The selectivity in selective autophagy pathways is achieved via the selective autophagy receptors (SARs) – proteins that bind a ligand on the substrate to be degraded and the Atg8-family protein on the growing autophagic membrane, phagophore, effectively bridging them. In mammals, the most common ligand of SARs is ubiquitin, a small protein modifier that tags substrates for their preferential degradation by autophagy. Consequently, the most common SARs are the ubiquitin-binding SARs, such as SQSTM1/p62 (sequestosome 1). Surprisingly, there is only one SAR of this type in yeast – Cue5, which acts as a receptor for aggrephagy and proteaphagy – pathways that remove the ubiquitinated protein aggregates and proteasomes, respectively. However, recent studies described the ubiquitin-dependent autophagic pathways that do not require Cue5, e.g. stationary phase lipophagy for intracellular lipid droplets and nitrogen starvation-induced mitophagy for mitochondria. What is the role of ubiquitin in these pathways? Here, we propose that the ubiquitinated lipid droplets and mitochondria are recognized by the alternative ubiquitin-binding SARs. Our analysis identifies the proteins that could potentially fulfill this role in yeast. We think that matching of the ubiquitin-dependent (but Cue5-independent) autophagic pathways with the ubiquitin-and-Atg8-binding proteins enlisted here might uncover the novel ubiquitin-binding SARs in yeast.

Monomeric Ubiquitin (Ub) is a 76-amino acid highly conserved protein found in eukaryotes. The first described biological activity of Ub in the 1970s was extracellular, but it quickly gained relevance due to its intracellular role, i.e., post-translational modification of intracellular proteins (ubiquitination) that regulate numerous eukaryotic cellular processes. In the following years, the extracellular role of Ub was relegated to the background, until a correlation between higher survival rate and increased serum Ub concentrations in patients with sepsis and burns was observed. Although the mechanism of action (MoA) of extracellular ubiquitin (eUb) is not yet well understood, further studies have shown that it may ameliorate the inflammatory response in tissue injury and multiple sclerosis diseases. These observations compounded with the high stability and low immunogenicity of eUb due to its high conservation in eukaryotes have made this small protein a relevant candidate for biotherapeutic development. Here, we review the in vitro and in vivo effects of eUb on immunologic, cardiovascular, and nervous systems, and discuss the potential MoAs of eUb as an anti-inflammatory, antimicrobial, cardio- and brain-protective agent.

SMAD ubiquitination regulatory factor 1 (Smurf1) is a Nedd4 family E3 ubiquitin ligase that regulates cell motility, polarity and TGFβ signaling. Smurf1 contains an N-terminal protein kinase C conserved 2 (C2) domain that targets cell membranes and is required for interactions with membrane-localized substrates such as RhoA. Here we investigated the lipid-binding mechanism of Smurf1 C2, revealing a general affinity for anionic membranes in addition to a selective affinity for phosphoinositides (PIPs). We found that Smurf1 C2 localizes not only to the plasma membrane but also to negatively charged intracellular sites, acting as an anionic charge sensor and selective PIP-binding domain. Site-directed mutagenesis combined with docking/molecular dynamics simulations revealed that the Smurf1 C2 domain loop region primarily interacts with PIPs and cell membranes, as opposed to the β-surface cationic patch employed by other C2 domains. By depleting PIPs from the inner leaflet of the plasma membrane, we found that PIP binding is necessary for plasma membrane localization. Finally, we used a Smurf1 cellular ubiquitination assay to show that the amount of ubiquitin at the plasma membrane interface depends on the lipid-binding properties of Smurf1. This study shows the mechanism by which Smurf1 C2 targets membrane-based substrates and reveals a novel interaction based on PI(4,5)P2 and PIP3 selectivity.

