Lipidic prodrug approach for improved oral drug delivery and therapy.

In the past, a prodrug design was used as a last option to improve bioavailability through controlling transport, distribution, metabolism, or other mechanisms. Prodrugs are currently used even in early stages of drug development, and a significant percentage of all drugs in the market are prodrugs. The focus of this article is lipidic prodrugs, a strategy whereby a lipid carrier is covalently bound to the drug moiety. The increased lipophilicity of the lipid-drug conjugate can improve the pharmacokinetic profile and provide meaningful advantages: increased absorption across biological barriers, prolonged circulation half-life, selective distribution profile (eg brain penetration), reduced hepatic first-pass metabolism, and overall enhanced bioavailability of the parent drug. Moreover, lipidic prodrugs may join the endogenous lipid trafficking pathways, thereby facilitate drug targeting, either by selective absorption pathway (eg lymphatic transport) or drug release at specific target site(s). The different lipid-drug conjugates (triglyceride-, fatty acids, phospholipid-, and steroid-based prodrugs), the physiological barriers that challenge the absorption of these conjugates, followed by their current utilization and potential clinical benefits are described and analyzed, and future opportunities this approach could provide are discussed. Altogether, lipidic prodrugs represent an exciting approach for improving different aspects of oral drug delivery/therapy and may provide solutions for various unmet needs; the use of this strategy is expected to grow.

Molecular Modeling-Guided Design of Phospholipid-Based Prodrugs.

The lipidic prodrug approach is an emerging field for improving a number of biopharmaceutical and drug delivery aspects. Owing to their structure and nature, phospholipid (PL)-based prodrugs may join endogenous lipid processing pathways, and hence significantly improve the pharmacokinetics and/or bioavailability of the drug. Additional advantages of this approach include drug targeting by enzyme-triggered drug release, blood-brain barrier permeability, lymphatic targeting, overcoming drug resistance, or enabling appropriate formulation. The PL-prodrug design includes various structural modalities-different conjugation strategies and/or the use of linkers between the PL and the drug moiety, which considerably influence the prodrug characteristics and the consequent effects. In this article, we describe how molecular modeling can guide the structural design of PL-based prodrugs. Computational simulations can predict the extent of phospholipase A2 (PLA2)-mediated activation, and facilitate prodrug development. Several computational methods have been used to facilitate the design of the pro-drugs, which will be reviewed here, including molecular docking, the free energy perturbation method, molecular dynamics simulations, and free density functional theory. Altogether, the studies described in this article indicate that computational simulation-guided PL-based prodrug molecular design correlates well with the experimental results, allowing for more mechanistic and less empirical development. In the future, the use of molecular modeling techniques to predict the activity of PL-prodrugs should be used earlier in the development process.

Crystal Structure of Cleaved Serp-1, a Myxomavirus-Derived Immune Modulating Serpin: Structural Design of Serpin Reactive Center Loop Peptides with Improved Therapeutic Function.

The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a "first-in-class" therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next-generation RCL peptides with improved therapeutic potential that is not reduced when the peptides are combined with antibiotic treatments. The crystal structure of cleaved Serp-1 was determined to 2.5 Å resolution and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and inhibit mammalian serpins, plasminogen activator inhibitor 1 (PAI-1), anti-thrombin III (ATIII), and α-1 antitrypsin (A1AT), and target proteases. Using in silico modeling of the Serp-1 RCL peptide, S-7, we designed several modified RCL peptides that were predicted to have stronger interactions with human serpins because of the larger number of stabilizing hydrogen bonds. Two of these peptides (MPS7-8 and -9) displayed extended activity, improving survival where activity was previously lost in antibiotic-treated MHV68-infected mice (P < 0.0001). Mass spectrometry and kinetic assays suggest interaction of the peptides with ATIII, A1AT, and target proteases in mouse and human plasma. In summary, we present the next step toward the development of a promising new class of anti-inflammatory serpin-based therapeutics.

Computational modeling and in-vitro/in-silico correlation of phospholipid-based prodrugs for targeted drug delivery in inflammatory bowel disease.

