Conformational Details Research Articles

Label-free detection and profiling of individual solution-phase molecules.

Most chemistry and biology occurs in solution, in which conformational dynamics and complexation underlie behaviour and function. Single-molecule techniques1 are uniquely suited to resolving molecular diversity and new label-free approaches are reshaping the power of single-molecule measurements. A label-free single-molecule method2-16 capable of revealing details of molecular conformation in solution17,18 would allow a new microscopic perspective of unprecedented detail. Here we use the enhanced light-molecule interactions in high-finesse fibre-based Fabry-Pérot microcavities19-21 to detect individual biomolecules as small as 1.2 kDa, a ten-amino-acid peptide, with signal-to-noise ratios (SNRs) >100, even as the molecules are unlabelled and freely diffusing in solution. Our method delivers 2D intensity and temporal profiles, enabling the distinction of subpopulations in mixed samples. Notably, we observe a linear relationship between passage time and molecular radius, unlocking the potential to gather crucial information about diffusion and solution-phase conformation. Furthermore, mixtures of biomolecule isomers of the same molecular weight and composition but different conformation can also be resolved. Detection is based on the creation of a new molecular velocity filter window and a dynamic thermal priming mechanism that make use of the interplay between optical and thermal dynamics22,23 and Pound-Drever-Hall (PDH) cavity locking24 to reveal molecular motion even while suppressing environmental noise. New in vitro ways of revealing molecular conformation, diversity and dynamics can find broad potential for applications in the life and chemical sciences.

SuperVidConform: Conformation detail-preserving network (CDPN) for video super-resolution

Video Super Resolution (VSR) applications extensively utilize deep learning-based methods. Several VSR methods primarily focus on improving the fine-patterns within reconstructed video frames. It frequently overlooks the crucial aspect of keeping conformation details, particularly sharpness. Therefore, reconstructed video frames often fail to meet expectations. In this paper, we propose a Conformation Detail-Preserving Network (CDPN) named as SuperVidConform. It focuses on restoring local region features and maintaining the sharper details of video frames. The primary focus of this work is to generate the high-resolution (HR) frame from its corresponding low-resolution (LR). It consists of two parts: (i) The proposed model decomposes confirmation details from the ground-truth HR frames to provide additional information for the super-resolution process, and (ii) These video frames pass to the temporal modelling SR network to learn local region features by residual learning that connects the network intra-frame redundancies within video sequences. The proposed approach is designed and validated using VID4, SPMC, and UDM10 datasets. The experimental results show the proposed model presents an improvement of 0.43 dB (VID4), 0.78 dB (SPMC), and 0.84 dB (UDM10) in terms of PSNR. Further, the CDPN model set new standards for the performance of self-generated surveillance datasets.

Polycatechols inhibit ferroptosis and modulate tau liquid-liquid phase separation to mitigate Alzheimer's disease.

Alzheimer's disease (AD) is a complex neurodegenerative disorder that affects learning, memory, and cognition. Current treatments targeting amyloid-β (Aβ) and tau have shown limited effectiveness, necessitating further research on the aggregation and toxicity mechanisms. One of these mechanisms involves the liquid-liquid phase separation (LLPS) of tau, contributing to the formation of pathogenic tau aggregates, although their conformational details remain elusive. Another mechanism is ferroptosis, a type of iron-dependent lipid peroxidation-mediated cell death, which has been implicated in AD. There is a lack of therapeutic strategies that simultaneously target amyloid toxicity and ferroptosis. This study aims to explore the potential of polycatechols, PDP and PLDP, consisting of dopamine and L-Dopa, respectively, as multifunctional agents to modulate the pathological nexus between ferroptosis and AD. Polycatechols were found to sequester the labile iron pool (LIP), inhibit Aβ and tau aggregation, scavenge free radicals, protect mitochondria, and prevent ferroptosis, thereby rescuing neuronal cell death. Interestingly, PLDP promotes tau LLPS, and modulates their intermolecular interactions to inhibit the formation of toxic tau aggregates, offering a conceptually innovative approach to tackle tauopathies. This is a first-of-its-kind polymer-based integrative approach that inhibits ferroptosis, counteracts amyloid toxicity, and modulates tau LLPS to mitigate the multifaceted toxicity of AD.

