Hit Identification Driven by Combining Artificial Intelligence and Computational Chemistry Methods: A PI5P4K-ß Case Study.

Computer-aided drug design (CADD), especially artificial intelligence-driven drug design (AIDD), is increasingly used in drug discovery. In this paper, a novel and efficient workflow for hit identification was developed within the ID4Inno drug discovery platform, featuring innovative artificial intelligence, high-accuracy computational chemistry, and high-performance cloud computing. The workflow was validated by discovering a few potent hit compounds (best IC50 is ∼0.80 µM) against PI5P4K-ß, a novel anti-cancer target. Furthermore, by applying the tools implemented in ID4Inno, we managed to optimize these hit compounds and finally obtained five hit series with different scaffolds, all of which showed high activity against PI5P4K-ß. These results demonstrate the effectiveness of ID4inno in driving hit identification based on artificial intelligence, computational chemistry, and cloud computing.

MolADI: A Web Server for Automatic Analysis of Protein-Small Molecule Dynamic Interactions.

Protein-ligand interaction analysis is important for drug discovery and rational protein design. The existing online tools adopt only a single conformation of the complex structure for calculating and displaying the interactions, whereas both protein residues and ligand molecules are flexible to some extent. The interactions evolved with time in the trajectories are of greater interest. MolADI is a user-friendly online tool which analyzes the protein-ligand interactions in detail for either a single structure or a trajectory. Interactions can be viewed easily with both 2D graphs and 3D representations. MolADI is available as a web application.

Free Energy Profile and Kinetics of Coupled Folding and Binding of the Intrinsically Disordered Protein p53 with MDM2.

Intrinsically disordered proteins (IDPs) exert their functions by binding to partner proteins via a complex process that includes coupled folding and binding. Because inhibiting the binding of the IDP p53 to its partner MDM2 has become a promising strategy for the design of anticancer drugs, we carried out metadynamics simulations to study the coupled folding and binding process linking the IDP p53 to MDM2 in atomic detail. Using bias-exchange metadynamics (BE-MetaD) and infrequent metadynamics (InMetaD), we estimated the binding free energy, the unbinding rate, and the binding rate. By analyzing the stable intermediates, we uncovered the role non-native interactions played in the p53-MDM2 binding/unbinding process. We used a three-state model to describe the whole binding/unbinding process and to obtain the corresponding rate constants. Our work shows that the binding of p53 favors an induced-fit mechanism which proceeds in a stepwise fashion. Our results can be helpful for gaining an in-depth understanding of the coupled folding and binding process needed for the design of MDM2 inhibitors.

Enhanced Sampling Simulations of Ligand Unbinding Kinetics Controlled by Protein Conformational Changes.

Understanding unbinding kinetics of protein-ligand systems is of great importance for the design of ligands with desired specificity and safety. In recent years, enhanced sampling techniques have emerged as effective tools for studying unbinding kinetics of protein-ligand systems at the atomistic level. However, in many protein-ligand systems, the ligand unbinding processes are strongly coupled to protein conformational changes and the disclosure of the hidden degrees of freedom closely related to the protein conformational changes so that sampling is enhanced over these degrees of freedom remains a great challenge. Here, we show how potential-scaled molecular dynamics (sMD) and infrequent metadynamics (InMetaD) simulation techniques can be combined to successfully reveal the unbinding mechanism of 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[18F]fluo-rodibenzo[b,d]thiophene 5,5-dioxide ([18F]ASEM) from a chimera structure of the α7-nicotinic acetylcholine receptor. By using sMD simulations, we disclosed that the "close" to "open" conformational change of loop C plays a key role in the ASEM unbinding process. By carrying out InMetaD simulations with this conformational change taken into account as an additional collective variable, we further captured the key states in the unbinding process and clarified the unbinding mechanism of ASEM from the protein. Our work indicates that combining sMD and InMetaD simulation techniques can be an effective approach for revealing the unbinding mechanism of a protein-ligand system where protein conformational changes control the unbinding process.

Activity of Antimicrobial Peptide Aggregates Decreases with Increased Cell Membrane Embedding Free Energy Cost.

