Analysis of N- and O-linked site-specific glycosylation by ion mobility mass spectrometry: State of the art and future directions.

Glycosylation, the major post-translational modification of proteins, significantly increases the diversity of proteoforms. Glycans are involved in a variety of pivotal structural and functional roles of proteins, and changes in glycosylation are profoundly connected to the progression of numerous diseases. Mass spectrometry (MS) has emerged as the gold standard for glycan and glycopeptide analysis because of its high sensitivity and the wealth of fragmentation information that can be obtained. Various separation techniques have been employed to resolve glycan and glycopeptide isomers at the front end of the MS. However, differentiating structures of isobaric and isomeric glycopeptides constitutes a challenge in MS-based characterization. Many reports described the use of various ion mobility-mass spectrometry (IM-MS) techniques for glycomic analyses. Nevertheless, very few studies have focused on N- and O-linked site-specific glycopeptidomic analysis. Unlike glycomics, glycoproteomics presents a multitude of inherent challenges in microheterogeneity, which are further exacerbated by the lack of dedicated bioinformatics tools. In this review, we cover recent advances made towards the growing field of site-specific glycosylation analysis using IM-MS with a specific emphasis on the MS techniques and capabilities in resolving isomeric peptidoglycan structures. Furthermore, we discuss commonly used software that supports IM-MS data analysis of glycopeptides.

Identification of the functional PD-L1 interface region responsible for PD-1 binding and initiation of PD-1 signaling.

The PD-1/PD-L1 checkpoint pathway is important for regulating immune responses and can be targeted by immunomodulatory drugs to treat a variety of immune disorders. However, the precise protein-protein interactions required for the initiation of PD-1/PD-L1 signaling are currently unknown. Previously, we designed a series of first-generation PD-1 targeting peptides based on the native interface region of programmed death ligand 1 (PD-L1) that effectively reduced PD-1/PD-L1 binding. In this work, we further characterized the previously identified lead peptide, MN1.1, to identify key PD-1 binding residues and design an optimized peptide, MN1.4. We show MN1.4 is significantly more stable than MN1.1 in serum and retains the ability to block PD-1/PD-L1 complex formation. We further characterized the immunomodulatory effects of MN1.4 treatment by measuring markers of T cell activation in a co-culture model with ovarian cancer cells and peripheral blood mononuclear cells. We found MN1.4 treatment reduced cytokine secretion and suppressed T cell responses in a similar manner as recombinant PD-L1. Therefore, the PD-L1 interface region used to design MN1.4 appeared sufficient to initiate PD-1 signaling and likely represents the minimum necessary region of PD-L1 required for PD-1 recognition. We propose a peptide agonist for PD-1, such as MN1.4, could have several applications for treating autoimmune disorders caused by PD-1 deficiencies such as type 1 diabetes, inflammatory arthritis, or autoimmune side effects arising from monoclonal antibody-based cancer immunotherapies.

Competitive Binding of Viral Nuclear Localization Signal Peptide and Inhibitor Ligands to Importin-α Nuclear Transport Protein.

Venezuelan equine encephalitis virus (VEEV) is a highly virulent pathogen whose nuclear localization signal (NLS) sequence from capsid protein binds to the host importin-α transport protein and blocks nuclear import. We studied the molecular mechanisms by which two small ligands, termed I1 and I2, interfere with the binding of VEEV's NLS peptide to importin-α protein. To this end, we performed all-atom replica exchange molecular dynamics simulations probing the competitive binding of the VEEV coreNLS peptide and I1 or I2 ligand to the importin-α major NLS binding site. As a reference, we used our previous simulations, which examined noncompetitive binding of the coreNLS peptide or the inhibitors to importin-α. We found that both inhibitors completely abrogate the native binding of the coreNLS peptide, forcing it to adopt a manifold of nonnative loosely bound poses within the importin-α major NLS binding site. Both inhibitors primarily destabilize the native coreNLS binding by masking its amino acids rather than competing with it for binding to importin-α. Because I2, in contrast to I1, binds off-site localizing on the edge of the major NLS binding site, it inhibits fewer coreNLS native binding interactions than I1. Structural analysis is supported by computations of the free energies of the coreNLS peptide binding to importin-α with or without competition from the inhibitors. Specifically, both inhibitors reduce the free energy gain from coreNLS binding, with I1 causing significantly larger loss than I2. To test our simulations, we performed AlphaScreen experiments measuring IC50 values for both inhibitors. Consistent with in silico results, the IC50 value for I1 was found to be lower than that for I2. We hypothesize that the inhibitory action of I1 and I2 ligands might be specific to the NLS from VEEV's capsid protein.