Targeting drugs to the inflamed intestinal tissue(s) represents a major advancement in the treatment of inflammatory bowel disease (IBD). In this work we present a powerful in-silico modeling approach to guide the molecular design of novel prodrugs targeting the enzyme PLA2, which is overexpressed in the inflamed tissues of IBD patients. The prodrug consists of the drug moiety bound to the sn-2 position of phospholipid (PL) through a carbonic linker, aiming to allow PLA2 to release the free drug. The linker length dictates the affinity of the PL-drug conjugate to PLA2, and the optimal linker will enable maximal PLA2-mediated activation. Thermodynamic integration and Weighted Histogram Analysis Method (WHAM)/Umbrella Sampling method were used to compute the changes in PLA2 transition state binding free energy of the prodrug molecule (∆∆Gtr) associated with decreasing/increasing linker length. The simulations revealed that 6-carbons linker is the optimal one, whereas shorter or longer linkers resulted in decreased PLA2-mediated activation. These in-silico results were shown to be in excellent correlation with experimental in-vitro data. Overall, this modern computational approach enables optimization of the molecular design of novel prodrugs, which may allow targeting the free drug specifically to the diseased intestinal tissue of IBD patients.

Varying the electronic structure of surface-bound ruthenium(II) polypyridyl complexes.

In the design of light-harvesting chromophores for use in dye-sensitized photoelectrosynthesis cells (DSPECs), surface binding to metal oxides in aqueous solutions is often inhibited by synthetic difficulties. We report here a systematic synthesis approach for preparing a family of Ru(II) polypyridyl complexes of the type [Ru(4,4'-R2-bpy)2(4,4'-(PO3H2)2-bpy)](2+) (4,4'(PO3H2)2-bpy = [2,2'-bipyridine]-4,4'-diylbis(phosphonic acid); 4,4'-R2-bpy = 4,4'-R2-2,2'-bipyridine; and R = OCH3, CH3, H, or Br). In this series, the nature of the 4,4'-R2-bpy ligand is modified through the incorporation of electron-donating (R = OCH3 or CH3) or electron-withdrawing (R = Br) functionalities to tune redox potentials and excited-state energies. Electrochemical measurements show that the ground-state potentials, E(o')(Ru(3+/2+)), vary from 1.08 to 1.45 V (vs NHE) when the complexes are immobilized on TiO2 electrodes in aqueous HClO4 (0.1 M) as a result of increased Ru dπ-π* back-bonding caused by the lowering of the π* orbitals on the 4,4'-R2-bpy ligand. The same ligand variations cause a negligible shift in the metal-to-ligand charge-transfer absorption energies. Emission energies decrease from λmax = 644 to 708 nm across the series. Excited-state redox potentials are derived from single-mode Franck-Condon analyses of room-temperature emission spectra and are discussed in the context of DSPEC applications.

Electronic and optical properties of polypyridylruthenium derivatized polystyrenes: multi-level computational analysis of metallo-polymeric chromophore assemblies.

Great effort is geared toward investigation of new materials for solar energy conversion in recent years. Polymeric chromophore assemblies consisting of [Ru(bpy)3](2+) complexes attached to a polystyrene backbone have gained considerable interest in recent years because of their structural flexibility combined with their ability to efficiently capture solar energy and transport the captured energy in the form of exciton or charges. We employ a combination of computational methods to examine how opto-electronic properties of [Ru(bpy)3](2+) complexes are influenced by the polymer dynamics in these polymeric chromophore assemblies. The covalent linker between the polymer and the light-absorbing Ru complex is thought to play an important role in optimizing the assemblies for solar energy conversion and transport. We find that the presence of -CH2- groups in the linker has a significant impact on the Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) energies of the pendants. Generally speaking, a longer linker leads to higher HOMO energies. Without the presence of -CH2- groups, a mixture of cis and trans amide bond in the covalent linker leads to a bimodal distribution for both HOMO and LUMO energies. Importantly, we find that distributions of orbital energies from individual [Ru(bpy)3](2+) pendants have the maximum overlap when there is only one -CH2- group in the linker. Such an isotropic energy distribution is likely to be important for charge transport within the assemblies. We also find that in contrast to the isolated [Ru(bpy)3](2+) complex, the HOMO is generally found on the linker rather than on Ru atom. This does not change the character of the metal-to-ligand charge transfer (MLCT) excited states, as these excitations in the pendants do not derive from HOMO/LUMO transitions but rather from HOMO - 2/LUMO transition since HOMO - 2 is located on the Ru atom.

Controlling ground and excited state properties through ligand changes in ruthenium polypyridyl complexes.

