key: cord-0750901-k9kn0kaz
authors: Sundaresan, Lakshmikirupa; Giri, Suvendu; Singh, Himanshi; Chatterjee, Suvro
title: Repurposing of thalidomide and its derivatives for the treatment of SARS‐coV‐2 infections: Hints on molecular action
date: 2021-03-15
journal: Br J Clin Pharmacol
DOI: 10.1111/bcp.14792
sha: 7dbdc892ca1f4c475e69849f70aa6481d611ecc8
doc_id: 750901
cord_uid: k9kn0kaz

AIMS: The SARS‐coV‐2 pandemic continues to cause an unprecedented global destabilization requiring urgent attention towards drug and vaccine development. Thalidomide, a drug with known anti‐inflammatory and immunomodulatory effects has been indicated to be effective in treating a SARS‐coV‐2 pneumonia patient. Here, we study the possible mechanisms through which thalidomide might affect coronavirus disease‐19 (COVID‐19). METHODS: The present study explores the possibility of repurposing thalidomide for the treatment of SARS‐coV‐2 pneumonia by reanalysing transcriptomes of SARS‐coV‐2 infected tissues with thalidomide and lenalidomide induced transcriptomic changes in transformed lung and haematopoietic models as procured from databases, and further comparing them with the transcriptome of primary endothelial cells. RESULTS: Thalidomide and lenalidomide exhibited pleiotropic effects affecting a range of biological processes including inflammation, immune response, angiogenesis, MAPK signalling, NOD‐like receptor signalling, Toll‐like receptor signalling, leucocyte differentiation and innate immunity, the processes that are aberrantly regulated in severe COVID‐19 patients. CONCLUSION: The present study indicates thalidomide analogues as a better fit for treating severe cases of novel viral infections, healing the damaged network by compensating the impairment caused by the COVID‐19.

followed by host immune system alert with active viral replication in the upper respiratory tract (Phase II). In Phase III, a minor cytokine storm occurs in the alveoli, releasing the inflammatory cytokines resulting in leaky blood vessels, which is followed by the second cytokine storm with uncontrolled inflammatory and life-threatening symptoms, acute respiratory distress syndrome (ARDS), seizure, severe hypoxia and severed organ toxicity (Phase IV). 1, 2 Manifestation of the biphasic cytokine storm occurs through the activation of a series of cytokines including monocyte chemoattractant protein 1 (CCL2), macrophage inflammatory protein 1α (MIP-1α), tumour necrosis factor α (TNF-α), interleukin (IL)-2R and IL-6 overwhelming the system leading to indiscriminate damages in multiple organs. [3] [4] [5] There is an increased amount of blood vessel growth in the lungs of COVID-19 patients compared to severe influenza. 6 As COVID-19 is a multilayer problem, researchers around the world are desperately in search for a drug, which would able to tackle all or few of these COVID-19 hallmarks.

Thalidomide, a small molecule drug, with many years of history known to cause misery, 7 became a game changer for its multifaceted pharmacological effects such as immunomodulation, antiinflammation, antiangiogenesis and antiviral effects. 8 At present, the world needs a smart solution. Thalidomide increases the hope of treating COVID-19 patients. 9 Chen et al. report successful treatment of SARS-coV-2 associated pneumonia with combinatory treatment of thalidomide and a low-dose glucocorticoid. 10 Two clinical trials, NCT04273581 and NCT04273529 have been registered to check the efficacy of thalidomide in treating COVID-19 patients. The adverse effects of thalidomide and its analogues are well documented. 11 Extensive information available on thalidomide's mechanisms, its efficacy and safety in haemophagocytic syndrome-induced cytokine storm 12 and idiopathic pulmonary fibrosis, 13 severe H1N1, and paraquat poisoning lung injury 14, 15 argues for the possible action of thalidomide on COVID-19 induced lung effects and cytokine storm.

Recent reviews on thalidomide in COVID-19 treatment endorse the possibility of thalidomide and its analogues for the treatment of COVID-19 symptoms. 16, 17 Transcriptome-based approach to connect diseases with drug responses is a recognized strategy in drug repurposing. 18 lenalidomide-treated PBMC (Accession ID: GSE84251) and CC-122 treated lymphoma cells (Accession ID: GSE75420) 25 were procured from GEO and differentially expressed genes (DEGs) were identified using limma. 26 Library of Integrated Network-Based Cellular Signatures (iLINCS) is a database that contains the gene expression signatures of >21, 000 compounds (http://www.ilincs.org/ilincs/). We obtained the gene expression signatures for A549 cells treated with 10 μM thalidomide for 6 hours (LINCSCP_4683) and 24 hours (LINCSCP_4463), 100 μM lenalidomide for 6 hours (LINCSCP_4650) and 24 h (LINCSCP_4427).

