Iterative machine learning-based chemical similarity search to identify novel chemical inhibitors.

Machine learning-based chemical screening has made substantial progress in recent years. However, these predictions often have low accuracy and high uncertainty when identifying new active chemical scaffolds. Hence, a high proportion of retrieved compounds are not structurally novel. In this study, we proposed a strategy to address this issue by iteratively optimizing an evolutionary chemical binding similarity (ECBS) model using experimental validation data. Various data update and model retraining schemes were tested to efficiently incorporate new experimental data into ECBS models, resulting in a fine-tuned ECBS model with improved accuracy and coverage. To demonstrate the effectiveness of our approach, we identified the novel hit molecules for the mitogen-activated protein kinase kinase 1 (MEK1). These molecules showed sub-micromolar affinity (Kd 0.1-5.3 µM) to MEKs and were distinct from previously-known MEK1 inhibitors. We also determined the binding specificity of different MEK isoforms and proposed potential docking models. Furthermore, using de novo drug design tools, we utilized one of the new MEK inhibitors to generate additional drug-like molecules with improved binding scores. This resulted in the identification of several potential MEK1 inhibitors with better binding affinity scores. Our results demonstrated the potential of this approach for identifying novel hit molecules and optimizing their binding affinities.

Domain-wise dissection of thermal stability enhancement in multidomain proteins.

Stability is critical for the proper functioning of all proteins. Optimization of protein thermostability is a key step in the development of industrial enzymes and biologics. Herein, we demonstrate that multidomain proteins can be stabilized significantly using domain-based engineering followed by the recombination of the optimized domains. Domain-level analysis of designed protein variants with similar structures but different thermal profiles showed that the independent enhancement of the thermostability of a constituent domain improves the overall stability of the whole multidomain protein. The crystal structure and AlphaFold-predicted model of the designed proteins via domain-recombination provided a molecular explanation for domain-based stepwise stabilization. Our study suggests that domain-based modular engineering can minimize the sequence space for calculations in computational design and experimental errors, thereby offering useful guidance for multidomain protein engineering.

Potent and Selective Inhibition of CYP1A2 Enzyme by Obtusifolin and Its Chemopreventive Effects.

Obtusifolin, a major anthraquinone component present in the seeds of Cassia tora, exhibits several biological activities, including the amelioration of memory impairment, prevention of breast cancer metastasis, and reduction of cartilage damage in osteoarthritis. We aimed to evaluate the inhibitory effects of obtusifolin and its analogs on CYP1A enzymes, which are responsible for activating procarcinogens, and investigate its inhibitory mechanism and chemopreventive effects. P450-selective substrates were incubated with human liver microsomes (HLMs) or recombinant CYP1A1 and CYP1A2 in the presence of obtusifolin and its four analogs. After incubation, the samples were analyzed using liquid chromatography-tandem mass spectrometry. Molecular docking simulations were performed using the crystal structure of CYP1A2 to identify the critical interactions between anthraquinones and human CYP1A2. Obtusifolin potently and selectively inhibited CYP1A2-mediated phenacetin O-deethylation (POD) with a Ki value of 0.031 µM in a competitive inhibitory manner in HLMs, whereas it exhibited negligible inhibitory effect against other P450s (IC50 > 28.6 µM). Obtusifolin also inhibited CYP1A1- and CYP1A2-mediated POD and ethoxyresorufin O-deethylation with IC50 values of <0.57 µM when using recombinant enzymes. Our molecular docking models suggested that the high CYP1A2 inhibitory activity of obtusifolin may be attributed to the combination of hydrophobic interactions and hydrogen bonding. This is the first report of selective and potent inhibitory effects of obtusifolin against CYP1A, indicating their potential chemopreventive effects.

New Monocyclic Terpenoid Lactones from a Brown Algae Sargassum macrocarpum as Monoamine Oxidase Inhibitors.