The mitochondrial network is a dynamic organization within eukaryotic cells that participates in a variety of essential cellular processes, such as ATP synthesis, central metabolism, apoptosis and inflammation. The mitochondrial network is balanced between rates of fusion and fission that respond to pathophysiologic signals to coordinate appropriate mitochondrial processes. Mitochondrial fusion and fission are regulated by proteins that either reside or translocate to the inner or outer mitochondrial membranes or are soluble in the inter-membrane space. Mitochondrial fission and fusion are performed by GTPases on the outer and inner mitochondrial membranes with the assistance of other mitochondrial proteins. Due to the essential nature of mitochondrial function for cellular homeostasis regulation of mitochondrial dynamics is under strict control. Some of the mechanisms used to regulate the function of these proteins are post-translational proteolysis and/or turnover and this review will discuss these mechanisms required for correct mitochondrial network organization.

Lysosomal degradation of tyrosinase, a pivotal enzyme in melanin synthesis, negatively impacts melanogenesis in melanocytes. Nevertheless, the precise molecular mechanisms by which lysosomes target tyrosinase have remained elusive. Here, we identify RING finger protein 152 (RNF152) as a membrane-associated ubiquitin ligase specifically targeting tyrosinase for the first time, utilizing AlphaScreen technology. We observed that modulating RNF152 levels in B16 cells, either via overexpression or siRNA knockdown, resulted in decreased or increased both tyrosinase and melanin levels, respectively. Notably, RNF152 and tyrosinase colocalized at the trans-Golgi network (TGN). However, upon treatment with lysosomal inhibitors, both proteins appeared in the lysosomes, indicating that tyrosinase undergoes RNF152-mediated lysosomal degradation. Through ubiquitination assays, we found the indispensable roles of both the RING and transmembrane (TM) domains of RNF152 in facilitating tyrosinase ubiquitination. In summary, our findings underscore RNF152 as a tyrosinase-specific ubiquitin ligase essential for regulating melanogenesis in melanocytes.

Chloroplasts are ancient organelles responsible for photosynthesis and various biosynthetic functions essential to most life on Earth. Many of these functions require tightly controlled regulatory processes to maintain homeostasis at the protein level. One such regulatory mechanism is the ubiquitin-proteasome system whose fundamental role is increasingly emerging in chloroplasts. In particular, the role of E3 ubiquitin ligases as determinants in the ubiquitination and degradation of specific intra-chloroplast proteins. Here, we highlight recent advances in understanding the roles of plant E3 ubiquitin ligases in chloroplast function.

A rapid and appropriate genetic and metabolic acclimation, which is crucial for plants’ survival in a changing environment, is maintained due to the coordinated action of plant hormones and cellular degradation mechanisms influencing proteostasis. The plant hormone abscisic acid (ABA) rapidly accumulates in plants in response to environmental stress and plays a pivotal role in the reaction to various stimuli. Increasing evidence demonstrates a significant role of autophagy in controlling ABA signaling. This field has been extensively investigated and new discoveries are constantly being provided. We present updated information on the components of the ABA signaling pathway, particularly on transcription factors modified by different E3 ligases. Then, we focus on the role of selective autophagy in ABA pathway control and review novel evidence on the involvement of autophagy in different parts of the ABA signaling pathway that are important for crosstalk with other hormones, particularly cytokinins and brassinosteroids.

In most Eukaryotes, ubiquitin either exists as free monoubiquitin or as a molecule that is covalently linked to other proteins. These two forms cycle between each other and due to the concerted antagonistic activity of ubiquitylating and deubiquitylating enzymes, an intracellular ubiquitin equilibrium is maintained that is essential for normal biological function. However, measuring the level and ratio of these forms of ubiquitin has been difficult and time consuming. In this paper, we have adapted a simple immunoblotting technique to monitor ubiquitin content and equilibrium dynamics in different developmental stages and tissues of Drosophila. Our data show that the level of total ubiquitin is distinct in different developmental stages, lowest at the larval-pupal transition and in three days old adult males, and highest in first instar larvae. Interestingly, the ratio of free mono-ubiquitin remains within 30-50% range of the total throughout larval development, but peaks to 70-80% at the larval-pupal and the pupal-adult transitions. It stays within the 70-80% range in adults. In developmentally and physiologically active tissues, the ratio of free ubiquitin is similarly high, most likely reflecting a high demand for ubiquitin availability. We also used this method to demonstrate the disruption of the finely tuned ubiquitin equilibrium by the abolition of proteasome function or the housekeeping deubiquitylase, Usp5. Our data support the notion that the ubiquitin equilibrium is regulated by tissue- and developmental stage-specific mechanisms.