Endothelitis is a common sign of COVID-19 6 and as thalidomide possesses well established vascular and anti-inflammatory effects, we

What is already known about this subject • Severe cases of COVID-19 infections show an aberrant surge in the host immune response and cytokine storm in lungs.

• There is an increased amount of angiogenesis observed in lungs of COVID-19 patients and the endothelium is heavily affected.

• A patient with severe pneumonia-associated with COVID-19 has been successfully treated with thalidomide.

• This study presents insights into the possible mechanisms by which thalidomide and lenalidomide would suppress the cytokine storm and immune response.

• Thalidomide and its derivative lenalidomide modulate expression of several genes and key pathways aberrantly regulated in SARS-coV-2 infected tissues. attempted to explore the effects of thalidomide and its derivatives on endothelium. HUVEC were subjected to 20 μM thalidomide or 20 μM lenalidomide or 20 μM pomalidomide or vehicle control treatment for 8 hours. RNA was isolated using TRIzol method and whole transcriptome sequencing was performed using Illumina HiSeq 2500 platform. The data can be accessed at GEO with the Accession ID GSE118979. The sequence reads were aligned with reference genome of Homo sapiens using TopHat2 (v2.0.8) and then followed by transcript compilation and gene identification was done using Cufflinks (v2.2.0). 27 The DEGs were identified using Cuffdiff program (v2.2.0). 27 

The raw counts from the SARS-coV-2 transcriptomic profiles were subjected to differential expression analysis by DESEq2 v1.26.0. 28 Subsequently the genes were pre-ranked using the P-values from DESeq2 analysis and subjected to pre-ranked gene set enrichment analysis. 29 Gene sets with false discovery rate < 0.05 were considered to be statistically significant and were visualized using EnrichmentMap plugin 30 of Cytoscape. 31 For drug signatures from iLINCS, differentially expressed genes with P < 0.05 were considered to be statistically significant. Enrichment of kinase perturbation was carried out using Enrichr. 32 

We analysed our previously published KINOMEscan kinase screening dataset of thalidomide 33 in order to investigate how thalidomide affects the immune system. Kinases whose activities were reduced at least by 60% were considered for further enrichment analysis.

The DEGs (Q < 0.05 for transcriptome and P < 0.05 for drug signatures) from all the gene expression profiles were compared for overlapping genes and over-represented pathways using Toppcluster 34 and the networks were visualized using in Cytoscape. 31 

For identifying the protein targets of thalidomide, we utilized the PharmMapper server (http://www.lilab-ecust.cn/pharmmapper/). 35 The server identifies possible physiological protein targets of any drug molecule by using a pharmacophore-based mapping approach. The 3-dimensional structure of thalidomide was obtained from PubChem and processed on the PharmMapper server choosing only human protein target sets. The top 100 target proteins were selected based on the ranking associated with a fit score (pKd value) for further enrichment analysis using Enrichr. 32 to be affected by thalidomide ( Figure 3D ). We studied the effects of thalidomide and its derivatives on endothelium and identified the downregulation of several angiogenic genes ( Figure 4B and Table 1 ). (Table 2 ).

Various genes aberrantly expressed in SARS-coV-2 affected lungs are known targets of thalidomide (Table 1) The expression profile of SARS-coV-2 infected lungs, PBMC as well as A549 cells show resemblance with profiles of lymphoma, multiple myeloma and SLE ( Figure 2A) ; however, we have focused on SLE as there was a striking similarity of the SLE expression profile and enriched pathways with that of lungs affected by COVID-19

