Alternative Binding Modes Research Articles

Induced fit, ensemble binding space docking and Monte Carlo simulations of MDMA ‘ecstasy’ and 3D pharmacophore design of MDMA derivatives on the human serotonin transporter (hSERT)

The popular recreational drug MDMA (3,4-methylenedioxy-methamphetamine) has a documented potential as a psychopharmacological clinical and research tool. This is due to its unique ability to promote reprocessing of traumatic memories, empathetic and pro-social states. Although it is established that MDMA exerts its behavioural effects via the serotonin transporter (SERT), the ligand-protein molecular interplay remains elusive. In order to shed light on the binding of MDMA and its primary congeneric entactogens (MDA, MBDB and MDAI), we first combined induced fit with Monte Carlo simulations. The computed interaction energies of the models correlated well with experimental activities (adjR2 = 0.78). Then we carried out ‘ensemble binding space docking’ on trajectories generated by interpolation of experimentally derived structures of the hSERT from the outward-open, and the occluded, to the inward-open states. This approach revealed low-energy alternative binding modes, suggesting high occupancy of the central site, yet considerable MDMA mobility within it, favouring the paroxetine-like orientation. Finally, we designed a pharmacophore that may be used to recognise hSERT-mediated serotonin releasers and uptake inhibitors of diverse chemical structure, identifying their active conformations and interacting residues. We conclude that the conserved amine-Asp98 ionic and edge-to-face π-π interactions are crucial to the mode of action of MDMA on the hSERT, underscoring the contributions of Tyr95 and gating residues Phe341, Tyr176 and Phe335. Amenable to experimental testing, our modelling may aid the rational design of novel entactogenic compounds and contribute to the understanding of an action mechanism, common and typical of psychotropic agents.

Harzianic Acid from Trichoderma afroharzianum Is a Natural Product Inhibitor of Acetohydroxyacid Synthase.

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthetic pathway and is a validated target for herbicide and fungicide development. Here we report harzianic acid (HA, 1) produced by the biocontrol fungus Trichoderma afroharzianum t-22 (Tht22) as a natural product inhibitor of AHAS. The biosynthetic pathway of HA was elucidated with heterologous reconstitution. Guided by a putative self-resistance enzyme in the genome, HA was biochemically demonstrated to be a selective inhibitor of fungal AHAS, including those from phytopathogenic fungi. In addition, HA can inhibit a common resistant variant of AHAS in which the active site proline is mutated. Structural analysis of AHAS complexed with HA revealed the molecular basis of competitive inhibition, which differs from all known commercial AHAS inhibitors. The alternative binding mode also rationalizes the selectivity of HA, as well as effectiveness toward resistant mutants. A proposed role of HA biosynthesis by Tht22 in the rhizosphere is discussed based on the data.

Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein.

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

The C‐terminus of VRK1 Governs Interactions with Nucleosomes to Influence Histone H3 Threonine 3 (H3T3) Phosphorylation

Vaccinia-related kinase 1 (VRK1) is a highly abundant nuclear kinase that has numerous functions during mitosis, including the phosphorylation of the histone H3 tail at threonine 3 (H3T3ph). This post-translational modification, attributed to both VRK1 and the haspin kinase, is crucial for ensuring appropriate chromosome segregation. Despite the importance of VRK1 to cell cycle regulation, little is known about the biochemical basis of its activity on nucleosomes. Because placement of histone phosphorylation during mitosis is tightly spatiotemporally regulated, we sought to determine the chromatin template itself serves as a regulatory mechanism for VRK1 activity. In this work, we characterize the interaction between VRK1 and nucleosomes and determine how nucleosome binding affects nucleosome-directed kinase activity at H3T3. We find that VRK1 directly and stably interacts with nucleosomes with an apparent Kd of ~200 nM. VRK1 binds nucleosomes via a C-terminal alternating arginine motif, mutations or truncations of which are found in rare cases of genetic spinal muscular atrophy. Mutation or truncation of this motif leads to a 4-50 fold reduction in nucleosome affinity that is correlated with mutation position and severity. C-terminal arginine mutants of VRK1 show reduced capacity for histone H3T3 phosphorylation, suggesting that stable interaction with nucleosomes is required for nucleosome-directed kinase activity at H3T3. The nucleosome acidic patch, a negatively charged region at the dimer interface of H2A/H2B, is a hot spot for arginine-mediated nucleosome interactions. Neutralization of the acidic patch impairs VRK1 binding but surprisingly increases the activity of VRK1 at H3T3. In contrast, the presence of extended lengths of linker DNA promotes both nucleosome binding and phosphorylation of H3T3. Cryo-EM single particle analysis of VRK1-bound nucleosome particles demonstrates both acidic patch binding and heterogenous DNA binding that concentrates around the nucleosome linker DNA. This evidence suggests that VRK1 adopts at least two alternative binding modes on the nucleosome (acidic patch-governed vs. linker DNA-governed) that either promote or inhibit nucleosome-directed kinase activity based on the position and distance of VRK1 from the H3 tail. We suggest a model wherein dynamic access to the nucleosome acidic patch regulates the activity of VRK1 towards H3T3 during mitosis.