Solution Structure of Poly(UG) RNA

Poly(UG) or “pUG” RNAs are UG or GU dinucleotide repeat sequences which are highly abundant in eukaryotes. Post-transcriptional addition of pUGs to RNA 3′ ends marks mRNAs as vectors for gene silencing in C. elegans. We previously determined the crystal structure of pUG RNA bound to the ligand N-methyl mesoporphyrin IX (NMM), but the structure of free pUG RNA is unknown. Here we report the solution structure of the free pUG RNA (GU)12, as determined by nuclear magnetic resonance spectroscopy and small and wide-angle x-ray scattering (NMR-SAXS-WAXS). The low complexity sequence and 4-fold symmetry of the structure result in overlapped NMR signals that complicate chemical shift assignment. We therefore utilized single site-specific deoxyribose modifications which did not perturb the structure and introduced well-resolved methylene signals that are easily identified in NMR spectra. The solution structure ensemble has a root mean squared deviation (RMSD) of 0.62 Å and is a compact, left-handed quadruplex with a Z-form backbone, or “pUG fold.” Overall, the structure agrees with the crystal structure of (GU)12 bound to NMM, indicating the pUG fold is unaltered by docking of the NMM ligand. The solution structure reveals conformational details that could not be resolved by x-ray crystallography, which explain how the pUG fold can form within longer RNAs.

Coil-Helix Block Copolymers Can Exhibit Divergent Thermodynamics in the Disordered Phase.

Chiral building blocks have the ability to self-assemble and transfer chirality to larger hierarchical length scales, which can be leveraged for the development of novel nanomaterials. Chiral block copolymers, where one block is made completely chiral, are prime candidates for studying this phenomenon, but fundamental questions regarding the self-assembly are still unanswered. For one, experimental studies using different chemistries have shown unexplained diverging shifts in the order-disorder transition temperature. In this study, particle-based molecular simulations of chiral block copolymers in the disordered melt were performed to uncover the thermodynamic behavior of these systems. A wide range of helical models were selected, and several free energy calculations were performed. Specifically, we aimed to understand (1) the thermodynamic impact of changing the conformation of one block in chemically identical block copolymers and (2) the effect of the conformation on the Flory-Huggins interaction parameter, χ, when chemical disparity was introduced. We found that the effective block repulsion exhibits diverging behavior, depending on the specific conformational details of the helical block. Commonly used conformational metrics for flexible or stiff block copolymers do not capture the effective block repulsion because helical blocks are semiflexible and aspherical. Instead, pitch can quantitatively capture the effective block repulsion. Quite remarkably, the shift in χ for chemically dissimilar block copolymers can switch sign with small changes in the pitch of the helix.

Dissecting the Effect of Temperature on Hyperthermophilic Pf2001 Esterase Dimerization by Molecular Dynamics.

Pf2001 esterase (Pf2001) from Pyrococcus furiosus has hyperthermophilic properties and exerts a biocatalytic function in a dimeric state. Crystal structures revealed that the structural rearrangement of the cap domain is responsible for the Pf2001 dimer formation. However, the details of the cap domain remodeling and the effects of temperature on the dimerization process remain elusive at the molecular level, taking into account that experimental methods are difficult to capture the dynamic process of dimerization to some extent. Herein, four dimer models based on the monomeric crystal structure (PDB ID: 5G59) were constructed to investigate the conformational transition details and temperature effects in the dimerization by conventional molecular dynamics and accelerated molecular dynamics simulations. Our simulation results indicate that the monomer undergoes a conformational change into a "preparatory state" at high temperatures, which is more favorable for its transformation into a stable dimer. The subsequent free energy landscape analysis further identifies four intermediate states (from separated state to dimeric state) and discloses that a more accessible α-helix driven by stronger hydrophobic interactions induces a rearrangement of the cap domain, displaying a "tic-tac-toe" activation feature that is important for stabilizing the dimer interface and facilitating the formation of hydrophobic pockets. In addition, the electrostatic potential surface analysis illustrates that the weaker electrostatic repulsion (Lys and Arg) in the dimer interface at high temperatures is also a key factor for dimer stabilization. Altogether, our results can provide molecular-level insight into the dimer formation process of hyperthermophilic esterase and would be useful to understand the enzymatic specificity of α/β-hydrolase.