The capture and storage of solar energy requires chromophores that absorb light throughout the solar spectrum. We report here the synthesis, characterization, electrochemical, and photophysical properties of a series of Ru(II) polypyridyl complexes of the type [Ru(bpy)2(N-N)](2+) (bpy = 2,2'-bipyridine; N-N is a bidentate polypyridyl ligand). In this series, the nature of the N-N ligand was altered, either through increased conjugation or incorporation of noncoordinating heteroatoms, as a way to use ligand electronic properties to tune redox potentials, absorption spectra, emission spectra, and excited state energies and lifetimes. Electrochemical measurements show that lowering the π* orbitals on the N-N ligand results in more positive Ru(3+/2+) redox potentials and more positive first ligand-based reduction potentials. The metal-to-ligand charge transfer absorptions of all of the new complexes are mostly red-shifted compared to Ru(bpy)3(2+) (λmax = 449 nm) with the lowest energy MLCT absorption appearing at λmax = 564 nm. Emission energies decrease from λmax = 650 nm to 885 nm across the series. One-mode Franck-Condon analysis of room-temperature emission spectra are used to calculate key excited state properties, including excited state redox potentials. The impacts of ligand changes on visible light absorption, excited state reduction potentials, and Ru(3+/2+) potentials are assessed in the context of preparing low energy light absorbers for application in dye-sensitized photoelectrosynthesis cells.

Controlled electropolymerization of ruthenium(II) vinylbipyridyl complexes in mesoporous nanoparticle films of TiO(2).

Surface-initiated, oligomeric assemblies of ruthenium(II) vinylpolypyridyl complexes have been grown within the cavities of mesoporous nanoparticle films of TiO2 by electrochemically controlled radical polymerization. Surface growth was monitored by cyclic voltammetry as well as UV/Vis and X-ray photoelectron spectroscopy. Polymerization occurs by a radical chain mechanism following cyclic voltammetry scans to negative potentials where reduction occurs at the π* levels of the polypyridyl ligands. Oligomeric growth within the cavities of the TiO2 films occurs until an average of six repeat units are added to the surface-bound initiator site, which is in agreement with estimates of the internal volumes of the pores in the nanoparticle films.

Atom transfer radical polymerization preparation and photophysical properties of polypyridylruthenium derivatized polystyrenes.

A ruthenium containing polymer featuring a short carbonyl-amino-methylene linker has been prepared by atom transfer radical polymerization (ATRP). The polymer was derived from ATRP of the N-hydroxysuccinimide (NHS) derivative of p-vinylbenzoic acid, followed by an amide coupling reaction of the NHS-polystyrene with Ru(II) complexes derivatized with aminomethyl groups (i.e., [Ru(bpy)2(CH3-bpy-CH2NH2)](2+) where bpy is 2,2'-bipyridine, and CH3-bpy-CH2NH2 is 4-methyl-4'-aminomethyl-2,2'-bipyridine). The Ru-functionalized polymer structure was confirmed by using nuclear magnetic resonance and infrared spectroscopy, and the results suggest that a high loading ratio of polypyridylruthenium chromophores on the polystyrene backbone was achieved. The photophysical properties of the polymer were characterized in solution and in rigid ethylene glycol glasses. In solution, emission quantum yield and lifetime studies reveal that the polymer's metal-to-ligand charge transfer (MLCT) excited states are quenched relative to a model Ru complex chromophore. In rigid media, the MLCT-ground state band gap and lifetime are both increased relative to solution with time-resolved emission measurements revealing fast energy transfer hopping within the polymer. Molecular dynamics studies of the polymer synthesized here as well as similar model systems with various spatial arrangements of the pendant Ru complex chromophores suggest that the carbonyl-amino-methylene linker probed in our target polymer provides shorter Ru-Ru nearest-neighbor distances leading to an increased Ru*-Ru energy hopping rate, compared to those with longer linkers in counterpart polymers.

Effect of backbone flexibility on charge transfer rates in peptide nucleic acid duplexes.