Antimicrobial peptides (AMPs) are a promising alternative to antibiotics for mitigating bacterial infections, in light of increasing bacterial resistance to antibiotics. However, predicting, understanding, and controlling the antibacterial activity of AMPs remain a significant challenge. While peptide intramolecular interactions are known to modulate AMP antimicrobial activity, peptide intermolecular interactions remain elusive in their impact on peptide bioactivity. Herein, we test the relationship between AMP intermolecular interactions and antibacterial efficacy by controlling AMP intermolecular hydrophobic and hydrogen bonding interactions. Molecular dynamics simulations and Gibbs free energy calculations in concert with experimental assays show that increasing intermolecular interactions via interpeptide aggregation increases the energy cost for the peptide to embed into the bacterial cell membrane, which in turn decreases the AMP antibacterial activity. Our findings provide a route for predicting and controlling the antibacterial activity of AMPs against Gram-negative bacteria via reductions of intermolecular AMP interactions.

Peptide self-assembly triggered by metal ions.

Through their unique and specific interactions with various metal ions, naturally occurring proteins control structures and functions of many biological processes and functions in organisms. Inspired by natural metallopeptides, chemists have developed artificial peptides which coordinate with metal ions through their functional groups either for introducing a special reactivity or for constructing nanostructures. However, the design of new coordination peptides requires a deep understanding of the structures, assembly properties, and dynamic behaviours of such peptides. This review briefly discusses strategies of peptide self-assembly induced by metal coordination to different natural and non-natural binding sites in the peptide. The structures and functions of the obtained aggregates are described as well. We also highlight some examples of a metal-induced peptide self-assembly with relevance to biotechnology applications.

Exploring the Interactions between two Ligands, UCB-J and UCB-F, and Synaptic Vesicle Glycoprotein 2 Isoforms.

In silico modeling was applied to study the efficiency of two ligands, namely, UCB-J and UCB-F, to bind to isoforms of the synaptic vesicle glycoprotein 2 (SV2) that are involved in the regulation of synaptic function in the nerve terminals, with the ultimate goal to understand the selectivity of the interaction between UCB-J and UCB-F to different isoforms of SV2. Docking and large-scale molecular dynamics simulations were carried out to unravel various binding patterns, types of interactions, and binding free energies, covering hydrogen bonding and nonspecific hydrophobic interactions, water bridge, π-π, and cation-π interactions. The overall preference for bonding types of UCB-J and UCB-F with particular residues in the protein pockets can be disclosed in detail. A unique interaction fingerprint, namely, hydrogen bonding with additional cation-π interaction with the pyridine moiety of UCB-J, could be established as an explanation for its high selectivity over the SV2 isoform A (SV2A). Other molecular details, primarily referring to the presence of π-π interactions and hydrogen bonding, could also be analyzed as sources of selectivity of the UCB-F tracer for the three isoforms. The simulations provide atomic details to support future development of new selective tracers targeting synaptic vesicle glycoproteins and their associated diseases.

Structural insights into drug transport by an aquaglyceroporin.

Pentamidine and melarsoprol are primary drugs used to treat the lethal human sleeping sickness caused by the parasite Trypanosoma brucei. Cross-resistance to these two drugs has recently been linked to aquaglyceroporin 2 of the trypanosome (TbAQP2). TbAQP2 is the first member of the aquaporin family described as capable of drug transport; however, the underlying mechanism remains unclear. Here, we present cryo-electron microscopy structures of TbAQP2 bound to pentamidine or melarsoprol. Our structural studies, together with the molecular dynamic simulations, reveal the mechanisms shaping substrate specificity and drug permeation. Multiple amino acids in TbAQP2, near the extracellular entrance and inside the pore, create an expanded conducting tunnel, sterically and energetically allowing the permeation of pentamidine and melarsoprol. Our study elucidates the mechanism of drug transport by TbAQP2, providing valuable insights to inform the design of drugs against trypanosomiasis.

Molecular basis of ligand selectivity for melatonin receptors.