( Figure 2B ). Interestingly, our findings showing the similarity of COVID-19 infected lung with SLE strongly support a recent study that identified the resemblance between severe cases of COVID-19 and SLE. 72 *Overlap = the number of genes enriched in the category vs the total no of proteins contributing to the particular pathway/biological process. **Adjusted P-value denotes the P-value obtained after multiple testing. multiple myeloma and certain forms of lymphoma. 49 Notably, hydroxychloroquine, an Food and Drug Administration-approved SLE drug is currently being used in the management of critically ill SARS-coV-2 patients. 73 CC-220, another thalidomide analogue shows very promising results in phase I/II clinical trials against SLE. 74, 75 CC-220 through suppression of Ikaros and Aiolos expression, 76 transcription factors that are essential for differentiation of leucocyte and NK cells, thus modulates the innate immune system. As innate immune system pathways are deregulated in SARS-coV-2 infected lung and PBMC, further studies are warranted to investigate the efficacy and safety of CC-220 in treating COVID- 19. 62 Any treatment strategy with thalidomide and its analogues including repurposing thalidomide for COVID-19, should consider thalidomide-induced adverse effects including neuropathy and venous thromboembolism. 77 There have been many reports on COVID-19

patients develop blood clots, 62 a dangerous issue that might be aggravated with the use of thalidomide and lenalidomide. In addition, lenalidomide might cause cytokine release syndrome in chronic lymphocytic leukaemia patients. 78 Therefore, a very careful dosage regimen has to be followed with all these drugs as serious adverse effects have been observed during dose escalation.

Our study sheds light on the possible mechanisms through which tha- 

The authors have none to declare.

The data generated in this study have been deposited to Gene 

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Analysis of clinical features of 29 patients with 2019 novel coronavirus pneumonia

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

The trinity of COVID-19: immunity, inflammation and intervention

Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19

Mechanism of action in thalidomide teratogenesis

The rise, fall and subsequent triumph of thalidomide: lessons learned in drug development

Potential Treatments for COVID-19; a Narrative Literature Review

Thalidomide Combined with Low-Dose Glucocorticoid in the Treatment of COVID-19 Pneumonia. Preprints; 2020. www.preprints.org

Thalidomide-Revisited: Are COVID-19 Patients Going to Be the Latest Victims of Yet Another Theoretical Drug-Repurposing? Front Immunol

Recurrent ECSIT mutation encoding V140A triggers hyperinflammation and promotes hemophagocytic syndrome in extranodal NK/T cell lymphoma

The therapy of idiopathic pulmonary fibrosis: what is next?

Anti-Inflammatory Effect of Thalidomide on H1N1 Influenza Virus-Induced Pulmonary Injury in Mice

Protective effects of thalidomide on pulmonary injuries in a rat model of paraquat intoxication

Proposed use of thalidomide for the cytokine storm of COVID-19

Thalidomide against coronavirus disease 2019 (Covid-19): A medicine with a thousand faces

A Next Generation Connectivity Map: L1000 Platform and the First 1,000,000 Profiles

Imbalanced Host Response to SARS-CoV-2 Drives Development of COVID-19

Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients

A potential association between IL-3 and type I and III interferons in systemic lupus erythematosus

IMiDs mobilize acute myeloid leukemia blasts to peripheral blood through downregulation of CXCR4 but fail to potentiate AraC/Idarubicin activity in preclinical models of non del5q/5q-AML

Immunomodulatory drugs target IKZF1-IRF4-MYC axis in primary effusion lymphoma in a cereblon-dependent manner and display synergistic cytotoxicity with BRD4 inhibitors

Expression profile of immune response genes in patients with severe acute respiratory syndrome

CC-122, a pleiotropic pathway modifier, mimics an interferon response and has antitumor activity in DLBCL

Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray Experiments

Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks

Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles

Enrichment map: a network-based method for gene-set enrichment visualization and interpretation

Cytoscape: A software Environment for integrated models of biomolecular interaction networks

Enrichr: a comprehensive gene set enrichment analysis web server 2016 update

Thalidomide and Its Analogs Differentially Target Fibroblast Growth Factor Receptors: Thalidomide Suppresses FGFR Gene Expression while Pomalidomide Dampens FGFR2 Activity

ToppCluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems

PharmMapper server: a web server for potential drug target identification using pharmacophore mapping approach

Synergistic Inhibition of Thalidomide and Icotinib on Human Non-Small Cell Lung Carcinomas Through ERK and AKT Signaling

Thalidomide Induces Limb Anomalies by PTEN Stabilization, Akt Suppression, and Stimulation of Caspase-Dependent Cell Death

Inhibition of 13-cis retinoic acid-induced gene expression of reactiveresistance genes by thalidomide in glioblastoma tumours in vivo

Immunomodulatory drugs reorganize cytoskeleton by modulating Rho GTPases

Thalidomide Prevented and Ameliorated Pathogenesis of Crohn's Disease in Mice via Regulation of Inflammatory Response and Fibrosis