Estimating the Roles of Protonation and Electronic Polarization in Absolute Binding Affinity Simulations

Accurate prediction of binding free energies is critical to streamlining the drug development and protein design process. With the advent of GPU acceleration, absolute alchemical methods, which simulate the removal of ligand electrostatics and van der Waals interactions with the protein, have become routinely accessible and provide a physically rigorous approach that enables full consideration of flexibility and solvent interaction. However, standard explicit solvent simulations are unable to model protonation or electronic polarization changes upon ligand transfer from water to the protein interior, leading to inaccurate prediction of binding affinities for charged molecules. Here, we perform extensive simulation totaling ∼540 μs to benchmark the impact of modeling conditions on predictive accuracy for absolute alchemical simulations. Binding to urokinase plasminogen activator (UPA), a protein frequently overexpressed in metastatic tumors, is evaluated for a set of 10 inhibitors with extended flexibility, highly charged character, and titratable properties. We demonstrate that the alchemical simulations can be adapted to utilize the MBAR/PBSA method to improve the accuracy upon incorporating electronic polarization, highlighting the importance of polarization in alchemical simulations of binding affinities. Comparison of binding energy prediction at various protonation states indicates that proper electrostatic setup is also crucial in binding affinity prediction of charged systems, prompting us to propose an alternative binding mode with protonated ligand phenol and Hid-46 at the binding site, a testable hypothesis for future experimental validation.

Computationally designed peptide macrocycle inhibitors of New Delhi metallo-β-lactamase 1

The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-β-lactamase 1 (NDM-1), a bacterial enzyme that degrades β-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of l- and d-amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.

Alternative binding modes in abortive NADH-alcohol complexes of horse liver alcohol dehydrogenase

Enzymes typically have high specificity for their substrates, but the structures of substrates and products differ, and multiple modes of binding are observed. In this study, high resolution X-ray crystallography of complexes with NADH and alcohols show alternative modes of binding in the active site. Enzyme crystallized with the good substrates NAD+ and 4-methylbenzyl alcohol was found to be an abortive complex of NADH with 4-methylbenzyl alcohol rotated to a “non-productive” mode as compared to the structures that resemble reactive Michaelis complexes with NAD+ and 2,2,2-trifluoroethanol or 2,3,4,5,6-pentafluorobenzyl alcohol. The NADH is formed by reduction of the NAD+ with the alcohol during the crystallization. The same structure was also formed by directly crystallizing the enzyme with NADH and 4-methylbenzyl alcohol. Crystals prepared with NAD+ and 4-bromobenzyl alcohol also form the abortive complex with NADH. Surprisingly, crystals prepared with NAD+ and the strong inhibitor 1H,1H-heptafluorobutanol also had NADH, and the alcohol was bound in two different conformations that illustrate binding flexibility. Oxidation of 2-methyl-2,4-pentanediol during the crystallization apparently led to reduction of the NAD+. Kinetic studies show that high concentrations of alcohols can bind to the enzyme-NADH complex and activate or inhibit the enzyme. Together with previous studies on complexes with NADH and formamide analogues of the carbonyl substrates, models for the Michaelis complexes with NAD+-alcohol and NADH-aldehyde are proposed.