The Mechanism of Channel Opening of Anion Channelrhodopsin GtACR1: A Molecular Dynamics Simulation

Guillardia theta anion channelrhodopsin 1 (GtACR1) is a widely used inhibitor of optogenetics with unique conductance mechanisms and photochemistry. However, the molecular mechanism of light-gated anion conduction is poorly understood without a crystal structure for the intermediate state. In this study, we built the dark-state model based on the crystal structure of retinal and isomerized the model by twisting the C12-C13=C14-C15 dihedral step by step using molecular dynamics simulation. The conformational changes revealed the all-trans to 13-cis photoisomerization of the retinal chromophore cannot open the channel. There is no water influx, and a pre-opened K-like intermediate after photoisomerization of retinal is formed. During the opening of the ion channel, proton transfer occurs between E68 and D234. Steered molecular dynamics (SMD) and umbrella sampling indicated that the E68 and D234 were the key residues for chloride-ion conducting. We propose a revised channel opening pathway model of GtACR1 after analyzing (de)protonation of E68 and D234. Reprotonation of D234 will result in two different early L intermediates, named L1-like and L1‘-like, which correspond to the L1 and L1‘ intermediates reported in a recent study. Simulation results showed that L1-like may convert by parallel paths into L1‘-like and L2-like states. This model provides conformational details for the intermediate as well.

Albumin-binding properties of an aromatic N-acylhydrazone

In this work, an aromatic N-acylhydrazone compound, N'-(4-hydroxybenzylidene) −4-methylbenzohydrazide (HMB), was designed as a potential active molecule. Its interaction with bovine serum albumin (BSA) was studied using multiple-spectroscopy, electrochemistry and molecular docking techniques. The fluorescence quenching theory, Arrhenius equation, Langmuir equation, and van’t Hoff equation were employed in analyzing the kinetics and thermodynamics. The results proved that HMB had moderate affinity with serum albumin at Sudlow's site II in mimetic physiological condition, and could be effectively bound and delivered by albumins. The interaction was an entropy-driven spontaneous process, with an activationenergy of 30.69 kJ·mol−1. H-bonding and hydrophobic effect between HMB and protein posed measurable influence on the contents of α-helix and β-fold in BSA. Molecular modeling disclosed the conformation details of BSA-HMB complex, including the residues that participated in the interactions, their spatial orientation, the monitored binding modes and binding energy. The obtained physical and chemical information could be used to evaluate the possibility of designing targeted drugs based on HMB since albumins could be preferentially taken in by tumor and inflammatory tissues.

Polymorphism and Perfection in Crystallization of Hard Sphere Polymers

We present results on polymorphism and perfection, as observed in the spontaneous crystallization of freely jointed polymers of hard spheres, obtained in an unprecedentedly long Monte Carlo (MC) simulation on a system of 54 chains of 1000 monomers. Starting from a purely amorphous configuration, after an initial dominance of the hexagonal closed packed (HCP) polymorph and a transitory random hexagonal close packed (rHCP) morphology, the system crystallizes in a final, stable, face centered cubic (FCC) crystal of very high perfection. An analysis of chain conformational characteristics, of the spatial distribution of monomers and of the volume accessible to them shows that the phase transition is caused by an increase in translational entropy that is larger than the loss of conformational entropy of the chains in the crystal, compared to the amorphous state. In spite of the significant local re-arrangements, as reflected in the bending and torsion angle distributions, the average chain size remains unaltered during crystallization. Polymers in the crystal adopt ideal random walk statistics as their great length renders local conformational details, imposed by the geometry of the FCC crystal, irrelevant.