Sleep disorders in adults are related to adverse health effects such as reduced quality of life and increased mortality. About 30-40% of adults are suffering from different sleep disorders. The human melatonin receptors (MT1 and MT2) are family A G protein-coupled receptors that respond to the neurohormone melatonin MEL which regulates circadian rhythm and sleep. Many efforts have been made to develop drugs targeting melatonin receptors to treat insomnia, circadian rhythm disorders, and even cancer. However, designing subtype-selective melatonergic drugs remains challenging due to their high similarities in both sequences and structures. MEL (a function-selective compound with a bulky ß-naphthyl group) behaves as an MT2-selective antagonist, whereas it is an agonist of MT1. Here, molecular dynamics simulations were used to investigate the ligand selectivity of MT receptors at the atomic level. We found that the binding conformation of MEL differs in different melatonin receptors. In MT1, the naphthalene ring of MEL forms a structure perpendicular to the membrane surface. In contrast, there is a 130° angle between the naphthalene ring of MEL and the membrane surface in MT2. Because of this conformational difference, the MEL leads to a constant water channel in MT1 which activates the receptor. However, MEL hinders the formation of continuous water channels, resulting in an inactive state of MT2. Furthermore, we found that A1173.29 in MT2 is a crucial amino acid capable of hindering the conformational flip of the MEL molecule. These results, coupled with previous functional data, reveal that although MT1 and MT2 share highly similar orthosteric ligand-binding pockets, they also display distinctive features that could be used to design selective compounds. Our findings provide new insights into functionally selective melatonergic drug development for sleep disorders.

Rutin, A Natural Inhibitor of IGPD Protein, Partially Inhibits Biofilm Formation in Staphylococcus xylosus ATCC700404 in vitro and in vivo.

Staphylococcus xylosus (S. xylosus) has become an emerging opportunistic pathogen due to its strong biofilm formation ability. Simultaneously, the biofilm of bacteria plays an important role in antibiotic resistance and chronic infection. Here, we confirmed that rutin can effectively inhibit biofilm formation in S. xylosus, of which the inhibition mechanism involves its ability to interact with imidazole glycerol phosphate dehydratase (IGPD), a key enzyme in the process of biofilm formation. We designed experiments to target IGPD and inhibited its activities against S. xylosus. Our results indicated that the activity of IGPD and the amount of histidine decreased significantly under the condition of 0.8 mg/ml rutin. Moreover, the expression of IGPD mRNA (hisB) and IGPD protein was significantly down-regulated. Meanwhile, the results from molecular dynamic simulation and Bio-layer interferometry (BLI) technique showed that rutin could bind to IGPD strongly. Additionally, in vivo studies demonstrated that rutin treatment reduced inflammation and protect mice from acute mastitis caused by S. xylosus. In summary, our findings provide new insights into the treatment of biofilm mediated persistent infections and chronic bacterial infections. It could be helpful to design next generation antibiotics to against resistant bacteria.

Secondary-structure switch regulates the substrate binding of a YopJ family acetyltransferase.

The Yersinia outer protein J (YopJ) family effectors are widely deployed through the type III secretion system by both plant and animal pathogens. As non-canonical acetyltransferases, the enzymatic activities of YopJ family effectors are allosterically activated by the eukaryote-specific ligand inositol hexaphosphate (InsP6). However, the underpinning molecular mechanism remains undefined. Here we present the crystal structure of apo-PopP2, a YopJ family member secreted by the plant pathogen Ralstonia solanacearum. Structural comparison of apo-PopP2 with the InsP6-bound PopP2 reveals a substantial conformational readjustment centered in the substrate-binding site. Combining biochemical and computational analyses, we further identify a mechanism by which the association of InsP6 with PopP2 induces an α-helix-to-ß-strand transition in the catalytic core, resulting in stabilization of the substrate recognition helix in the target protein binding site. Together, our study uncovers the molecular basis governing InsP6-mediated allosteric regulation of YopJ family acetyltransferases and further expands the paradigm of fold-switching proteins.

Asymmetric opening of the homopentameric 5-HT3A serotonin receptor in lipid bilayers.

Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop receptor family are key players in fast signal transduction throughout the nervous system. They have been shown to be modulated by the lipid environment, however the underlying mechanism is not well understood. We report three structures of the Cys-loop 5-HT3A serotonin receptor (5HT3R) reconstituted into saposin-based lipid bilayer discs: a symmetric and an asymmetric apo state, and an asymmetric agonist-bound state. In comparison to previously published 5HT3R conformations in detergent, the lipid bilayer stabilises the receptor in a more tightly packed, 'coupled' state, involving a cluster of highly conserved residues. In consequence, the agonist-bound receptor conformation adopts a wide-open pore capable of conducting sodium ions in unbiased molecular dynamics (MD) simulations. Taken together, we provide a structural basis for the modulation of 5HT3R by the membrane environment, and a model for asymmetric activation of the receptor.