Thalidomide decreases gelatinase production by malignant B lymphoid cell lines through disruption of multiple integrin-mediated signaling pathways

Anticancer effects of novel thalidomide analogs in A549 cells through inhibition of vascular endothelial growth factor and matrix metalloproteinase-2

Response to thalidomide in multiple myeloma: Impact of angiogenic factors

Expression of PIGF and Its receptor Flt-1 in Patients with Multiple Myeloma and their Correlation with Chemotherapeutic Efficacy

Thalidomide inhibits fibronectin production in TGF-β1-treated normal and keloid fibroblasts via inhibition of the p38/Smad3 pathway

Thalidomide inhibits interferon-γ-mediated nitric oxide production in mouse vascular endothelial cells

Effect of lenalidomide on the human gastric cancer cell line SGC7901/vincristine Notch signaling

Immunomodulation by thalidomide and thalidomide analogues

Thalidomide and lenalidomide: Mechanism-based potential drug combinations

Thalidomide exerts its inhibitory action on tumor necrosis factor alpha by enhancing mRNA degradation

Thalidomide as an Anti-TNF-a Inhibitor: Implications for Clinical Use

Thalidomide upregulates macrophage inflammatory protein-1? in a herpes simplex virus-induced Behçet?s disease-like animal model

Thalidomide attenuates nitric oxide mediated angiogenesis by blocking migration of endothelial cells

Thalidomide attenuates nitric oxide-driven angiogenesis by interacting with soluble guanylyl cyclase

Thalidomide's ability to augment the synthesis of IL-2 in vitro in HIV-infected patients is associated with the percentage of CD4+ cells in their blood

Lenalidomide inhibits the malignant clone and up-regulates the SPARC gene mapping to the commonly deleted region in 5q-syndrome patients

Thalidomide induce response in patients with corticosteroid-resistant or relapsed ITP by upregulating Neuropilin-1 expression

Clinical Outcome of an Multicentre, Randomized, Phase II Clinical Trial for Patients with Extranodal NK/T Cell Lymphoma Treated By P-Gemox or Aspametdex

Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells

A Novel Anticancer Effect of Thalidomide: Inhibition of Intercellular Adhesion Molecule-1-Mediated Cell Invasion and Metastasis through Suppression of Nuclear Factor-B

Systematic Quality Control Analysis of LINCS Data

Atherosclerosis as Pathogenetic Substrate for Sars-Cov2 Cytokine Storm

Clinical characteristics of 2019 novel coronavirus infection in China

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the neuroendocrine stress axis

Effects of taurine on ACE, ACE2 and HSP70 expression of hypothalamic-pituitary-adrenal axis in stressinduced hypertensive rats

Post-SARS sickness syndrome manifestations and endocrinopathy: How, why, and so what?

Neuroinflammation as a Factor of Neurodegenerative Disease: Thalidomide Analogs as Treatments

COVID-19: Immunology and treatment options

Inhibition of Beta Interferon Induction by Severe Acute Respiratory Syndrome Coronavirus Suggests a Two-Step Model for Activation of Interferon Regulatory Factor 3

Rabex-5 is a lenalidomide target molecule that negatively regulates TLR-induced type 1 IFN production

Immunomodulatory drugs inhibit TLR4-induced type-1 interferon production independently of Cereblon via suppression of the TRIF/IRF3 pathway

Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19

Hydroxychloroquine in the management of critically ill patients with COVID-19: the need for an evidence base

Cereblon modulator iberdomide induces degradation of the transcription factors Ikaros and Aiolos: immunomodulation in healthy volunteers and relevance to SLE

First clinical (phase 1b/2a) study of iberdomide (CC-220), a CELMoD, in combination with dexamethasone (DEX) in patients (pts) with relapsed/refractory multiple myeloma (RRMM)

Suppression of Aiolos and Ikaros expression by lenalidomide reduces human ILC3−ILC1/NK cell transdifferentiation

Venous and arterial thrombotic risks with thalidomide: Evidence and practical guidance

Lenalidomide-induced upregulation of CD80 on tumor cells correlates with T-cell activation, the rapid onset of a cytokine release syndrome and leukemic cell clearance in chronic lymphocytic leukemia

Repurposing of thalidomide and its derivatives for the treatment of SARS-coV-2 infections: Hints on molecular action