Molecular interaction mechanism and structure–activity relationships of protein–polyphenol complexes revealed by side-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy

Deciphering interactions between bioactive protein and polyphenols are critical for designing and controlling functional protein–polyphenol complexes. Herein, using the site-directed spin labeled T4 lysozyme (T4L) and rosmarinic acid (RA) as a model system, we combined electron paramagnetic resonance spectra to investigate molecular interaction mechanism of the protein–polyphenol complexes in structural or conformational details. Experimental results show that molecular interactions between T4L and RA are a process from order to disorder. TEM images display that the complexes finally assemble into quasi-spherical colloidal particles. When T4L/RA ratio is 1:1, the complexes exhibit the optimized enzymatic and antioxidant dual-functionalities due to the synergetic effect and protection mechanism. However, with excess addition of RA, the enzymatic and antioxidant activities of the complexes started to attenuate because the catalytic active site and bioactive hydroxyl groups were buried. The revealed high-resolution interaction process could help better understand the corresponding alterations between structure and functionalities.

Abstract 148: New conformational details help in clarifying Raf activation by dimerization

Abstract Raf is a family of serine/threonine kinases in the mitogen-activated protein kinase (MAPK) pathway for cell proliferation. The full activation of Raf requires the dimerization of the kinase domain. The dimer interface of Raf has been regarded as an important therapeutic target against cancer. The next generation paradox breakers target the Raf dimerization. Despite the clinical importance, it remains unclear that how the transverse side-to-side dimerization promotes the OFF-to-ON transition of Raf’s kinase domain, and how the activated ON-state kinase domain can be stabilized in the dimer for Raf signaling. In this work, we uncover an atomic-level mechanism of Raf activation through dimerization, addressing these questions. The mechanism shows that the replacement of the intramolecular π-π stacking by the intermolecular π-π stacking at the dimer interface promotes the Raf activation. It releases the structural constraint of the αC-helix, inducing a series of conformational changes for the OFF-to-ON transition, such as the disruption of the inhibitory hydrophobic interactions and the approach of phosphorylation sites in A-loop to HRD motif for cis-autophosphorylation. Once activated, the ON-state kinase domain can be stabilized by the N-terminal basic (NtB) motif in the dimer for Raf signaling. This work provides the high-resolution structural insights into the Raf activation, suggesting a new structural strategy for drug discovery against Raf dimerization. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261201500003I. Citation Format: Mingzhen Zhang, Ryan Maloney, Hyunbum Jang, Ruth Nussinov. New conformational details help in clarifying Raf activation by dimerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 148.

MD Simulations Revealing Special Activation Mechanism of Cannabinoid Receptor 1.

Cannabinoid receptor 1 (CB1) is a G protein-coupled receptor (GPCR) that is gaining much interest for its regulating role in the central nervous system and its value as a drug target. Structures of CB1 in inactive and active states have revealed conformational change details that are not common in other GPCRs. Here, we performed molecular dynamics simulations of CB1 in different ligand binding states and with mutations to reveal its activation mechanism. The conformational change of the “twin toggle switch” residues F2003.36 and W3566.48 that correlates with ligand efficacy is identified as a key barrier step in CB1 activation. Similar conformational change of residues 3.36/6.48 is also observed in melanocortin receptor 4, showing this “twin toggle switch” residue pair is crucial for the activation of multiple GPCR members.

Photoinduced reaction mechanisms in prototypical and bathy phytochromes.