Binding of viral nuclear localization signal peptides to importin-α nuclear transport protein.

Using all-atom replica-exchange molecular dynamics simulations, we mapped the mechanisms of binding of the nuclear localization signal (NLS) sequence from Venezuelan equine encephalitis virus (VEEV) capsid protein to importin-α (impα) transport protein. Our objective was to identify the VEEV NLS sequence fragment that confers native, experimentally resolved binding to impα as well as to study associated binding energetics and conformational ensembles. The two selected VEEV NLS peptide fragments, KKPK and KKPKKE, show strikingly different binding mechanisms. The minNLS peptide KKPK binds non-natively and nonspecifically by adopting five diverse conformational clusters with low similarity to the x-ray structure 3VE6 of NLS-impα complex. Despite the prevalence of non-native interactions, the minNLS peptide still largely binds to the impα major NLS binding site. In contrast, the coreNLS peptide KKPKKE binds specifically and natively, adopting a largely homogeneous binding ensemble with a dominant, highly native-like conformational cluster. The coreNLS peptide retains most of native binding interactions, including π-cation contacts and a tryptophan cage. While KKPK binding is governed by a complex multistate free energy landscape featuring transitions between multiple binding poses, the coreNLS peptide free energy map is simple, exhibiting a single dominant native-like bound basin. We argue that the origin of the coreNLS peptide binding specificity is several electrostatic interactions formed by the two C-terminal amino acids, Lys10 and Glu11, with impα. The coreNLS sequence is then sufficient for native binding, but none of the amino acids flanking minNLS, including Lys10 and Glu11, are strictly necessary for the native pose. Our analyses indicate that the VEEV coreNLS sequence is virtually unique among human and viral proteins interacting with impα making it a potential target for VEEV-specific inhibitors.

Can Free Energy Perturbation Simulations Coupled with Replica-Exchange Molecular Dynamics Study Ligands with Distributed Binding Sites?

Free energy perturbation coupled with replica exchange with solute tempering (FEP/REST) offers a rigorous approach to compute relative free energy changes for ligands. To determine the applicability of FEP/REST for the ligands with distributed binding poses, we considered two alchemical transformations involving three putative inhibitors I0, I1, and I2 of the Venezuelan equine encephalitis virus nuclear localization signal sequence binding to the importin-α (impα) transporter protein. I0 → I1 and I0 → I2 transformations, respectively, increase or decrease the polarity of the parent molecule. Our objective was three-fold─(i) to verify FEP/REST technical performance and convergence, (ii) to estimate changes in binding free energy ΔΔG, and (iii) to determine the utility of FEP/REST simulations for conformational binding analysis. Our results are as follows. First, our FEP/REST implementation properly follows FEP/REST formalism and produces converged ΔΔG estimates. Due to ligand inherent unbinding, the better FEP/REST strategy lies in performing multiple independent trajectories rather than extending their length. Second, I0 → I1 and I0 → I2 transformations result in overall minor changes in inhibitor binding free energy, slightly strengthening the affinity of I1 and weakening that of I2. Electrostatic interactions dominate binding interactions, determining the enthalpic changes. The two transformations cause opposite entropic changes, which ultimately govern binding affinities. Importantly, we confirm the validity of FEP/REST free energy estimates by comparing them with our previous REST simulations, directly probing binding of three ligands to impα. Third, we established that FEP/REST simulations can sample binding ensembles of ligands. Thus, FEP/REST can be applied (i) to study the energetics of the ligand binding without defined poses and showing minor differences in affinities |ΔΔG| ≲ 0.5 kcal/mol and (ii) to collect ligand binding conformational ensembles.

Skipped dienes in natural product synthesis.

