ß-sheets mediate the conformational change and allosteric signal transmission between the AsLOV2 termini.

Avena sativa phototropin 1 light-oxygen-voltage 2 domain (AsLOV2) is a model protein of Per-Arnt-Sim (PAS) superfamily, characterized by conformational changes in response to external environmental stimuli. This conformational change begins with the unfolding of the N-terminal A'α helix in the dark state followed by the unfolding of the C-terminal Jα helix. The light state is characterized by the unfolded termini and the subsequent modifications in hydrogen bond patterns. In this photoreceptor, ß-sheets are identified as crucial components for mediating allosteric signal transmission between the two termini. Through combined experimental and computational investigations, the Hß and Iß strands are recognized as the most critical and influential ß-sheets in AsLOV2's allosteric mechanism. To elucidate the role of these ß-sheets, we introduced 13 distinct mutations (F490L, N492A, L493A, F494L, H495L, L496F, Q497A, R500A, F509L, Q513A, L514A, D515V, and T517V) and conducted comprehensive molecular dynamics simulations. In-depth hydrogen bond analyses emphasized the role of two hydrogen bonds, Asn482-Leu453 and Gln479-Val520, in the observed distinct behaviors of L493A, L496F, Q497A, and D515V mutants. This illustrates the role of ß-sheets in the transmission of the allosteric signal upon the photoactivation of the light state.

Homology modeling of Forkhead box protein C2: identification of potential inhibitors using ligand and structure-based virtual screening.

Overexpression of Forkhead box protein C2 (FOXC2) has been associated with different types of carcinomas. FOXC2 plays an important role in the initiation and maintenance of the epithelial-mesenchymal transition (EMT) process, which is essential for the development of higher-grade tumors with an enhanced ability for metastasis. Thus, FOXC2 has become a therapeutic target for the development of anticancer drugs. MC-1-F2, the only identified experimental inhibitor of FOXC2, interacts with the full length of FOXC2. However, only the DNA-binding domain (DBD) of FOXC2 has resolved crystal structure. In this work, a three-dimensional (3D) structure of the full-length FOXC2 using homology modeling was developed and used for structure-based drug design (SBDD). The quality of this 3D model of the full-length FOXC2 was evaluated using MolProbity, ERRAT, and ProSA modules. Molecular dynamics (MD) simulation was also carried out to verify its stability. Ligand-based drug design (LBDD) was carried out to identify similar analogues for MC-1-F2 against 15 million compounds from ChEMBL and ZINC databases. 792 molecules were retrieved from this similarity search. De novo SBDD was performed against the full-length 3D structure of FOXC2 through homology modeling to identify novel inhibitors. The combination of LBDD and SBDD helped in gaining a better insight into the binding of MC-1-F2 and its analogues against the full length of the FOXC2. The binding free energy of the top hits was further investigated using MD simulations and MM/GBSA calculations to result in eight promising hits as lead compounds targeting FOXC2.

Novel Allosteric Effectors Targeting Human Transcription Factor TEAD.

The Hippo pathway is an evolutionary conserved signaling network involved in several cellular regulatory processes. Dephosphorylation and overexpression of Yes-associated proteins (YAPs) in the Hippo-off state are common in several types of solid tumors. YAP overexpression results in its nuclear translocation and interaction with transcriptional enhanced associate domain 1-4 (TEAD1-4) transcription factors. Covalent and non-covalent inhibitors have been developed to target several interaction sites between TEAD and YAP. The most targeted and effective site for these developed inhibitors is the palmitate-binding pocket in the TEAD1-4 proteins. Screening of a DNA-encoded library against the TEAD central pocket was performed experimentally to identify six new allosteric inhibitors. Inspired by the structure of the TED-347 inhibitor, chemical modification was performed on the original inhibitors by replacing secondary methyl amide with a chloromethyl ketone moiety. Various computational tools, including molecular dynamics, free energy perturbation, and Markov state model analysis, were employed to study the effect of ligand binding on the protein conformational space. Four of the six modified ligands were associated with enhanced allosteric communication between the TEAD4 and YAP1 domains indicated by the relative free energy perturbation to original molecules. Phe229, Thr332, Ile374, and Ile395 residues were revealed to be essential for the effective binding of the inhibitors.