Charge transfer (CT) properties are compared between peptide nucleic acid structures with an aminoethylglycine backbone (aeg-PNA) and those with a γ-methylated backbone (γ-PNA). The common aeg-PNA is an achiral molecule with a flexible structure, whereas γ-PNA is a chiral molecule with a significantly more rigid structure than aeg-PNA. Electrochemical measurements show that the CT rate constant through an aeg-PNA bridging unit is twice the CT rate constant through a γ-PNA bridging unit. Theoretical calculations of PNA electronic properties, which are based on a molecular dynamics structural ensemble, reveal that the difference in the CT rate constant results from the difference in the extent of backbone fluctuations of aeg- and γ-PNA. In particular, fluctuations of the backbone affect the local electric field that broadens the energy levels of the PNA nucleobases. The greater flexibility of the aeg-PNA gives rise to more broadening, and a more frequent appearance of high-CT rate conformations than in γ-PNA.

Interfacial hydration, dynamics and electron transfer: multi-scale ET modeling of the transient [myoglobin, cytochrome b5] complex.

Formation of a transient [myoglobin (Mb), cytochrome b(5) (cyt b(5))] complex is required for the reductive repair of inactive ferri-Mb to its functional ferro-Mb state. The [Mb, cyt b(5)] complex exhibits dynamic docking (DD), with its cyt b(5) partner in rapid exchange at multiple sites on the Mb surface. A triple mutant (Mb(3M)) was designed as part of efforts to shift the electron-transfer process to the simple docking (SD) regime, in which reactive binding occurs at a restricted, reactive region on the Mb surface that dominates the docked ensemble. An electrostatically-guided brownian dynamics (BD) docking protocol was used to generate an initial ensemble of reactive configurations of the complex between unrelaxed partners. This ensemble samples a broad and diverse array of heme-heme distances and orientations. These configurations seeded all-atom constrained molecular dynamics simulations (MD) to generate relaxed complexes for the calculation of electron tunneling matrix elements (T(DA)) through tunneling-pathway analysis. This procedure for generating an ensemble of relaxed complexes combines the ability of BD calculations to sample the large variety of available conformations and interprotein distances, with the ability of MD to generate the atomic level information, especially regarding the structure of water molecules at the protein-protein interface, that defines electron-tunneling pathways. We used the calculated T(DA) values to compute ET rates for the [Mb(wt), cyt b(5)] complex and for the complex with a mutant that has a binding free energy strengthened by three D/E → K charge-reversal mutations, [Mb(3M), cyt b(5)]. The calculated rate constants are in agreement with the measured values, and the mutant complex ensemble has many more geometries with higher T(DA) values than does the wild-type Mb complex. Interestingly, water plays a double role in this electron-transfer system, lowering the tunneling barrier as well as inducing protein interface remodeling that screens the repulsion between the negatively-charged propionates of the two hemes.

Evidence for a near-resonant charge transfer mechanism for double-stranded peptide nucleic acid.

We present evidence for a near-resonant mechanism of charge transfer in short peptide nucleic acid (PNA) duplexes obtained through electrochemical, STM break junction (STM-BJ), and computational studies. A seven base pair (7-bp) PNA duplex with the sequence (TA)(3)-(XY)-(TA)(3) was studied, in which XY is a complementary nucleobase pair. The experiments showed that the heterogeneous charge transfer rate constant (k(0)) and the single-molecule conductance (σ) correlate with the oxidation potential of the purine base in the XY base pair. The electrochemical measurements showed that the enhancement of k(0) is independent, within experimental error, of which of the two PNA strands contains the purine base of the XY base pair. 7-bp PNA duplexes with one or two GC base pairs had similar measured k(0) and conductance values. While a simple superexchange model, previously used to rationalize charge transfer in single stranded PNA (Paul et al. J. Am. Chem. Soc. 2009, 131, 6498-6507), describes some of the experimental observations, the model does not explain the absence of an enhancement in the experimental k(0) and σ upon increasing the G content in the duplexes from one to two. Moreover, the superexchange model is not consistent with other studies (Paul et al. J. Phys. Chem. B 2010, 114, 14140), that showed a hopping charge transport mechanism is likely important for PNA duplexes longer than seven base pairs. A quantitative computational analysis shows that a near-resonant charge transfer regime, wherein a mix of superexchange and hopping mechanisms are expected to coexist, can rationalize all of the experimental results.

Nucleic Acid Charge Transfer: Black, White and Gray.