Covering: 2000-2020The 1,4-diene motif, also known as a skipped diene, is widespread across various classes of natural products including alkaloids, fatty acids, terpenoids, and polyketides as part of either the finalized structure or a biosynthetic intermediate. The prevalence of this nonconjugated diene system in nature has resulted in numerous encounters in the total synthesis literature. However, skipped dienes have not been extensively reviewed, which could be attributed to overshadowing by the more recognized 1,3-diene system. In this review, we aim to highlight the relevance of skipped dienes in natural products through the lens of total synthesis. Subjects that will be covered include nomenclature, structural properties, prevalence in natural products, synthetic strategies and the future direction of the field.

Protein painting, an optimized MS-based technique, reveals functionally relevant interfaces of the PD-1/PD-L1 complex and the YAP2/ZO-1 complex.

Protein-protein interactions lie at the heart of many biological processes and therefore represent promising drug targets. Despite this opportunity, identification of protein-protein interfaces remains challenging. We have previously developed a method that relies on coating protein surfaces with small-molecule dyes to discriminate between solvent-accessible protein surfaces and hidden interface regions. Dye-bound, solvent-accessible protein regions resist trypsin digestion, whereas hidden interface regions are revealed by denaturation and sequenced by MS. The small-molecule dyes bind promiscuously and with high affinity, but their binding mechanism is unknown. Here, we report on the optimization of a novel dye probe used in protein painting, Fast Blue B + naphthionic acid, and show that its affinity for proteins strongly depends on hydrophobic moieties that we call here "hydrophobic clamps." We demonstrate the utility of this probe by sequencing the protein-protein interaction regions between the Hippo pathway protein Yes-associated protein 2 (YAP2) and tight junction protein 1 (TJP1 or ZO-1), uncovering interactions via the known binding domain as well as ZO-1's MAGUK domain and YAP's N-terminal proline-rich domain. Additionally, we demonstrate how residues predicted by protein painting are present exclusively in the complex interface and how these residues may guide the development of peptide inhibitors using a case study of programmed cell death protein 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1). Inhibitors designed around the PD-1/PD-L1 interface regions identified via protein painting effectively disrupted complex formation, with the most potent inhibitor having an IC50 of 5 µm.

Identification of a binding site of the human immunodeficiency virus envelope protein gp120 to neuronal-specific tubulin.

Human immunodeficiency virus-1 (HIV) promotes synaptic simplification and neuronal apoptosis, and causes neurological impairments termed HIV-associated neurological disorders. HIV-associated neurotoxicity may be brought about by acute and chronic mechanisms that still remain to be fully characterized. The HIV envelope glycoprotein gp120 causes neuronal degeneration similar to that observed in HIV-associated neurocognitive disorders subjects. This study was undertaken to discover novel mechanisms of gp120 neurotoxicity that could explain how the envelope protein promotes neurite pruning. Gp120 has been shown to associate with various intracellular organelles as well as microtubules in neurons. We then analyzed lysates of neurons exposed to gp120 with liquid chromatography mass spectrometry for potential protein interactors. We found that one of the proteins interacting with gp120 is tubulin ß-3 (TUBB3), a major component of neuronal microtubules. We then tested the hypothesis that gp120 binds to neuronal microtubules. Using surface plasmon resonance, we confirmed that gp120 binds with high affinity to neuronal-specific TUBB3. We have also identified the binding site of gp120 to TUBB3. We then designed a small peptide (Helix-A) that displaced gp120 from binding to TUBB3. To determine whether this peptide could prevent gp120-mediated neurotoxicity, we cross-linked Helix-A to mesoporous silica nanoparticles (Helix-A nano) to enhance the intracellular delivery of the peptide. We then tested the neuroprotective property of Helix-A nano against three strains of gp120 in rat cortical neurons. Helix-A nano prevented gp120-mediated neurite simplification as well as neuronal loss. These data propose that gp120 binding to TUBB3 could be another mechanism of gp120 neurotoxicity. We propose a novel direct mechanism of human immunodeficiency virus neurotoxicity. Our data show that the viral protein gp120 binds to neuronal specific tubulin ß-3 and blocks microtubule transport. Displacing gp120 from binding to tubulin by a small peptide prevents gp120-mediated neuronal loss. Our study reveals a novel target for developing adjunct therapies against viral infection that promotes neurocognitive disorders.

Aminopeptidase P Mediated Targeting for Breast Tissue Specific Conjugate Delivery.