<I>Saccharomyces cerevisiae</I> proliferates by budding, which includes the formation of a cytoplasmic protrusion called the ‘bud’, into which DNA, RNA, proteins, organelles, and other materials are transported. The transport of organelles into the growing bud must be strictly regulated for proper inheritance of organelles by daughter cells. In yeast, E3 ubiquitin ligases are involved in inheritance of mitochondria, vacuoles, and peroxisomes. These organelles are transported toward the tip of the growing bud by the myosin motor protein Myo2 along actin filaments. During organelle transport, adaptor proteins bridge the organelles and myosin. After reaching the bud, the adaptor proteins are ubiquitinated by E3 ubiquitin ligases and then degraded by the proteasome. Targeted degradation of the adaptor proteins is necessary to unload to unload the organelles from the actin–myosin machinery. Impairment of the ubiquitination of adaptor proteins results in a failure of organelle release from myosin, which in turn leads to abnormal dynamics, morphology, and function of the inherited organelles. Here, we summarize the role and regulation of E3 ubiquitin ligases during organelle inheritance in yeast.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that instigates several signaling cascades, including the NF-kB signaling pathway, to induce cell differentiation and proliferation. Overexpression and mutations of EGFR are found in up to 30% of solid tumors and correlate with a poor prognosis. Although it is known that EGFR-mediated NF-kB activation is involved in tumor development, the signaling axis is not well elucidated. Here, we found that PKP2 and the LUBAC complex were required for EGFR-mediated NF-kB activation. Upon EGF stimulation, EGFR recruited PKP2 to the plasma membrane, and PKP2 bridged HOIP, the catalytic E3 ubiquitin ligase in the LUBAC, to the EGFR complex. The recruitment activated the LUBAC complex and the linear ubiquitination of NEMO, leading to IkB phosphorylation and subsequent NF-kB activation. Furthermore, EGF-induced linear ubiquitination was critical for tumor cell proliferation and tumor development. Knockout of HOIP impaired EGF-induced NF-kB activity and reduced cell proliferation. HOIP knockout also abrogated the growth of A431 epidermal xenograft tumors in nude mice by more than 70%. More importantly, the HOIP inhibitor, HOIPIN-8, inhibited EGFR-mediated NF-kB activation and cell proliferation of A431, MCF-7, and MDA-MB-231 cancer cells. Overall, our study reveals a novel linear ubiquitination signaling axis of EGFR, and perturbation of HOIP E3 ubiquitin ligase activity is potential targeted cancer therapy.

Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44 (Leu-8, Ile-44, Val-70). Nevertheless, a variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD recognizes ubiquitin. Here, we describe the structure of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis and NMR spectroscopy of the ¹⁵N isotopically labelled protein, we demonstrate that a Phe-Pro motif in N4BP1 recognizes the canonical hydrophobic patch of ubiquitin. This recognition mode resembles the molecular mechanism evolved in the CUE (Coupling of ubiquitin conjugation to ER degradation) domain family, where an invariant proline, usually following a phenylalanine, is required for binding to ubiquitin. Interestingly, the UBD of N4BP1 is evolutionary related to CUBAN (Cullin binding domain associating with NEDD8) (40% identity and 47% similarity), a protein module that also recognizes the ubiquitin-like NEDD8, which is the closest relative of ubiquitin (58% identity and 80% similarity). By performing circular dichroism and ¹⁵N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the UBD of N4BP1 lacks the NEDD8 binding properties observed in CUBAN and it recognizes the Ile44-centered patch of ubiquitin through a dedicated binding site, which share some of the features observed in the CUE domain family. Moreover, we show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both the CUBAN and the UBD of N4BP1 are poly-ubiquitinated in cells. This modification is dependent on the presence of a functional domain. We believe that the structural and functional characterization of this novel UBD will allow a deeper understanding of the molecular mechanisms governing N4BP1 function, while at the same time providing a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.