How \u03bc-opioid receptor recognizes fentanyl

Roughly half of the drug overdose-related deaths in the United States are related to synthetic opioids represented by fentanyl which is a potent agonist of mu-opioid receptor (mOR). In recent years, X-ray crystal structures of mOR in complex with morphine derivatives have been determined; however, structural basis of mOR activation by fentanyl-like opioids remains lacking. Exploiting the X-ray structure of BU72-bound mOR and several molecular simulation techniques, we elucidated the detailed binding mechanism of fentanyl. Surprisingly, in addition to the salt-bridge binding mode common to morphinan opiates, fentanyl can move deeper and form a stable hydrogen bond with the conserved His2976.52, which has been suggested to modulate mOR’s ligand affinity and pH dependence by previous mutagenesis experiments. Intriguingly, this secondary binding mode is only accessible when His2976.52 adopts a neutral HID tautomer. Alternative binding modes may represent a general mechanism in G protein-coupled receptor-ligand recognition.

Cohesin-dockerin code in cellulosomal dual binding modes and its allosteric regulation by proline isomerization

Cellulose is the most abundant organic molecule on Earth and represents a renewable and practically everlasting feedstock for the production of biofuels and chemicals. Self-assembled owing to the high-affinity cohesin-dockerin interaction, cellulosomes are huge multi-enzyme complexes with unmatched efficiency in the degradation of recalcitrant lignocellulosic substrates. The recruitment of diverse dockerin-borne enzymes into a multicohesin protein scaffold dictates the three-dimensional layout of the complex, and interestingly two alternative binding modes have been proposed. Using single-molecule fluorescence resonance energy transfer and molecular simulations on a range of cohesin-dockerin pairs, we directly detect varying distributions between these binding modes that follow a built-in cohesin-dockerin code. Surprisingly, we uncover a prolyl isomerase-modulated allosteric control mechanism, mediated by the isomerization state of a single proline residue, which regulates the distribution and kinetics of binding modes. Overall, our data provide a novel mechanistic understanding of the structural plasticity and dynamics of cellulosomes.

Characterization of Beta-2-Glycoprotein I Domain V interaction with Plasma Membrane

Beta-2-Glycoprotein I (β2GPI), also known as apolipoprotein H, is a 48kDa protein which circulates in blood at a high concentration [0.2 mg/ml]. β2GPI is the main target of autoantibodies in antiphospholipid syndrome (APS) which results in thrombosis and/or recurrent miscarriage. Therefore, investigating membrane binding mechanism of β2GPI is important to elucidate how this protein become immunogenic upon binding to plasma membrane and to help target specific drug development for APS. It is known that β2GPI binds to anionic phospholipids in the plasma membrane via its positively charged domain V (DV) but the mechanism of binding is still elusive. in this study, we performed multiple 500-ns molecular dynamics simulations of β2GPI-DV in the presence of membranes containing PC and PS in varying compositions employing the HMMM (highly mobile membrane mimetic) model. We observed stable membrane binding in eight out of ten replicas. Our results show that in seven out of eight replicas, β2GPI-DV interacts with the membrane via its flexible loop (311-317) which insert into the membrane below the PO4 level. interestingly, in one replica we observe insertion of the loop carrying three lysine residues, a mode which closely corresponds to an alternative binding mode reported previously. The orientation of membrane-bound states was characterized by measuring the tilt angle between the protein and the membrane. After insertion of the flexible loop, β2GPI-DV held an average tilt angle of 92 with respect to the membrane normal. This study establishes the significance of the flexible loop for membrane binding of β2GPI which could lead to a more rational approach in drug design for treatment of APS.

Potent and selective A3 adenosine receptor antagonists bearing aminoesters as heterobifunctional moieties.

A3 adenosine receptors were found to have a role in different pathological states, such as glaucoma, renal fibrosis, neuropathic pain and cancer. Consequently, it is important to utilize any molecular tool which could help to study these conditions. In the present study we continue our search for potent A3 adenosine receptor ligands which could be successively conjugated to other molecules with the aim of obtaining more potent (e.g. allosteric ligand conjugation) or detectable ligands (e.g. fluorescent molecule or biotin conjugation). Specifically, different aminoester moieties were introduced at the 5 position of the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine core. The ester functionalization represents the candidate for the subsequent conjugation. All the reported compounds are potent hA3 adenosine receptor antagonists and some of them exhibited high selectivity against the other adenosine receptors. The main structural terms of ligand recognition and selectivity were disclosed by molecular modelling studies. Molecular docking results led to the characterization of an alternative binding mode for antagonists at the orthosteric binding site of the hA3 adenosine receptor, evaluated and assessed by classical molecular dynamics simulations.

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*.