Molecular Engineered Squaraine Nanoprobe for NIR-II/Photoacoustic Imaging and Photothermal Therapy of Metastatic Breast Cancer.

Various squaraine dyes have been developed for biological imaging. Nevertheless, squaraine dyes with emission in the second window (NIR-II, 1000-1700 nm) have few reports largely due to the short of a simple and universal design strategy. In this contribution, molecular engineering strategy is explored to develop squaraine dyes with NIR-II emission. First, NIR-I squaraine dye SQ2 is constructed by the ethyl-grafted 1,8-naphtholactam as donor units and square acid as acceptor unit in a donor-acceptor-donor (D-A-D) structure. To red-shift the fluorescence emission into NIR-II window, malonitrile, as a forceful electron-withdrawing group, is introduced to strengthen square acid acceptor. As a result, the fluorescence spectrum of acceptor-engineered squaraine dye SQ1 exhibits a significant red-shift into NIR-II window. To translate NIR-II fluorophores SQ1 into effective theranostic agents, fibronectin-targeting SQ1 nanoprobe was constructed and showed excellent NIR-II imaging performance in angiography and tumor imaging, including lung metastatic foci in deep tissue. Furthermore, SQ1 nanoprobe can be used for photoacoustic imaging and photothermal ablation of tumors. This research demonstrates that the donor-acceptor engineering strategy is feasible and effective to develop NIR-II squaraine dyes.

Clinical HDAC Inhibitors Are Effective Drugs to Prevent the Entry of SARS-CoV2.

The outbreak of COVID-19 by the end of 2019 has posed serious health threats to humanity and jeopardized the global economy. However, no effective drugs are available to treat COVID-19 currently and there is a great demand to fight against it. Here, we combined computational screening and an efficient cellular pseudotyped virus system, confirming that clinical HDAC inhibitors can efficiently prevent SARS-CoV-2 and potentially be used to fight against COVID-19.

Free Energy Profile for Penetration of Pittsburgh Compound-B into the Amyloid ß Fibril.

The amyloid ß (Aß) fibril is a hallmark of Alzheimer's disease (AD) and has therefore served as an important target for early diagnosis of AD. The Pittsburgh Compound-B (PiB) is one of the most famous positron emission tomography (PET) tracers commonly used for in vivo detection of Aß fibrils. Many theoretical studies have predicted the existence of various core binding sites with different microenvironments for probes binding to the Aß fibril. However, little attention has been devoted to how the probes actually penetrate into the different core binding sites. In this study, an integrated molecular modeling scheme is used to study the penetration of PiB into the core binding sites of the Aß1-42 fibril structure recently obtained by cryogenic electron microscopy. We find that there are two core binding sites for PiB with dramatic differences in cavity size and microenvironment properties, and furthermore that the penetration of PiB into site-1 is energetically prohibitive, whereas the penetration into site-2 is much more favorable. Therefore, the binding capacity at site-2 may be larger than that at site-1 despite its lower binding affinity. Our results thus suggest that site-2 may be a major binding site for PiB binding to Aß fibril and emphasize the importance to adopt a full dynamical picture when studying tracer-fibril binding problems in general, something that in turn can be used to guide the development of tracers with higher affinity and selectivity for the Aß fibril.

A Probe for the Detection of Hypoxic Cancer Cells.

Hypoxia is a common feature of tumor cells. Nitroreductase (NTR), a common biomarker of hypoxia, has been widely used to evaluate the extent of tumor hypoxia. In this study, three fluorescent probes (FBN-1-3) were synthesized to monitor the extent of hypoxia in cancer cells in real time. FBN-1-3 were composed of a fluorescein analogue and one of three different aromatic nitro groups. Of these probes, FBN-1 showed excellent sensitivity and selectivity in detecting hypoxia via a reduction in O2 concentration. Confocal fluorescence imaging and flow cytometry demonstrated that HepG-2, A549, and SKOV-3 cells incubated with FBN-1 under reduced oxygen conditions showed significantly enhanced fluorescence. A mouse HepG-2 tumor model confirmed that FBN-1 responds rapidly to NTR and can be used to evaluate the degree of tumor hypoxia. The changes in intra- and extracellular NTR in tumor cells were also concurrently monitored, which did not reveal a link between NTR concentration and degree of hypoxia. Our work provides a functional probe for tumor hypoxia, and our results suggest the fluorescent response of our probe is due to a decrease in O2 concentration, and not NTR concentration.