Cytotoxic chemotherapies are used to treat breast cancer, but are limited by systemic toxicity. The key to addressing this important issue is the development of a nontoxic, tissue selective, and molecular specific delivery system. In order to potentially increase the therapeutic index of clinical reagents, we designed an Aminopeptidase P (APaseP) targeting tissue-specific construct conjugated to a homing peptide for selective binding to human breast-derived cancer cells. Homing peptides are short amino acid sequences derived from phage display libraries that have the unique property of localizing to specific organs. Our molecular construct allows for tissue-specific drug delivery, by binding to APaseP in the vascular endothelium. The breast homing peptide evaluated in our studies is a cyclic nine-amino-acid peptide with the sequence CPGPEGAGC, referred to as PEGA. We show by confocal microscopy that the PEGA peptide and similar peptide conjugates distribute to human breast tissue xenograft specifically and evaluate the interaction with the membrane-bound proline-specific APaseP (KD = 723 ± 3 nM) by binding studies. To achieve intracellular breast cancer cell delivery, the incorporation of the Tat sequence, a cell-penetrating motif derived from HIV, was conjugated with the fluorescently labeled PEGA peptide sequence. Ultimately, tissue specific peptides and their conjugates can enhance drug delivery and treatment by their ability to discriminate between tissue types. Tissue specific conjugates as we have designed may be valuable tools for drug delivery and visualization, including the potential to treat breast cancer, while simultaneously minimizing systemic toxicity.

Role of leukotriene A4 hydrolase aminopeptidase in the pathogenesis of emphysema.

The leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme with epoxy hydrolase and aminopeptidase activities. We hypothesize that the LTA4H aminopeptidase activity alleviates neutrophilic inflammation, which contributes to cigarette smoke (CS)-induced emphysema by clearing proline-glycine-proline (PGP), a triamino acid chemokine known to induce chemotaxis of neutrophils. To investigate the biological contributions made by the LTA4H aminopeptidase activity in CS-induced emphysema, we exposed wild-type mice to CS over 5 mo while treating them with a vehicle or a pharmaceutical agent (4MDM) that selectively augments the LTA4H aminopeptidase without affecting the bioproduction of leukotriene B4. Emphysematous phenotypes were assessed by premortem lung physiology with a small animal ventilator and by postmortem histologic morphometry. CS exposure acidified the airspaces and induced localization of the LTA4H protein into the nuclei of the epithelial cells. This resulted in accumulation of PGP in the airspaces by suppressing the LTA4H aminopeptidase activity. When the LTA4H aminopeptidase activity was selectively augmented by 4MDM, the levels of PGP in the bronchoalveolar lavage fluid and infiltration of neutrophils into the lungs were significantly reduced without affecting the levels of leukotriene B4. This protected murine lungs from CS-induced emphysematous alveolar remodeling. In conclusion, CS exposure promotes the development of CS-induced emphysema by suppressing the enzymatic activities of the LTA4H aminopeptidase in lung tissues and accumulating PGP and neutrophils in the airspaces. However, restoring the leukotriene A4 aminopeptidase activity with a pharmaceutical agent protected murine lungs from developing CS-induced emphysema.

Synthesis and pharmacological characterization of new neuronal nicotinic acetylcholine receptor ligands derived from Sazetidine-A.

The enantiomers of two analogs of Sazetidine-A as well as several other novel biosteric analogues were synthesized. Their binding affinities at three major nAChRs subtypes and selectivity profiles were determined. Though many (S)-enantiomers of Sazetidine-A analogs have high binding affinities and good subtype selectivities, it is not a general rule that (S)-enantiomers are better than their (R) counterparts. Compound 11, of which the ethynyl group was replaced by its' bioisostere-the triazole via click chemistry, showed a high binding affinity to α4ß2 subtype (Ki=1.3 nM) and better selectivity to the α4ß2 subtype over α3ß4 subtype with that of Sazetidine-A. The azide compound 15, a potential photoaffinity label, showed improved high selectivity and similar binding property profile with that of Sazetidine-A. The biaryl analog 17 exhibited a much lower affinity as compared to Sazetidine-A indicating the importance of a 'long tail' side chain for α4ß2 nAChR binding.

Design, synthesis and biological evaluation of ezrin inhibitors targeting metastatic osteosarcoma.