Phytochromes, found in plants, fungi, and bacteria, exploit light as a source of information to control physiological processes via photoswitching between two states of different physiological activity, i.e. a red-absorbing Pr and a far-red-absorbing Pfr state. Depending on the relative stability in the dark, bacterial phytochromes are divided into prototypical and bathy phytochromes, where the stable state is Pr and Pfr, respectively. In this work we studied representatives of these groups (prototypical Agp1 and bathy Agp2 from Agrobacterium fabrum) together with the bathy-like phytochrome XccBphP from Xanthomonas campestris by resonance Raman and IR difference spectroscopy. In all three phytochromes, the photoinduced conversions display the same mechanistic pattern as reflected by the chromophore structures in the various intermediate states. We also observed in each case the secondary structure transition of the tongue, which is presumably crucial for the function of phytochrome. The three phytochromes differ in details of the chromophore conformation in the various intermediates and the energetic barrier of their respective decay reactions. The specific protein environment in the chromophore pocket, which is most likely the origin for these small differences, also controls the proton transfer processes concomitant to the photoconversions. These proton translocations, which are tightly coupled to the structural transition of the tongue, presumably proceed via the same mechanism along the Pr → Pfr conversion whereas the reverse Pfr → Pr photoconversion includes different proton transfer pathways. Finally, classification of phytochromes in prototypical and bathy (or bathy-like) phytochromes is discussed in terms of molecular structure and mechanistic properties.

Probing the adsorption behavior and free energy landscape of single-stranded DNA oligonucleotides on single-layer MoS2 with molecular dynamics

Interfacing single-stranded DNA (ssDNA) with 2D transition metal dichalcogenides are important for numerous technological advancements. However, the molecular mechanism of this process, including the nature of intermolecular association and conformational details of the self-assembled hybrids is still not well understood. Here, atomistic molecular dynamics simulation is employed to study the distinct adsorption behavior of ssDNA on a single-layer MoS2 in aqueous environment. The ssDNA sequences [T10, G10, A10, C10, U10, (GT)5, and (AC)5] are chosen on the basis that short ssDNA segments can undergo a spontaneous conformational change upon adsorption and allow efficient sampling of the conformational landscape. Differences in hybridization is attributed to the inherent molecular recognition ability of the bases. While the binding appears to be primarily driven by energetically favorable van der Waals π-stacking interactions, equilibrium structures are modulated by the ssDNA conformational changes. The poly-purines demonstrate two concurrently competing π-stacking interactions: nucleobase–nucleobase (intramolecular) and nucleobase–MoS2 (intermolecular). The poly-pyrimidines, on the other hand, reveal enhanced π-stacking interactions, thereby maximizing the number of contacts. The results provide new molecular-level understanding of ssDNA adsorption on the MoS2 surface and facilitate future studies in design of functional DNA/MoS2 structure-based platforms for DNA sequencing, biosensing (optical, electrochemical, and electronic), and drug delivery.

Cyclosporin A: Conformational Complexity and Chameleonicity.

The chameleonic behavior of cyclosporin A (CsA) was investigated through conformational ensembles employing multicanonical molecular dynamics simulations that could sample the cis and trans isomers of N-methylated amino acids; these assessments were conducted in explicit water, dimethyl sulfoxide, acetonitrile, methanol, chloroform, cyclohexane (CHX), and n-hexane (HEX) using AMBER ff03, AMBER10:EHT, AMBER12:EHT, and AMBER14:EHT force fields. The conformational details were discussed employing the free-energy landscapes (FELs) at T = 300 K; it was observed that the experimentally determined structures of CsA were only a part of the conformational space. Comparing the ROESY measurements in CHX-d12 and HEX-d14, the major conformations in those apolar solvents were essentially the same as that in CDCl3 except for the observation of some sidechain rotamers. The effects of the metal ions on the conformations, including the cis/trans isomerization, were also investigated. Based on the analysis of FELs, it was concluded that the AMBER ff03 force field best described the experimentally derived conformations, indicating that CsA intrinsically formed membrane-permeable conformations and that the metal ions might be the key to the cis/trans isomerization of N-methylated amino acids before binding a partner protein.