Algae are unique natural products that can produce various types of biologically active compounds. The 70% ethanol extract of brown algae Sargassum macrocarpum collected from the East Sea of Korea inhibited human monoamine oxidases A and B enzymes (hMAO-A and hMAO-B) at a 50 µg/mL concentration. The bioassay-guided isolation was performed through solid-phase extraction and the Sepbox system followed by serial high-performance liquid chromatography on the reverse phase condition, resulting in the identification of two new monocyclic terpenoid lactones, sargassumins A and B (1 and 2). The planar structures of the compounds were determined by a combination of spectroscopic data. The absolute configurations were determined by the interpretation of circular dichroism data. Compound 1 exhibited mild hMAO-A inhibition (42.18 ± 2.68% at 200 µM) and docked computationally into the active site of hMAO-A (-8.48 kcal/mol). Although compound 2 could not be tested due to insufficient quantity, it docked better into hMAO-A (-9.72 kcal/mol). Therefore, the above results suggest that this type of monocyclic terpenoid lactone could be one of the potential lead compounds for the treatment of psychiatric or neurological diseases.

Characterization of interaction between blood coagulation factor VIII and LRP1 suggests dynamic binding by alternating complex contacts.

BACKGROUND: Deficiency in blood coagulation factor VIII (FVIII) results in life-threating bleeding (hemophilia A) treated by infusions of FVIII concentrates. To improve disease treatment, FVIII has been modified to increase its plasma half-life, which requires understanding mechanisms of FVIII catabolism. An important catabolic actor is hepatic low density lipoprotein receptor-related protein 1 (LRP1), which also regulates many other clinically significant processes. Previous studies showed complexity of FVIII site for binding LRP1. OBJECTIVES: To characterize binding sites between FVIII and LRP1 and suggest a model of the interaction. METHODS: A series of recombinant ligand-binding complement-type repeat (CR) fragments of LRP1 including mutated variants was generated in a baculovirus system and tested for FVIII interaction using surface plasmon resonance, tissue culture model, hydrogen-deuterium exchange mass spectrometry, and in silico. RESULTS: Multiple CR doublets within LRP1 clusters II and IV were identified as alternative FVIII-binding sites. These interactions follow the canonical binding mode providing major binding energy, and additional weak interactions are contributed by adjacent CR domains. A representative CR doublet was shown to have multiple contact sites on FVIII. CONCLUSIONS: FVIII and LRP1 interact via formation of multiple complex contacts involving both canonical and non-canonical binding combinations. We propose that FVIII-LRP1 interaction occurs via switching such alternative binding combinations in a dynamic mode, and that this mechanism is relevant to other ligand interactions of the low-density lipoprotein receptor family members including LRP1.

A Novel Competitive Binding Screening Assay Reveals Sennoside B as a Potent Natural Product Inhibitor of TNF-α.

Natural products (NPs) have played a significant role in drug discovery for diverse diseases, and numerous attempts have been made to discover promising NP inhibitors of tumor necrosis factor α (TNF-α), a major therapeutic target in autoimmune diseases. However, NP inhibitors of TNF-α, which have the potential to be developed as new drugs, have not been reported for over a decade. To facilitate the search for new promising inhibitors of TNF-α, we developed an efficient competitive binding screening assay based on analytical size exclusion chromatography coupled with liquid chromatography-tandem mass spectrometry. Application of this screening method to the NP library led to the discovery of a potent inhibitor of TNF-α, sennoside B, with an IC50 value of 0.32 µM in TNF-α induced HeLa cell toxicity assays. Surprisingly, the potency of sennoside B was 5.7-fold higher than that of the synthetic TNF-α inhibitor SPD304. Molecular docking was performed to determine the binding mode of sennoside B to TNF-α. In conclusion, we successfully developed a novel competition binding screening method to discover small molecule TNF-α inhibitors and identified the natural compound sennoside B as having exceptional potency.

Janus Nanosheets with Face-Selective Molecular Recognition Properties from DNA-Peptide Conjugates.

Chemical and functional anisotropy in Janus materials offer intriguing possibilities for constructing complex nanostructures and regulating chemical and biological reactions. Here, the authors report the fabrication of Janus nanosheets from molecular building blocks composed of two information-carrying biopolymers, DNA and peptides. Experimental and structural modeling studies reveal that DNA-peptide diblock conjugates assemble into Janus nanosheets with distinct DNA and peptide faces. The surprising level of structural control is attributed to the exclusive parallel ß-sheet formation of phenylalanine-rich peptides. This approach is extended to triblock DNA1-peptide-DNA2 conjugates, which assemble into nanosheets presenting two different DNA on opposite faces. The Janus nanosheets with independently addressable faces are utilized to organize an enzyme pair for concerted enzymatic reactions, where enhanced catalytic activities are observed. These results demonstrate that the predictable and designable peptide interaction is a promising tool for creating Janus nanostructures with regio-selective and sequence-specific molecular recognition properties.