Respiratory failure due to pulmonary metastasis is the major cause of death for patients with osteosarcoma. However, the molecular basis for metastasis of osteosarcoma is poorly understood. Recently, ezrin, a member of the ERM family of proteins, has been associated with osteosarcoma metastasis to the lungs. The small molecule NSC 668394 was identified to bind to ezrin, inhibit in vitro and in vivo cell migration, invasion, and metastatic colony survival. Reported herein are the design and synthesis of analogues of NSC 668394, and subsequent functional ezrin inhibition studies. The binding affinity was characterized by surface plasmon resonance technique. Cell migration and invasion activity was determined by electrical cell impedance methodology. Optimization of a series of heterocyclic-dione analogues led to the discovery of compounds 21k and 21m as potential novel antimetastatic agents.

A bipolar polymer cathode for sodium-ion batteries.

A bipolar polymer cathode material, containing redox-active azo benzene and diamine moieties, was synthesized for sodium-ion batteries. The n-type azo group and p-type amine group enable a wide cutoff window with an initial capacity of 93 mA h g-1 at 50 mA g-1 and a high voltage plateau at ∼3.3 V.

Binding of Inhibitors to Nuclear Localization Signal Peptide from Venezuelan Equine Encephalitis Virus Capsid Protein Explored with All-Atom Replica Exchange Molecular Dynamics.

Several small molecule inhibitors have been designed to block binding of the Venezuelan equine encephalitis virus (VEEV) nuclear localization signal (NLS) sequence to the importin-α nuclear transport protein. To probe the inhibition mechanism on a molecular level, we used all-atom explicit water replica exchange molecular dynamics to study the binding of two inhibitors, I1 and I2, to the coreNLS peptide, representing the core fragment of the VEEV NLS sequence. Our objective was to evaluate the possibility of masking wherein binding of these inhibitors to the coreNLS occurs prior to its binding to importin-α. We found that the free energy of I1 and I2 binding to the coreNLS is less favorable than that to importin-α. This outcome argues against preemptive inhibitor binding to the coreNLS prior to importin-α. Instead, both inhibitors are expected to compete with the coreNLS peptide for binding to importin-α. The two factors responsible for the low affinities of the inhibitors to the coreNLS peptide are (i) the low cooperativity of binding to the peptide and (ii) the strong hydrophobic effect associated with binding to importin-α. Our results further show that upon binding to the coreNLS peptide, the inhibitors form multiple diverse binding poses. The coreNLS peptide coincubated with I1 and I2 adopts several conformational states, including open and collapsed, which underscores the fluidity of the coreNLS conformational ensemble as a target for inhibitors. Taken together with our prior investigations, this study sheds light on the molecular mechanism by which I1 and I2 ligands inhibit binding of the VEEV capsid protein to importin-α.

Protein Painting Mass Spectrometry in the Discovery of Interaction Sites within the Acetylcholine Binding Protein.

Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channel receptors that contribute to cognition, memory, and motor control in many organisms. The pharmacological targeting of these receptors, using small molecules or peptides, presents an important strategy for the development of drugs that can treat important human diseases, including neurodegenerative disorders. The Aplysia californica acetylcholine binding protein (Ac-AChBP) is a structural surrogate of the nAChR with high homology to the extracellular ligand binding domain of homopentameric nAChRs. In this study, we optimized protein-painting-based mass spectrometry to identify regions of interaction between the Ac-AChBP and several nAChR ligands. Using molecular dyes that adhere to the surface of a solubilized Ac-AChBP complex, we identified amino acid residues that constitute a contact site within the Ac-AChBP for α-bungarotoxin, choline, nicotine, and amyloid-ß 1-42. By integrating innovation in protein painting mass spectrometry with computational structural modeling, we present a new experimental tool for analyzing protein interactions of the nAChR.

Total Synthesis of Homoseongomycin Enantiomers and Evaluation of Their Optical Rotation.