Cholesterol-directed nanoparticle assemblies based on single amino acid peptide mutations activate cellular uptake and decrease tumor volume.

Peptide drugs have been difficult to translate into effective therapies due to their low in vivo stability. Here, we report a strategy to develop peptide-based therapeutic nanoparticles by screening a peptide library differing by single-site amino acid mutations of lysine-modified cholesterol. Certain cholesterol-modified peptides are found to promote and stabilize peptide α-helix formation, resulting in selectively cell-permeable peptides. One cholesterol-modified peptide self-assembles into stable nanoparticles with considerable α-helix propensity stabilized by intermolecular van der Waals interactions between inter-peptide cholesterol molecules, and shows 68.3% stability after incubation with serum for 16 h. The nanoparticles in turn interact with cell membrane cholesterols that are disproportionately present in cancer cell membranes, inducing lipid raft-mediated endocytosis and cancer cell death. Our results introduce a strategy to identify peptide nanoparticles that can effectively reduce tumor volumes when administered to in in vivo mice models. Our results also provide a simple platform for developing peptide-based anticancer drugs.

A peptide probe for the detection of neurokinin-1 receptor by disaggregation enhanced fluorescence and magnetic resonance signals.

We report a novel peptide probe for the detection of neurokinin-1 receptor using disaggregation-caused signal enhancement. The probe was obtained via the aggregation of a modified substance P in a terpyridine-Fe (II) complex with Gd (III)-DOTA into well-defined nanostructures, which effectively weaken ligand fluorescence and slow the exchange rate of inner-sphere water molecules. This probe disaggregates upon binding to the neurokinin-1 receptor and activates the contrast agents to generate a fluorescent signal that positively enhances magnetic resonance imaging contrast and allows for the detection of overexpressed receptors on tumor cells and the identification of lung cancer using serum samples.

Synthesis and antibacterial activities of antibacterial peptides with a spiropyran fluorescence probe.

In this report, antibacterial peptides 1-3 were prepared with a spiropyran fluorescence probe. The probe exhibits a change in fluorescence when isomerized from a colorless spiro-form (spiropyran, Sp) to a colored open-form (merocyanine, Mc) under different chemical environments, which can be used to study the mechanism of antimicrobial activity. Peptides 1-3 exhibit a marked decrease in antimicrobial activity with increasing alkyl chain length. This is likely due to the Sp-Mc isomers in different polar environments forming different aggregate sizes in TBS, as demonstrated by time-dependent dynamic light scattering (DLS). Moreover, peptides 1-3 exhibited low cytotoxicity and hemolytic activity. These probe-modified peptides may provide a novel approach to study the effect of structural changes on antibacterial activity, thus facilitating the design of new antimicrobial agents to combat bacterial infection.

Magainin II modified polydiacetylene micelles for cancer therapy.

Polydiacetylene (PDA) micelles have been widely used to deliver anticancer drugs in the treatment of a variety of tumours and for imaging living cells. In this study, we developed an effective strategy to directly conjugate magainin II (MGN-II) to the surface of PDA micelles using a fluorescent dye. These stable and well-defined PDA micelles had high cytotoxicity in cancer cell lines, and were able to reduce the tumour size in mice. The modified PDA micelles improved the anticancer effects of MGN-II in the A549 cell line only at a concentration of 16.0 µg mL(-1) (IC50). In addition, following irradiation with UV light at 254 nm, the PDA micelles gave rise to an energy transfer from the fluorescent dye to the backbone of PDA micelles to enhance the imaging of living cells. Our results demonstrate that modified PDA micelles can not only be used in the treatment of tumors in vitro and in vivo in a simple and directed way, but also offer a new platform for designing functional liposomes to act as anticancer agents.