Identification of Tyrosinase Inhibitors and Their Structure-Activity Relationships via Evolutionary Chemical Binding Similarity and Structure-Based Methods.

Tyrosinase is an enzyme that plays a crucial role in the melanogenesis of humans and the browning of food products. Thus, tyrosinase inhibitors that are useful to the cosmetic and food industries are required. In this study, we have used evolutionary chemical binding similarity (ECBS) to screen a virtual chemical database for human tyrosinase, which resulted in seven potential tyrosinase inhibitors confirmed through the tyrosinase inhibition assay. The tyrosinase inhibition percentage for three of the new actives was over 90% compared to 61.9% of kojic acid. From the structural analysis through pharmacophore modeling and molecular docking with the human tyrosinase model, the pi-pi interaction of tyrosinase inhibitors with conserved His367 and the polar interactions with Asn364, Glu345, and Glu203 were found to be essential for tyrosinase-ligand interactions. The pharmacophore features and the docking models showed high consistency, revealing the possible essential binding interactions of inhibitors to human tyrosinase. We have also presented the activity cliff analysis that successfully revealed the chemical features related to substantial activity changes found in the new tyrosinase inhibitors. The newly identified inhibitors and their structure-activity relationships presented here will help to identify or design new human tyrosinase inhibitors.

Evolutionary chemical binding similarity approach integrated with 3D-QSAR method for effective virtual screening.

BACKGROUND: Despite continued efforts using chemical similarity methods in virtual screening, currently developed approaches suffer from time-consuming multistep procedures and low success rates. We recently developed a machine learning-based chemical binding similarity model considering common structural features from molecules binding to the same, or evolutionarily related targets. The chemical binding similarity measures the resemblance of chemical compounds in terms of binding site similarity to better describe functional similarities that arise from target binding. In this study, we have shown how the chemical binding similarity could be used in virtual screening together with the conventional structure-based methods. RESULTS: The chemical binding similarity, receptor-based pharmacophore, chemical structure similarity, and molecular docking methods were evaluated to identify an effective virtual screening procedure for desired target proteins. When we tested the chemical binding similarity method with test sets of 51 kinases, it outperformed the traditional structural similarity-based methods as well as structure-based methods, such as molecular docking and receptor-based pharmacophore modeling, in terms of finding active compounds. We further validated the results by performing virtual screening (using the chemical binding similarity and receptor-based pharmacophore methods) against a completely blind dataset for mitogen-activated protein kinase kinase 1 (MEK1), ephrin type-B receptor 4 (EPHB4) and wee1-like protein kinase (WEE1). The in vitro kinase binding assay confirmed that 6 out of 13 (46.2%) for MEK1 and 2 out of 12 (16.7%) for EPHB4 were newly identified only by the chemical binding similarity model. CONCLUSIONS: We report that the virtual screening results could further be improved by combining the chemical binding similarity model with 3D-QSAR pharmacophore and molecular docking models. Not only the new inhibitors are identified in this study, but also many of the identified molecules have low structural similarity scores against already reported inhibitors and that show the revelation of novel scaffolds.

Machine learning-based chemical binding similarity using evolutionary relationships of target genes.

Chemical similarity searching is a basic research tool that can be used to find small molecules which are similar in shape to known active molecules. Despite its popularity, the retrieval of local molecular features that are critical to functional activity related to target binding often fails. To overcome this limitation, we developed a novel machine learning-based chemical binding similarity score by using various evolutionary relationships of binding targets. The chemical similarity was defined by the probability of chemical compounds binding to identical targets. Comprehensive and heterogeneous multiple target-binding chemical data were integrated into a paired data format and processed using multiple classification similarity-learning models with various levels of target evolutionary information. Encoding evolutionary information to chemical compounds through their binding targets substantially expanded available chemical-target interaction data and significantly improved model performance. The output probability of our integrated model, referred to as ensemble evolutionary chemical binding similarity (ensECBS), was effective for finding hidden chemical relationships. The developed method can serve as a novel chemical similarity tool that uses evolutionarily conserved target binding information.

Phloretin as a Potent Natural TLR2/1 Inhibitor Suppresses TLR2-Induced Inflammation.