A total synthesis of each homoseongomycin enantiomer was accomplished in 17 total steps (longest linear sequence = 12 steps) and 10 chromatographic purifications. Several schemes were attempted to forge the key 5-membered ring, but only a Suzuki coupling-intramolecular Friedel-Crafts acylation sequence proved viable. Challenges encountered during the optical rotation characterization of the natural product left us with two important takeaways. First, highly colored compounds like homoseongomycin that absorb near/at the sodium d-line may require optical rotation measurements at other wavelengths. Second, high dilution of such compounds to obtain measurement at the sodium d-line could result in artificially large and incorrectly assigned specific rotations. To verify the optical rotation, electronic circular dichroism spectra were acquired for both homoseongomycin enantiomers and were transformed into optical rotary dispersions via the Kramers-Kronig transform. We note the wavelength dependency on rotation, and at the sodium d-line 589 nm, we reassign the optical rotation of L-homoseongomycin from (-) to (+).

Synthesis and Evaluation of diaryl ether modulators of the leukotriene A4 hydrolase aminopeptidase activity.

Activation of the aminopeptidase (AP) activity of leukotriene A4 hydrolase (LTA4H) presents a potential therapeutic strategy for resolving chronic inflammation. Previously, ARM1 and derivatives were found to activate the AP activity using the alanine-p-nitroanilide (Ala-pNA) as a reporter group in an enzyme kinetics assay. As an extension of this previous work, novel ARM1 derivatives were synthesized using a palladium-catalyzed Ullmann coupling reaction and screened using the same assay. Analogue 5, an aminopyrazole (AMP) analogue of ARM1, was found to be a potent AP activator with an AC50 of 0.12 µM. An X-ray crystal structure of LTA4H in complex with AMP was refined at 2.7 Å. Despite its AP activity with Ala-pNA substrate, AMP did not affect hydrolysis of the previously proposed natural ligand of LTA4H, Pro-Gly-Pro (PGP). This result highlights a discrepancy between the hydrolysis of more conveniently monitored chromogenic synthetic peptides typically employed in assays and endogenous peptides. The epoxide hydrolase (EH) activity of AMP was measured in vivo and the compound significantly reduced leukotriene B4 (LTB4) levels in a murine bacterial pneumonia model. However, AMP did not enhance survival in the murine pneumonia model over a 14-day period. A liver microsome stability assay showed metabolic stability of AMP. The results suggested that accelerated Ala-pNA cleavage is not sufficient for predicting therapeutic potential, even when the full mechanism of activation is known.

Species-Selective Detection of Volatile Organic Compounds by Ionic Liquid-Based Electrolyte Using Electrochemical Methods.

The detection of volatile organic compounds (VOCs) is an important topic for environmental safety and public health. However, the current commercial VOC detectors suffer from cross-sensitivity and low reproducibility. In this work, we present species-selective detection for VOCs using an electrochemical cell based on ionic liquid (IL) electrolytes with features of high selectivity and reliability. The voltammograms measured with the IL-based electrolyte absorbing different VOCs exhibited species-selective features that were extracted and classified by linear discriminant analysis (LDA). The detection system could identify as many as four types of VOCs, including methanol, ethanol, acetone, formaldehyde, and additional water. A mixture of methanol and formaldehyde was detected as well. The sample required for the VOCs classification system was 50 µL, or 1.164 mmol, on average. The response time for each VOC measurement is as fast as 24 s. The volume of VOCs such as formaldehyde in solution could also be quantified by LDA and electrochemical impedance spectroscopy techniques, respectively. The system showed a tunable detection range for 1.6 and 16% (w/v) CH2O solution by adjusting the composition of the electrolyte. The limit of detection was as low as 1 µL. For the 1.6% CH2O solution, the linearity calibration range was determined to be from 5.30 to 53.00 µmol with a limit of detection at 0.53 µmol. The mechanisms for VOCs determination and quantification are also thoroughly discussed. It is expected that this work could provide a new insight into the concept of electrochemical detection of VOCs with machine learning analysis and be applied to both VOCs gas monitoring and fluid detection.

Binding of Venezuelan Equine Encephalitis Virus Inhibitors to Importin-α Receptors Explored with All-Atom Replica Exchange Molecular Dynamics.