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio‐ and stereoselectivity at the 16α‐position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21‐hydroxylated product 11‐deoxycorticosterone in addition to 16α‐hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α‐hydroxylation was observed for wild‐type CYP154C5 in the conversion of 5α‐androstan‐3‐one, lacking an oxy‐functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.

Structural Modeling and Molecular Dynamics of the Immune Checkpoint Molecule HLA-G.

HLA-G is considered to be an immune checkpoint molecule, a function that is closely linked to the structure and dynamics of the different HLA-G isoforms. Unfortunately, little is known about the structure and dynamics of these isoforms. For instance, there are only seven crystal structures of HLA-G molecules, being all related to a single isoform, and in some cases lacking important residues associated to the interaction with leukocyte receptors. In addition, they lack information on the dynamics of both membrane-bound HLA-G forms, and soluble forms. We took advantage of in silico strategies to disclose the dynamic behavior of selected HLA-G forms, including the membrane-bound HLA-G1 molecule, soluble HLA-G1 dimer, and HLA-G5 isoform. Both the membrane-bound HLA-G1 molecule and the soluble HLA-G1 dimer were quite stable. Residues involved in the interaction with ILT2 and ILT4 receptors (α3 domain) were very close to the lipid bilayer in the complete HLA-G1 molecule, which might limit accessibility. On the other hand, these residues can be completely exposed in the soluble HLA-G1 dimer, due to the free rotation of the disulfide bridge (Cys42/Cys42). In fact, we speculate that this free rotation of each protomer (i.e., the chains composing the dimer) could enable alternative binding modes for ILT2/ILT4 receptors, which in turn could be associated with greater affinity of the soluble HLA-G1 dimer. Structural analysis of the HLA-G5 isoform demonstrated higher stability for the complex containing the peptide and coupled β2-microglobulin, while structures lacking such domains were significantly unstable. This study reports for the first time structural conformations for the HLA-G5 isoform and the dynamic behavior of HLA-G1 molecules under simulated biological conditions. All modeled structures were made available through GitHub (https://github.com/KavrakiLab/), enabling their use as templates for modeling other alleles and isoforms, as well as for other computational analyses to investigate key molecular interactions.

Development of autotaxin inhibitors: A series of tetrazole cinnamides

A series of inhibitors of Autotaxin (ATX) have been developed from a high throughput screening hit, 1a, which shows an alternative binding mode to known catalytic site inhibitors. Selectivity over the hERG channel and microsomal clearance were dependent on the lipophilicity of the compounds, and this was optimised by reduction of clogD whilst maintaining high affinity ATX inhibition. Compound 15a shows good oral exposure, and concentration dependent inhibition of formation of LPA in vivo, as shown in pharmacokinetic-pharmacodynamic (PK/PD) experiments.

Structural Analysis of Different LINC Complexes Reveals Distinct Binding Modes

Linker of nucleoskeleton and cytoskeleton (LINC) complexes are molecular tethers that span the nuclear envelope (NE) and physically connect the nucleus to the cytoskeleton. They transmit mechanical force across the NE in processes such as nuclear anchorage, nuclear migration, and homologous chromosome pairing during meiosis. LINC complexes are composed of KASH proteins traversing the outer nuclear membrane, and SUN proteins crossing the inner nuclear membrane. Humans have several SUN- and KASH-containing proteins, yet what governs their proper engagement is poorly understood. To investigate this question, we solved high resolution crystal structures of human SUN2 in complex with the KASH-peptides of Nesprin3, Nesprin4, and KASH5. In comparison to the published structures of SUN2-KASH1/2 we observe alternative binding modes for these KASH peptides. While the core interactions between SUN and the C-terminal residues of the KASH peptide are similar in all five complexes, the extended KASH-peptide adopts at least two different conformations. The much-improved resolution allows for a more detailed analysis of other elements critical for KASH interaction, including the KASH-lid and the cation loop, and a possible self-locked state for unbound SUN. In summary, we observe distinct differences between the examined SUN-KASH complexes. These differences may have an important role in regulating the SUN-KASH network.

Ligand binding free-energy calculations with funnel metadynamics.