Toll-like receptor 2 (TLR2) responses are involved in various inflammatory immune disorders. Phloretin is a naturally occurring dietary flavonoid that is abundant in fruit. Here, we investigated whether the anti-inflammatory activity of phloretin is mediated through TLR2 pathways, and whether phloretin acts as an inhibitor of TLR2/1 heterodimerization using the TLR2/1 agonist Pam3CSK4. We tested the effects of phloretin on tumor necrosis factor (TNF)-α production induced by various TLRs using known TLR-specific agonists. Phloretin significantly inhibited Pam3CSK4-induced TRL2/1 signaling in Raw264.7 cells compared to TLR signaling induced by the other agonists tested. Therefore, we further tested the effects of phloretin in human embryonic kidney (HEK) 293-hTLR2 cells induced by Pam3CSK4, and confirmed that phloretin has comparable inhibition of TLR2/1 heterodimerization to that induced by the known TLR2 inhibitor CU-CPT22. Moreover, phloretin reduced the secretion of the inflammatory cytokines TNF-α and interleukin (IL)-8 in Pam3CSK4-induced HEK293-hTLR2 cells, whereas it did not significantly reduce these cytokines under Pam2CSK4-induced activation. Western blot results showed that phloretin significantly suppressed Pam3CSK4-induced TLR2 and NF-κB p65 expression. The molecular interactions between phloretin and TLR2 were investigated using bio-layer interferometry and in silico docking. Phloretin bound to TLR2 with micromolar binding affinity, and we proposed a binding model of phloretin at the TLR2⁻TLR1 interface. Overall, we confirmed that phloretin inhibits the heterodimerization of TLR2/1, highlighting TLR2 signaling as a therapeutic target for treating TLR2-mediated inflammatory immune diseases.

Tyr51: Key Determinant of the Low Thermostability of the Colwellia psychrerythraea Cold-Shock Protein.

Cold-shock proteins (Csps) are expressed at lower-than-optimum temperatures, and they function as RNA chaperones; however, no structural studies on psychrophilic Csps have been reported. Here, we aimed to investigate the structure and dynamics of the Csp of psychrophile Colwellia psychrerythraea 34H, ( Cp-Csp). Although Cp-Csp shares sequence homology, common folding patterns, and motifs, including a five ß-stranded barrel, with its thermophilic counterparts, its thermostability (37 °C) was markedly lower than those of other Csps. Cp-Csp binds heptathymidine with an affinity of 10-7 M, thereby increasing its thermostability to 50 °C. Nuclear magnetic resonance spectroscopic analysis of the Cp-Csp structure and backbone dynamics revealed a flexible structure with only one salt bridge and 10 residues in the hydrophobic cavity. Notably, Cp-Csp contains Tyr51 instead of the conserved Phe in the hydrophobic core, and its phenolic hydroxyl group projects toward the surface. The Y51F mutation increased the stability of hydrophobic packing and may have allowed for the formation of a K3-E21 salt bridge, thereby increasing its thermostability to 43 °C. Cp-Csp exhibited conformational exchanges in its ribonucleoprotein motifs 1 and 2 (754 and 642 s-1), and heptathymidine binding markedly decreased these motions. Cp-Csp lacks salt bridges and has longer flexible loops and a less compact hydrophobic cavity resulting from Tyr51 compared to mesophilic and thermophilic Csps. These might explain the low thermostability of Cp-Csp. The conformational flexibility of Cp-Csp facilitates its accommodation of nucleic acids at low temperatures in polar oceans and its function as an RNA chaperone for cold adaptation.

Biophysical Studies Reveal Key Interactions between Papiliocin-Derived PapN and Lipopolysaccharide in Gram-Negative Bacteria.

Papiliocin, isolated from the swallowtail butterfly (Papilio xuthus), is an antimicrobial peptide with high selectivity against gram-negative bacteria. We previously showed that the N-terminal helix of papiliocin (PapN) plays a key role in the antibacterial and anti-inflammatory activity of papiliocin. In this study, we measured the selectivity of PapN against multidrug-resistant gram-negative bacteria, as well as its anti-inflammatory activity. Interactions between Trp2 of PapN and lipopolysaccharide (LPS), which is a major component of the outer membrane of gram-negative bacteria, were studied using the Trp fluorescence blue shift and quenching in LPS micelles. Furthermore, using circular dichroism, we investigated the interactions between PapN and LPS, showing that LPS plays critical roles in peptide folding. Our results demonstrated that Trp2 in PapN was buried deep in the negatively charged LPS, and Trp2 induced the α-helical structure of PapN. Importantly, docking studies determined that predominant electrostatic interactions of positively charged arginine residues in PapN with phosphate head groups of LPS were key factors for binding. Similarly, hydrophobic interactions by aromatic residues of PapN with fatty acid chains in LPS were also significant for binding. These results may facilitate the development of peptide antibiotics with anti-inflammatory activity.