Mechanistic physicochemical insights into glycation and drug binding by serum albumin: Implications in diabetic conditions

The drug binding ability of serum albumin might get affected as a result of its glycation under diabetic conditions. It requires not only an understanding of the effect of glycation of the protein upon association with the drug, but also calls for an assessment of structure-property-energetics relationships. A combination of ultrasensitive calorimetric, spectroscopic and chromatographic approach has been employed to correlate thermodynamic signatures with recognition, conformation and mechanistic details of the processes involved. An important observation from this work is that 3-(dansylamino) phenyl boronic acid (DnsPBA) assay cannot always determine the extent of glycation as evidenced by MALDI-TOF mass spectra of glycated HSA due to its selectivity for 1,2 or 1,3 cis-diol structures which may be absent in certain AGEs. Protein gets modified post glycation with the formation of advanced glycation end products (AGEs), which are monitored to be targeted by the guanidine group present in anti-diabetic drugs. AGEs formed in the third and fourth week of glycation are significant in the recognition of anti-diabetic drugs. The results with metformin and aminoguanidine suggest that the extent of binding depends upon the number of guanidine group(s) in the drug molecule. Open chain molecules having guanidine group(s) exhibit stronger affinity towards glycated HSA than closed ring entities like naphthalene or pyridine moiety. The observation that the drug binding ability of HSA is not adversely affected, rather strengthened upon glycation, has implications in diabetic conditions. A rigorous structure-property-energetics correlation based on thermodynamic signatures and identification of functional groups on drugs for recognition by HSA are essential in deriving guidelines for rational drug design addressing diabetes.

Regulation and drug modulation of a voltage-gated sodium channel: Pivotal role of the S4–S5 linker in activation and slow inactivation

Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4-S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4-S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4-S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed "door-wedge" action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.

Composition-dependent energetic contribution of complex salt bridges to collagen stability

Complex salt bridges, on which three or more charged residues interplay simultaneously, cannot be considered as addition of individual salt bridges. This is still an intriguing problem in protein folding and stability. Here, we used an obligated ABC-type collagen heterotrimer as a platform to study the relationship between energetic contributions and conformational details of three-body complex salt bridges anchored by positively charged residues, K and R. Eight complex salt bridges were constructed by engineering point mutations in the heterotrimer. The circular dichroism measurements showed that the K-anchored complex salt bridges were stronger than the R-anchored ones. The molecular dynamics simulation revealed that both types of salt bridges had distinct dynamic features. The energetic contribution of K-anchored salt bridges was mainly determined by strong single bridges. In the R-anchored complex salt bridges, both side-chain electrostatic interactions and side-chain-backbone hydrogen bonding were involved. An empirical equation was proposed to predict the energetic contributions with high accuracy (R2 = 0.93). This work could help us take insights into the mechanisms of composition-dependent behaviors of the complex salt bridges on protein surface.

Localization atomic force microscopy.

Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM)1 has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules2. Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms3 to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset.

Virtual screening by targeting proteolytic sites of furin and TMPRSS2 to propose potential compounds obstructing the entry of SARS-CoV-2 virus into human host cells

Background and aimThe year 2020 begins with the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that cause the disease COVID-19, and continue till today. As of March 23, 2021, the outbreak has infected 124,313,054 worldwide with a total death of 2,735,707. The use of traditional medicines as an adjuvant therapy with western drugs can lower the fatality rate due to the COVID-19. Therefore, in silico molecular docking study was performed to search potential phytochemicals and drugs that can block the entry of SARS-CoV-2 into host cells by inhibiting the proteolytic cleavage activity of furin and TMPRSS2. Experimental procedureThe protein-protein docking of the host proteases furin and TMPRSS2 was carried out with the virus spike (S) protein to examine the conformational details and residues involved in the complex formation. Subsequently, a library of 163 ligands containing phytochemicals and drugs was virtually screened to propose potential hits that can inhibit the proteolytic cleavage activity of furin and TMPRSS2. Results and conclusionThe phytochemicals like limonin, gedunin, eribulin, pedunculagin, limonin glycoside and betunilic acid bind at the active site of both furin and TMPRSS2. Limonin and gedunin found mainly in the citrus fruits and neem showed the highest binding energy at the active site of furin and TMPRSS2, respectively. The polyphenols found in green tea can also be useful in suppressing the furin activity. Among the drugs, the drug nafamostat may be more beneficial than the camostat in suppressing the activity of TMPRSS2.