to the EGFR-GAK inhibitors, erlotinib and gefitinib), lapatinib monotherapy has not been associated with acute or chronic lung injury. Importantly, we will probe the utility of other pan-ErbB inhibitors as back up strategies. While lapatinib has not been studied clinically, ibrutinib was protective in a small series of cancer patients with COVID-19. Since this effect was attributed solely to its anti-inflammatory effect via its cancer target BTK, our work will provide insight into its mechanism of action (MOA). Long-Term Impact: A pan-ErbB inhibitor will position us to combat new coronaviruses and possibly other viral pathogens, providing readiness for future outbreaks. It may also find broader indications for treating noninfectious ARDS/ALI and/or lung fibrosis. Military Relevance: Coronavirus outbreaks present military and global threat. COVID-19 has infected a large number of troops across military installations globally and impacted our military readiness by virus presence in our military training facilities. Even though COVID-19 is typically non-lethal/debilitating due to the younger age of the majority of the military force, the necessity to quarantine COVID-19 positive Soldiers strains our capacity and readiness state. This work will yield drugs to protect from COVID-19, future coronaviral infections, and other viral infections that threaten our military personnel. FY21 Topic Areas: (1) Emerging Viral Diseases; (2) Respiratory Health. Areas of Encouragement: (1) Research focused on acute and chronic lung injury/disorders due to viral infections, such as SARS-CoV-2; (2) development and/or testing of innovative treatments to prevent, or delay the progression of ALI/ARDS. Less