Although Venezuelan equine encephalitis virus (VEEV) is a life-threatening pathogen with a capacity for epidemic outbreaks, there are no FDA-approved VEEV antivirals for humans. VEEV cytotoxicity is partially attributed to the formation of a tetrameric complex between the VEEV capsid protein, the nuclear import proteins importin-α and importin-ß, and the nuclear export protein CRM1, which together block trafficking through the nuclear pore complex. Experimental studies have identified small molecules from the CL6662 scaffold as potential inhibitors of the viral nuclear localization signal (NLS) sequence binding to importin-α. However, little is known about the molecular mechanism of CL6662 inhibition. To address this issue, we employed all-atom replica exchange molecular dynamics simulations to probe, in atomistic detail, the binding mechanism of CL6662 ligands to importin-α. Three ligands, including G281-1485 and two congeners with varying hydrophobicities, were considered. We investigated the distribution of ligand binding poses, their locations, and ligand specificities measured by the strength of binding interactions. We found that G281-1485 binds nonspecifically without forming well-defined binding poses throughout the NLS binding site. Binding of the less hydrophobic congener becomes strongly on-target with respect to the NLS binding site but remains nonspecific. However, a more hydrophobic congener is a strongly specific binder and the only ligand out of three to form a well-defined binding pose, while partially overlapping with the NLS binding site. On the basis of free energy estimates, we argue that all three ligands weakly compete with the viral NLS sequence for binding to importin-α in an apparent compromise to preserve host NLS binding. We further show that all-atom replica exchange binding simulations are a viable tool for studying ligands binding nonspecifically without forming well-defined binding poses.

Drug discovery efforts at George Mason University.

With over 39,000 students, and research expenditures in excess of $200 million, George Mason University (GMU) is the largest R1 (Carnegie Classification of very high research activity) university in Virginia. Mason scientists have been involved in the discovery and development of novel diagnostics and therapeutics in areas as diverse as infectious diseases and cancer. Below are highlights of the efforts being led by Mason researchers in the drug discovery arena. To enable targeted cellular delivery, and non-biomedical applications, Veneziano and colleagues have developed a synthesis strategy that enables the design of self-assembling DNA nanoparticles (DNA origami) with prescribed shape and size in the 10 to 100 nm range. The nanoparticles can be loaded with molecules of interest such as drugs, proteins and peptides, and are a promising new addition to the drug delivery platforms currently in use. The investigators also recently used the DNA origami nanoparticles to fine tune the spatial presentation of immunogens to study the impact on B cell activation. These studies are an important step towards the rational design of vaccines for a variety of infectious agents. To elucidate the parameters for optimizing the delivery efficiency of lipid nanoparticles (LNPs), Buschmann, Paige and colleagues have devised methods for predicting and experimentally validating the pKa of LNPs based on the structure of the ionizable lipids used to formulate the LNPs. These studies may pave the way for the development of new LNP delivery vehicles that have reduced systemic distribution and improved endosomal release of their cargo post administration. To better understand protein-protein interactions and identify potential drug targets that disrupt such interactions, Luchini and colleagues have developed a methodology that identifies contact points between proteins using small molecule dyes. The dye molecules noncovalently bind to the accessible surfaces of a protein complex with very high affinity, but are excluded from contact regions. When the complex is denatured and digested with trypsin, the exposed regions covered by the dye do not get cleaved by the enzyme, whereas the contact points are digested. The resulting fragments can then be identified using mass spectrometry. The data generated can serve as the basis for designing small molecules and peptides that can disrupt the formation of protein complexes involved in disease processes. For example, using peptides based on the interleukin 1 receptor accessory protein (IL-1RAcP), Luchini, Liotta, Paige and colleagues disrupted the formation of IL-1/IL-R/IL-1RAcP complex and demonstrated that the inhibition of complex formation reduced the inflammatory response to IL-1B. Working on the discovery of novel antimicrobial agents, Bishop, van Hoek and colleagues have discovered a number of antimicrobial peptides from reptiles and other species. DRGN-1, is a synthetic peptide based on a histone H1-derived peptide that they had identified from Komodo Dragon plasma. DRGN-1 was shown to disrupt bacterial biofilms and promote wound healing in an animal model. The peptide, along with others, is being developed and tested in preclinical studies. Other research by van Hoek and colleagues focuses on in silico antimicrobial peptide discovery, screening of small molecules for antibacterial properties, as well as assessment of diffusible signal factors (DFS) as future therapeutics. The above examples provide insight into the cutting-edge studies undertaken by GMU scientists to develop novel methodologies and platform technologies important to drug discovery.