G protein-coupled receptors (GPCRs) comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and β-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

Ubiquitin-specific protease 14 (USP14) is a member of the Deubiquitinating enzymes (DUBs) involved in disrupting the regulation of the ubiquitin-proteasome system, responsible for the degradation of impaired and misfolded proteins which is an essential mechanism in eukaryotic cells. The involvement of USP14 in cancer progression and neurodegenerative disorders has been reported. Thereof USP14 is a prime therapeutic target; hence, designing efficacious inhibitors of USP14 is central in curbing these conditions. Herein, we relied on structural bioinformatics methods incorporating molecular docking, Molecular Mechanics Generalized Born Surface Area (MM-GBSA), Molecular dynamics simulation (MD simulation) and ADME to identify potential allosteric USP14 inhibitors. A library of over 733 compounds from the PubChem repository with >90% match to the IU1 chemical structure was screened in a multi-step framework to attain prospective drug-like inhibitors. Two potential lead compounds (CID 43013232 and CID 112370349) were shown to record better binding affinity compared to IU1, but with subtle difference to IU1-47, a 10-fold potent compound when compared to IU1. The stability of the lead molecules complexed with USP14 was studied via MD simulations. The molecules were found to be stable within the binding site throughout the 50ns simulation time. Moreover, the protein-ligand interactions across the simulation run time suggest Phe331, Tyr476, and Gln197 as crucial residues for USP14 inhibition. Furthermore, in silico pharmacological evaluation revealed the lead compounds as pharmacological sound molecules. Overall, the methods deployed in this study revealed two novel candidates that may show selective inhibitory activity against USP14, which could be exploited to produce potent and harmless USP14 inhibitors.

The size of seeds is particularly important for agricultural development, as it is a key trait that determines yield. It is controlled by the coordinated development of the integument, endosperm, and embryo. Large seeds are one of the important ways to improve the ultimate “sink strength” of crops, providing more nutrients for early plant growth and showing certain tolerance to abiotic stresses. There are several pathways for regulating plant seed size, including the HAIKU (IKU) pathway, ubiquitin-proteasome pathway, G (Guanosine triphosphate) protein regulatory pathway, mitogen-activated protein kinase (MAPK) pathway, transcriptional regulators pathway, phytohormone regulatory pathways including auxin, brassinosteroid (BR), gibberellin (GA), jasmonic acid (JA), cytokinin (CK), Abscisic acid (ABA), and MicroRNAs (miRNAs) regulatory pathway. This article summarized the seed size regulatory network and prospected ways to improve yield. We expect to provide valuable reference to researcher in the related field.

Injury to the central nervous system (CNS), in most vertebrate animals, results in permanent damage and lack of function, due to their limited regenerative capacities. In contrast, echinoderms can fully regenerate their radial nerve cord (RNC) following transection, with little or no scarring. Investigators have associated the regenerative capacity of some organisms with the stress response and inflammation produced by the injury. Here we explore the gene activation profile of the stressed holothurian CNS. To do this, we performed RNA sequencing on isolated RNC explants submitted to the stress of transection and enzyme dissection and compared them to explants kept in culture for 3 days following dissection. We describe stress-associated genes, including members of heat-shock families, ubiquitin-related pathways, transposons, and apoptosis that were differentially expressed. Surprisingly, the stress response does not induce apoptosis in this system. Other genes associated with stress in other animal models, such as hero proteins and those associated with the integrated stress response, were not found to be differentially expressed either. Our results provide a new viewpoint on the stress response in the nervous system of an organism with an amazing regenerative capacity. This is the first step to deciphering the molecular processes that allow echinoderms to undergo fully functional CNS regeneration while also providing a comparative view for students of the stress response in other organisms.

Proteolysis Targeting Chimeras (PROTACs) describe compounds that bind to and induce degradation of a target by simultaneously binding to a ubiquitin ligase. More generally referred to as bifunctional degraders, PROTACs have led the way in the field of targeted protein degradation (TPD) with several compounds currently undergoing clinical testing. Alongside bifunctional degraders, single-moiety compounds, or molecular glue degraders (MGDs), are increasingly being considered as a viable approach for development of therapeutics, driven by advances in rational discovery approaches. This review focusses on drug discovery with respect to bifunctional and molecular glue degraders within the ubiquitin proteasome system, including analysis of mechanistic concepts and discovery approaches, with an overview of current clinical and pre-clinical degrader status in oncology, neurodegenerative and inflammatory disease.