Toll-like receptor 2 antagonists identified through virtual screening and experimental validation.

Toll-like receptor 2 (TLR2) antagonists are key therapeutic targets because they inhibit several inflammatory diseases caused by surplus TLR2 activation. In this study, we identified two novel nonpeptide TLR2 antagonists, C11 and C13, through pharmacophore-based virtual screening. At 10 µm, the level of interleukin (IL)-8 inhibition by C13 and C11 in human embryonic kidney TLR2 overexpressing cells was comparable to the commercially available TLR2 inhibitor CU-CPT22. In addition, C11 and C13 acted in mouse macrophage-like RAW 264.7 cells as TLR2-specific inhibitors and did not suppress the tumor necrosis factor-α induction by TLR3 and TLR4 activators. Moreover, the two identified compounds bound directly to the human recombinant TLR2 ectodomain, during surface plasmon resonance analysis, and did not affect cell viability in a 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay. In total, two virtually screened molecules, C11 and C13, were experimentally proven to be effective as TLR2 antagonists, and thus will provide new insights into the structure of TLR2 antagonists, and pave the way for the development of TLR2-targeted drug molecules.

Structural Mechanism behind Distinct Efficiency of Oct4/Sox2 Proteins in Differentially Spaced DNA Complexes.

The octamer-binding transcription factor 4 (Oct4) and sex-determining region Y (SRY)-box 2 (Sox2) proteins induce various transcriptional regulators to maintain cellular pluripotency. Most Oct4/Sox2 complexes have either 0 base pairs (Oct4/Sox2(0bp)) or 3 base pairs (Oct4/Sox2(3bp)) separation between their DNA-binding sites. Results from previous biochemical studies have shown that the complexes separated by 0 base pairs are associated with a higher pluripotency rate than those separated by 3 base pairs. Here, we performed molecular dynamics (MD) simulations and calculations to determine the binding free energy and per-residue free energy for the Oct4/Sox2(0bp) and Oct4/Sox2(3bp) complexes to identify structural differences that contribute to differences in induction rate. Our MD simulation results showed substantial differences in Oct4/Sox2 domain movements, as well as secondary-structure changes in the Oct4 linker region, suggesting a potential reason underlying the distinct efficiencies of these complexes during reprogramming. Moreover, we identified key residues and hydrogen bonds that potentially facilitate protein-protein and protein-DNA interactions, in agreement with previous experimental findings. Consequently, our results confess that differential spacing of the Oct4/Sox2 DNA binding sites can determine the magnitude of transcription of the targeted genes during reprogramming.

Middle East respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes high fever, cough, acute respiratory tract infection and multiorgan dysfunction that may eventually lead to the death of the infected individuals. MERS-CoV is thought to be transmitted to humans through dromedary camels. The occurrence of the virus was first reported in the Middle East and it subsequently spread to several parts of the world. Since 2012, about 1368 infections, including ~487 deaths, have been reported worldwide. Notably, the recent human-to-human 'superspreading' of MERS-CoV in hospitals in South Korea has raised a major global health concern. The fatality rate in MERS-CoV infection is four times higher compared with that of the closely related severe acute respiratory syndrome coronavirus infection. Currently, no drug has been clinically approved to control MERS-CoV infection. In this study, we highlight the potential drug targets that can be used to develop anti-MERS-CoV therapeutics.

A Combined Molecular Docking/Dynamics Approach to Probe the Binding Mode of Cancer Drugs with Cytochrome P450 3A4.

Cytarabine, daunorubicin, doxorubicin and vincristine are clinically used for combinatorial therapies of cancers in different combinations. However, the knowledge about the interaction of these drugs with the metabolizing enzyme cytochrome P450 is limited. Therefore, we utilized computational methods to predict and assess the drug-binding modes. In this study, we performed docking, MD simulations and free energy landscape analysis to understand the drug-enzyme interactions, protein domain motions and the most populated free energy minimum conformations of the docked protein-drug complexes, respectively. The outcome of docking and MD simulations predicted the productive, as well as the non-productive binding modes of the selected drugs. Based on these interaction studies, we observed that S119, R212 and R372 are the major drug-binding residues in CYP3A4. The molecular mechanics Poisson-Boltzmann surface area analysis revealed the dominance of hydrophobic forces in the CYP3A4-drug association. Further analyses predicted the residues that may contain favorable drug-specific interactions. The probable binding modes of the cancer drugs from this study may extend the knowledge of the protein-drug interaction and pave the way to design analogs with reduced toxicity. In addition, they also provide valuable insights into the metabolism of the cancer drugs.

