Self-assembled peptide nanofibers can form biomimetic hydrogels at physiological pH and ionic strength through noncovalent and reversible interactions. Inspired by natural antimicrobial peptides, we designed a class of cationic amphiphilic self-assembled peptides (CASPs) that self-assemble into thixotropic nanofibrous hydrogels. These constructs employ amphiphilicity and high terminal charge density to disrupt bacterial membranes. Here, we focus on three aspects of the self-assembly of these hydrogels: (a) the material properties of the individual self-assembled nanofibers, (b) emergence of bulk-scale elasticity in the nanofibrous hydrogel, and (c) trade-off between the desirable material properties and antimicrobial efficacy. The design of the supramolecular nanofibers allows for higher-order noncovalent ionic cross-linking of the nanofibers into a viscoelastic network. We determine the stiffness of the self-assembled nanofibers via the peak force quantitative nanomechanical atomic force microscopy and the bulk-scale rheometry. The storage moduli depend on peptide concentration, ionic strength, and concentration of multivalent ionic cross-linker. CASP nanofibers are demonstrated to be effective against Pseudomonas aeruginosa colonies. We use nanomechanical analysis and microsecond-time scale coarse-grained simulation to elucidate the interaction between the peptides and bacterial membranes. We demonstrate that the membranes stiffen, contract, and buckle after binding to peptide nanofibers, allowing disruption of osmotic equilibrium between the intracellular and extracellular matrix. This is further associated with dramatic changes in cell morphology. Our studies suggest that self-assembled peptide nanofibrils can potentially acts as membrane-disrupting antimicrobial agents, which can be formulated as injectable hydrogels with tunable material properties.
Plasmodium parasites are reliant on the Apicomplexan AP2 (ApiAP2) transcription factor family to regulate gene expression programs. AP2 DNA binding domains have no homologs in the human or mosquito host genomes, making them potential antimalarial drug targets. Using an in-silico screen to dock thousands of small molecules into the crystal structure of the AP2-EXP (Pf3D7_1466400) AP2 domain (PDB:3IGM), we identified putative AP2-EXP interacting compounds. Four compounds were found to block DNA binding by AP2-EXP and at least one additional ApiAP2 protein. Our top ApiAP2 competitor compound perturbs the transcriptome of P . falciparum trophozoites and results in a decrease in abundance of log 2 fold change > 2 for 50% (46/93) of AP2-EXP target genes. Additionally, two ApiAP2 competitor compounds have multi-stage anti- Plasmodium activity against blood and mosquito stage parasites. In summary, we describe a novel set of antimalarial compounds that interact with AP2 DNA binding domains. These compounds may be used for future chemical genetic interrogation of ApiAP2 proteins or serve as starting points for a new class of antimalarial therapeutics.
Short-chain synthetically designed biomimetic peptides have recently emerged as the practical alternatives of naturally occurring antimicrobial peptides. A pertinent question in this regard is: how does the distinct molecular architecture of short synthetic peptides, compared to their relatively long and flexible natural antimicrobial counterpart, lead to potent membrane disruption ability ? Here, we address this question via computationally investigating the action of 10-residue-long {beta}-peptide, a low-molecular weight synthetically designed antimicrobial foldamer, with a bacterial membrane-mimicking phospholipid bilayer. The investigation demonstrates that, beyond a threshold peptide-concentration, this short biomimetic peptide undergoes spontaneous self-aggregation and membrane-adsorption and subsequently forms stable transmembrane pore inside the membrane via a cooperative fashion, leading to membrane-disruption via water-leakage. Interestingly, the pore-inducing ability is found to be elusive in a non-globally amphiphilic sequence isomer, displaying its strong sequence-selective action on membrane. The analysis reveals that, despite having a short helical frame-work, these synthetic peptides, once inside the membrane, are able to stretch themselves towards favourable potential contact with polar head groups and interfacial water layer, thereby facilitating membrane-spanning pore formation process. Taken together, this work brings out a distinct mechanism of membrane-activity of minimally designed synthetic biomimetic oligomers relative to the natural antimicrobial peptides.
Progesterone receptor, nuclear receptor subfamily 3, group C, member 3 (PR, NR3C3) is a member of the steroid receptor subfamily of nuclear receptors (NR3), i.e. ligand-regulated transcription factors. PR regulates multiple biological processes in response to binding of steroid-derived hormones. PR is promiscuously activated by various steroid hormones, including estrogens, androgens, and corticosteroids. Currently, it is not understood how steroid hormones differentially modulate the ligand binding domain (LBD) of PR to achieve various transcriptional outcomes. Here, we use a computational approach to investigate how dynamics of PR are altered by ligand binding to achieve distinct transcriptional properties. Previous studies on the PR LBD revealed that residues in helices 3, 5 and 12 are functionally conserved in nuclear receptors and play important roles in stabilizing agonistic conformations. To probe the roles of these residues in discriminating between steroid hormones, we performed long MD simulations, using a library of 34 steroidal ligands with EC50 values ranging from inactive to 1E-02 nM. To investigate ligand-specific allosteric signaling, we investigated communication pathways between the ligand binding pocket and key regulatory surfaces on the PR LBD surface to determine the specificity with which ligands modulate allosteric coupling between two sites. Our weighted dynamic network analysis revealed that the active and inactive steroids modulated key regions of PR distinctly. Additional analysis of communication paths allowed us to further categorize agonists into three groups, consistent with their potency of activation. We used in silico mutagenesis to investigate roles for specific residues in achieving ligand-selective PR signaling. Combined, the results of the study provide structural and dynamic insight into how PR achieves ligand specificity. Further, these studies can illuminate strategies for the design of novel PR ligands for therapeutical uses.
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