The devastating impact of the ongoing coronavirus disease 2019 (COVID-19) on public health, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has made fighting of the COVID-19 pandemic is a top priority in medical research and pharmaceutical development. Surveillance of SARS-CoV-2 mutations is essential for the comprehension of SARS-CoV-2 variant diversity and their impact on virulence and pathogenicity. The SARS-CoV-2 open reading frame 10 (ORF10) protein interacts with multiple human proteins CUL2, ELOB, ELOC, MAP7D1, PPT1, RBX1, THTPA, TIMM8B, and ZYG11B expressed in the lung tissues. Mutations and co-mutations in the emerging SARS-CoV-2 ORF10 variants are expected to impact the severity of the virus and its associated consequences. In this article, We highlight 128 single mutations and 35 co-mutations in the unique SARS-CoV-2 ORF10 variants in this article. The possible predicted effects of these mutations and co-mutations on the secondary structure of ORF10 variants and host protein interactomes are presented. The findings highlight the possible effects of mutations and co-mutations on the emerging 140 ORF10 unique variants from secondary structure and intrinsic protein disorder perspectives.

Mitochondria are constantly subjected to stressful conditions due to their unique physiology and organization. The resulting damage leads to mitochondrial dysfunction, which underlies many pathophysiological conditions. Hence, constant surveillance is required to closely monitor mitochondrial health for sound maintenance of cellular metabolism and thus, for viability. In addition to internal mitochondrial chaperones and proteases, mitochondrial health is also governed by host cell protein quality control systems. The Ubiquitin-Proteasome System (UPS) and autophagy constitute the main pathways for removal of damaged or superfluous proteins in the cytosol, nucleus, and from certain organelles such as the ER and mitochondria. Although stress-induced ubiquitin-dependent degradation of mitochondrial outer membrane proteins has been widely studied, mechanisms of intramitochondrial protein ubiquitination have remained largely elusive due to the predominantly cytosolic nature of UPS components, separated from internal mitochondrial proteins by a double membrane. However, recent research has illuminated examples of intramitochondrial protein ubiquitination pathways and highlighted their importance under basal and stressful conditions. Owing to the dependence of mitochondria on the error-prone process of protein import from the cytosol, it is imperative that the cell eliminate any accumulated proteins in the event of mitochondrial import deficiency. Apparently, a significant portion of this activity involves ubiquitination in one way or another. In the present review article, following a brief introduction to mitochondrial protein quality control mechanisms, we discuss our recent understanding of intramitochondrial protein ubiquitination, its importance for the basal function of mitochondria, metabolic implications, and possible therapeutic applications.

Plants are immobile, and, to overcome harsh environmental conditions, such as drought, salt, and cold, they have evolved complex signaling pathways. Abscisic acid (ABA), an isoprenoid phytohormone, is a critical signaling mediator that regulates diverse biological processes in various organisms. Significant progress has been made in the determination and characterization of key ABA-mediated molecular factors involved in different stress responses, including stomatal closure and developmental processes, such as seed germination and bud dormancy. Since ABA-signaling is a complex signaling network that integrates with other signaling pathways, the dissection of its intricate regulatory network is necessary to understand the function of essential regulatory genes involved in ABA signaling. In the present review, we focus on two aspects of ABA signaling. First, the perception of the stress signal (abiotic and biotic) and the response network of ABA-signaling components that transduce the signal to the downstream pathway to respond to stress tolerance, regulation of stomata, and ABA signaling component ubiquitination. Second, ABA-signaling in plant development processes, such as lateral root growth regulation, seed germination, and flowering time regulation. Examining such diverse signal integration dynamics could enhance our understanding of the underlying genetic, biochemical, and molecular mechanisms of ABA signaling networks in plants.