Microsecond Molecular Dynamics Simulations and Markov State Models of Mutation-Induced Allosteric Mechanisms for the Light-Oxygen-Voltage 2 Protein : Revealing Structural Basis of Signal Transmission Induced by Photoactivation of the Light Protein State.

Avena Sativa phototropin 1 Light-oxygen-voltage 2 domain (AsLOV2) is the model protein of Per-Arnt-Sim (PAS) superfamily, characterized by conformational changes in response to external environmental stimuli. This conformational change is initiated by the unfolding of the N-terminal helix in the dark state followed by the unfolding of the C-terminal helix. The light state is defined by the unfolded termini and the subsequent modifications in hydrogen bond patterns. In this photoreceptor, ß-sheets have been identified as crucial components for mediating allosteric signal transmission between the two termini. In this study, we combined microsecond all-atm molecular dynamics simulations and Markov state modeling of conformational states to quantify molecular basis of mutation-induced allostery in the AsLOV2 protein. Through a combination of computational investigations, we determine that the Hß and Iß strands are the most critical structural elements involved in the allosteric mechanism. To elucidate the role of these ß-sheets, we introduced 13 distinct mutations (F490L, N492A, L493A, F494L, H495L, L496F, Q497A, R500A, F509L, Q513A, L514A, D515V, and T517V) and conducted comprehensive simulation analysis. The results highlighted the role of two hydrogen bond Asn482-Leu453 and Gln479-Val520 in the observed distinct behaviors of L493A, L496F, Q497A, and D515V mutants. The comprehensive atomistic-level analysis of the conformational landscapes revealed the critical functional role of ß-sheet segments in the transmission of the allosteric signal upon the photoactivation of the light state.

Dynamics of hydrogen bonds in the secondary structures of allosteric protein Avena Sativa phototropin 1.

The Light-Oxygen-Voltage 2 (LOV2) domain of Avena Sativa phototropin 1 (AsLOV2) protein is one of the most studied domains in the field of designing photoswitches. This is due to the several unique features in the AsLOV2, such as the monomeric structure of the protein in both light and dark states and the relatively short transition time between the two states. Despite that, not many studies focus on the effect of the secondary structures on the drastic conformational change between the light and dark states. In this study, we focus on the role of A' α helix as a key player in the transition between both states using various computational tools as: 1.5 µ s molecular dynamics simulations for each configuration, Markov state model, different machine learning techniques, and community analysis. The impact of the A' α helix was studied on the atomistic level by introducing two groups of mutations, helicity enhancing mutations (T406A and T407A) and helicity disrupting mutations (L408D and R410P), as well as on the overall secondary structure by using the community analysis. Maintaining the N-terminal hydrogen bond network was found to be essential for the transition between the two states. Via in-depth hydrogen bonding and contact analysis we were able to identify key residues (Thr407 and Arg410) involved in the functional conformational switch and their impact on the overall protein dynamics. Moreover, the community analysis highlighted the significant role of the ß sheets in the overall protein allosteric process.

Cancer immunotherapy from biology to nanomedicine.

With the significant drawbacks of conventional cancer chemotherapeutics, cancer immunotherapy has demonstrated the ability to eradicate cancer cells and circumvent multidrug resistance (MDR) with fewer side effects than traditional cytotoxic therapies. Various immunotherapeutic agents have been investigated for that purpose including checkpoint inhibitors, cytokines, monoclonal antibodies and cancer vaccines. All these agents aid immune cells to recognize and engage tumor cells by acting on tumor-specific pathways, antigens or cellular targets. However, immunotherapeutics are still associated with some concerns such as off-target side effects and poor pharmacokinetics. Nanomedicine may resolve some limitations of current immunotherapeutics such as localizing delivery, controlling release and enhancing the pharmacokinetic profile. Herein, we discuss recent advances of immunotherapeutic agents with respect to their development and biological mechanisms of action, along with the advantages that nanomedicine strategies lend to immunotherapeutics by possibly improving therapeutic outcomes and minimizing side effects.