The accurate resolution of the binding mechanism of a ligand to its molecular target is fundamental to develop a successful drug design campaign. Free-energy calculations, which provide the energy value of the ligand-protein binding complex, are essential for resolving the binding mode of the ligand. The accuracy of free-energy calculation methods is counteracted by their poor user-friendliness, which hampers their broad application. Here we present the Funnel-Metadynamics Advanced Protocol (FMAP), which is a flexible and user-friendly graphical user interface (GUI)-based protocol to perform funnel metadynamics, a binding free-energy method that employs a funnel-shape restraint potential to reveal the ligand binding mode and accurately calculate the absolute ligand-protein binding free energy. FMAP guides the user through all phases of the free-energy calculation process, from preparation of the input files, to production simulation, to analysis of the results. FMAP delivers the ligand binding mode and the absolute protein-ligand binding free energy as outputs. Alternative binding modes and the role of waters are also elucidated, providing a detailed description of the ligand binding mechanism. The entire protocol on the paradigmatic system benzamidine-trypsin, composed of ~105 k atoms, took ~2.8 d using the Cray XC50 piz Daint cluster at the Swiss National Supercomputing Centre.

X‐Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A2A Adenosine Receptor Antagonists

AbstractWe present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X‐ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2AAR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2AAR were experimentally determined and investigated through a cycle of ligand‐FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X‐ray crystallography of the A2AAR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2AAR, an emerging target in immuno‐oncology.

X-Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A2A Adenosine Receptor Antagonists.

We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X‐ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2AAR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2AAR were experimentally determined and investigated through a cycle of ligand‐FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X‐ray crystallography of the A2AAR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2AAR, an emerging target in immuno‐oncology.

Conformational selection in the flaviviral NS2B-NS3 protease

The first x-ray structures of flaviviral proteases defined two conformational states, open and closed, depending on the relative position of NS2B with respect to NS3, a feature that affects the shape of the binding site. The degree of flexibility in the active site was limited to changes in the fold of NS2B rather than NS3 and an induced-fit mechanism was regarded as the main factor for ligand binding. A minor degree of conformational plasticity in NS3 is observed in the two protein chains in the asymmetric unit for the structure of Zika protease with a dipeptide boronate, synthesized in our group. We hypothesize that the NS3 fold has a crucial influence on the shape of the binding site and that a reevaluation of the induced-fit interpretation is warranted. A comparison of flaviviral protease structures identifies conformational dynamics of NS3 and their unexpected role in controlling the depth of the, otherwise shallow, active site. The structural changes of NS3 are mediated by conserved residues and reveal a subpocket, which we denote as subpocket B, extending beyond the catalytic aspartate 75 towards the allosteric binding site, providing a unique connection between the orthosteric and allosteric sites in the protease. The structural evidence supports a molecular recognition based primarily on conformational selection and population shift rather than induced-fit. Besides the implications on protease studies and drug development, this hypothesis provides an interpretation for the alternate binding modes with respect to the catalytic serine, which are observed for recently developed beta-lactam inhibitors incorporating benzyloxyphenylglycine.

Identification and validation of a novel dual small-molecule TLR2/8 antagonist

Toll-like receptor 2 (TLR2) and TLR8 are involved in the recognition of bacterial and viral components and are linked not only to protective antimicrobial immunity but also to inflammatory diseases. Recently, increasing attention has been paid to the receptor crosstalk between TLR2 and TLR8 to fine-tune innate immune responses. In this study, we report a novel dual TLR2/TLR8 antagonist, compound 24 that was developed by a modeling-guided synthesis approach. The modulator was optimized from the previously reported 1,3-benzothiazole derivative, compound 8. Compound 24 was pharmacologically characterized for the ability to inhibit TLR2- and TLR8-mediated responses in TLR-overexpressing reporter cells and THP-1 macrophages. The modulator showed high efficacy with IC50 values in the low micromolar range for both TLRs, selectivity towards other TLRs and low cytotoxicity. At TLR2, a slight predominance for the TLR2/1 heterodimer was found in reporter cells selectively expressing TLR2/1 or TLR2/6 heterodimers. Concentration ratio analysis in the presence of Pam3CSK4 or Pam2CSK4 indicated non-competitive antagonist behavior at hTLR2. In computational docking studies, a plausible alternative binding mode of compound 24 was predicted for both TLR2 and TLR8. Our results provide evidence that it is feasible to simultaneously and selectively target endosomal- and surface-located TLRs. We identified a small-molecule dual TLR2/8 antagonist that may serve as a valuable pharmacological tool to decipher the role of TLR2/8 co-signaling in inflammation.