Structure and Effects of Cyanobacterial Lipopolysaccharides.

Lipopolysaccharide (LPS) is a component of the outer membrane of mainly Gram-negative bacteria and cyanobacteria. The LPS molecules from marine and terrestrial bacteria show structural variations, even among strains within the same species living in the same environment. Cyanobacterial LPS has a unique structure, since it lacks heptose and 3-deoxy-d-manno-octulosonic acid (also known as keto-deoxyoctulosonate (KDO)), which are present in the core region of common Gram-negative LPS. In addition, the cyanobacterial lipid A region lacks phosphates and contains odd-chain hydroxylated fatty acids. While the role of Gram-negative lipid A in the regulation of the innate immune response through Toll-like Receptor (TLR) 4 signaling is well characterized, the role of the structurally different cyanobacterial lipid A in TLR4 signaling is not well understood. The uncontrolled inflammatory response of TLR4 leads to autoimmune diseases such as sepsis, and thus the less virulent marine cyanobacterial LPS molecules can be effective to inhibit TLR4 signaling. This review highlights the structural comparison of LPS molecules from marine cyanobacteria and Gram-negative bacteria. We discuss the potential use of marine cyanobacterial LPS as a TLR4 antagonist, and the effects of cyanobacterial LPS on humans and marine organisms.

Structure and dynamic behavior of Toll-like receptor 2 subfamily triggered by malarial glycosylphosphatidylinositols of Plasmodium falciparum.

Proinflammatory responses by Toll-like receptors (TLRs) to malaria infection are considered to be a significant factor in suppressing pathogen growth and in disease control. The key protozoan parasite Plasmodium falciparum causes malaria through glycosylphosphatidylinositols (GPIs), which induce the host immune response mainly via TLR2 signalling. Experimental studies have suggested that malarial GPIs from P. falciparum are recognized by the TLR2 subfamily. However, the interaction site and their involvement in the activation mechanism are still unknown. A better understanding of the detailed structure of the TLR-GPI interaction is important for the design of more effective anti-malarial therapeutics. We used a molecular docking method to predict the binding regions of malarial GPIs with the TLR2 subfamily members. We also employed molecular dynamics simulations and principal component analysis to understand ligand-induced conformational changes of the TLR2 subfamily. We observed the expected structural changes upon ligand binding, and significant movements were found in loop regions located in the ligand-binding site of the TLR2 subfamily. We further propose that the binding modes of malarial GPIs are similar to lipopeptides, and that the lipid portions of the ligands could play an essential role in selective dimerization of the TLR2 subfamily.

Molecular evolution and structural features of IRAK family members.

The interleukin-1 receptor-associated kinase (IRAK) family comprises critical signaling mediators of the TLR/IL-1R signaling pathways. IRAKs are Ser/Thr kinases. There are 4 members in the vertebrate genome (IRAK1, IRAK2, IRAKM, and IRAK4) and an IRAK homolog, Pelle, in insects. IRAK family members are highly conserved in vertebrates, but the evolutionary relationship between IRAKs in vertebrates and insects is not clear. To investigate the evolutionary history and functional divergence of IRAK members, we performed extensive bioinformatics analysis. The phylogenetic relationship between IRAK sequences suggests that gene duplication events occurred in the evolutionary lineage, leading to early vertebrates. A comparative phylogenetic analysis with insect homologs of IRAKs suggests that the Tube protein is a homolog of IRAK4, unlike the anticipated protein, Pelle. Furthermore, the analysis supports that an IRAK4-like kinase is an ancestral protein in the metazoan lineage of the IRAK family. Through functional analysis, several potentially diverged sites were identified in the common death domain and kinase domain. These sites have been constrained during evolution by strong purifying selection, suggesting their functional importance within IRAKs. In summary, our study highlighted the molecular evolution of the IRAK family, predicted the amino acids that contributed to functional divergence, and identified structural variations among the IRAK paralogs that may provide a starting point for further experimental investigations.