The retinoblastoma (RB) transcriptional corepressor 1 (RB1) is a critical tumor suppressor gene, governing diverse cellular processes implicated in cancer biology. Dysregulation or deletion in RB1 contributes to the development and progression of various cancers, making it a prime target for therapeutic intervention. RB1's canonical function in cell cycle control and DNA repair mechanisms underscores its significance in restraining aberrant cell growth and maintaining genomic stability. Understanding the complex interplay between RB1 and cellular pathways is beneficial to fully elucidate its tumor-suppressive role across different cancer types and for therapeutic development. As a result, investigating vulnerabilities arising from RB1 deletion-associated mechanisms offers promising avenues for targeted therapy. Recently, several findings highlighted multiple methods as a promising strategy for combating tumor growth driven by RB1 loss, offering potential clinical benefits in various cancer types. This review summarizes the multifaceted role of RB1 in cancer biology and its implications for targeted therapy.

Epidemiological evidence points to an inverse association between Parkinson's disease (PD) and almost all cancers except melanoma, for which this association is positive. The results of many studies have found that patients with PD are at reduced risk of the majority of neoplasm occurrence and death. Several potential biological explanations exist for the inverse relationship between cancer and PD. Recent results identified several PD-associated proteins and factors mediating cancer development and cancer-associated factors affecting PD. Accumulating data points to the role of genetic traits, members of the synuclein family, neurotrophic factors, ubiquitin-proteasome system, circulating melatonin, and transcription factors as such mediators. We present recent data about shared pathogenetic factors and mediators that might be involved in the association between these two diseases. We also discuss how these factors, individually or in combination, may be involved in pathology, serve as links between PD and cancer, and affect the prevalence of these disorders. Identification of these factors and investigations of their mechanisms will lead to the discovery of new targets for the treatment of both diseases

Ubiquitin-Specific-peptidase 22 (Usp22) cleaves ubiquitin moieties from numerous proteins, in particular transcription factors. Recently, it was reported that Usp22 acts as negative regulator of interferon-dependent responses. In the current study, we investigated the role of Usp22 deficiency upon acute viral infection with lymphocytic choriomeningitis (LCMV) virus. We found that lack of Usp22 on bone marrow derived cells (Usp22 fl/fl Vav1-Cre mice) reduced induction of type I and II interferons. Limited type I interferon response did not influence virus replication. However, restricted expression of PD-L1 led to increased frequencies of functional virus-specific CD8+ T cells and rapid death of Usp22 deficient mice. CD8+ T cell depletion experiments revealed that accelerated CD8+ T cells were responsible for enhanced lethality in Usp22 deficient mice. In conclusion, we found that the lack of Usp22 generated a pathological CD8+ T cell response, which gave rise to severe disease in mice.

The ubiquitin-specific protease 2 (USP) belongs to the family of deubiquitinases and plays a critical role in tumors cells’ survival and therefore signifies an important therapeutic target. Previous studies have indicated promising efficacies of potent human USP2 inhibitors including, thiopurine analogues against SARS-CoV papain-like proteases (PLpro). The PLpro have significant functional implications in the innate immune response during SARS-CoV-2 infection and considered an important antiviral target. Both proteases share strikingly similar USP fold with right-handed thumb–palm–fingers structural scaffold and conserved catalytic triad Cys-His-Asp/Asn. In this urgency situation of COVID-19 outbreak, there is a lack of in-vitro facilities readily available to test SARS-CoV-2 inhibitors in whole-cell assays. Therefore, we adopted an alternate route to identify potential USP2 inhibitor through integrated structure-based virtual screening efforts. After a subsequent virtual screening protocol, the best compounds were selected and tested. The compound Z93 showed significant IC₅₀ value against Jurkat (9.67 µM) and MOTL-4 cells (11.8 µM). The binding mode of Z93 was extensively analyzed through molecular docking, followed by MD simulations, and molecular interactions were compared with SARS-CoV-2. The relative binding poses of Z93 fitted well in the binding site of both proteases and showed consensus π-π stacking and H-bond interactions with histidine and aspartate/asparagine residues of the catalytic triad. These results led us to speculate that compound Z93 might be the first potential chemical lead against SARS-CoV-2 PLpro, which warrants in-vitro evaluations.