Theoretical studies of charge transport in deoxyribonucleic acid (DNA) and peptide nucleic acid (PNA) indicate that structure and dynamics modulate the charge transfer rates, and that different members of a structural ensemble support different charge transport mechanisms. Here, we review the influences of nucleobase geometry, electronic structure, solvent environment, and thermal conformational fluctuations on the charge transfer mechanism. We describe an emerging framework for understanding the diversity of charge transport mechanisms seen in nucleic acids.

Deriving Immune-Modulating Peptides from Viral Serine Protease Inhibitors (Serpins).

Viruses have devised highly effective approaches that modulate the host immune response, blocking immune responses that are designed to eradicate viral infections. Over millions of years of evolution, virus-derived immune-modulating proteins have become extraordinarily potent, in some cases working at picomolar concentrations when expressed into surrounding tissues and effectively blocking host defenses against viral invasion and replication. The marked efficiency of these immune-modulating proteins is postulated to be due to viral engineering of host immune modulators as well as design and development of new strategies (i.e., some derived from host proteins and some entirely unique). Two key characteristics of viral immune modulators confer both adaptive advantages and desirable functions for therapeutic translation. First, many virus-derived immune modulators have evolved structures that are not readily recognized or regulated by mammalian immune pathways, ensuring little to no neutralizing antibody responses or proteasome-mediated degradation. Second, these immune modulators tend to target early steps in central immune responses, producing a powerful downstream inhibitory "domino effect" which may alter cell activation and gene expression.We have proposed that peptide metabolites of these immune-modulating proteins can enhance and extend protein function. Active immunomodulating peptides have been derived from both mammalian and viral proteins. We previously demonstrated that peptides derived from computationally predicted cleavage sites in the reactive center loop (RCL) of a viral serine proteinase inhibitor (serpin ) from myxoma virus, Serp-1 , can modify immune response activation. We have also demonstrated modulation of host gut microbiota produced by Serp-1 and RCL-derived peptide , S7, in a vascular inflammation model. Of interest, generation of derived peptides that maintain therapeutic function from a serpin can act by a different mechanism. Whereas Serp-1 has canonical serpin-like function to inhibit serine proteases, S7 instead targets mammalian serpins. Here we describe the derivation of active Serp- RCL peptides and their testing in inflammatory vasculitis models.

Revealing noncovalent interactions.

Molecular structure does not easily identify the intricate noncovalent interactions that govern many areas of biology and chemistry, including design of new materials and drugs. We develop an approach to detect noncovalent interactions in real space, based on the electron density and its derivatives. Our approach reveals the underlying chemistry that compliments the covalent structure. It provides a rich representation of van der Waals interactions, hydrogen bonds, and steric repulsion in small molecules, molecular complexes, and solids. Most importantly, the method, requiring only knowledge of the atomic coordinates, is efficient and applicable to large systems, such as proteins or DNA. Across these applications, a view of nonbonded interactions emerges as continuous surfaces rather than close contacts between atom pairs, offering rich insight into the design of new and improved ligands.

Optimizing single-molecule conductivity of conjugated organic oligomers with carbodithioate linkers.

In molecular electronics, the linker group, which attaches the functional molecular core to the electrode, plays a crucial role in determining the overall conductivity of the molecular junction. While much focus has been placed on optimizing molecular core conductivity, there have been relatively few attempts at designing optimal linker groups to metallic or semiconducting electrodes. The vast majority of molecular electronic studies use thiol linker groups; work probing alternative amine linker systems has only recently been explored. Here, we probe single-molecule conductances in phenylene-ethynylene molecules terminated with thiol and carbodithioate linkers, experimentally using STM break-junction methods and theoretically using a nonequilibrium Green's function approach. Experimental studies demonstrate that the carbodithioate linker augments electronic coupling to the metal electrode and lowers the effective barrier for charge transport relative to the conventional thiol linker, thus enhancing the conductance of the linker-phenylene-ethynylene-linker unit; these data underscore that phenylene-ethynylene-based structures are more highly conductive than originally appreciated in molecular electronics applications. The theoretical analysis shows that the nature of sulfur hybridization in these species is responsible for the order-of-magnitude increased conductance in carbodithioate-terminated systems relative to identical conjugated structures that feature classic thiol linkers, independent of the mechanism of charge transport. Interestingly, in these systems, the tunneling current is not dominated by the frontier molecular orbitals. While barriers >k(B)T to produce the low beta values seen in our experiments. Taken together, these experimental and theoretical studies indicate a promising role for carbodithioate-based connectivity in molecular-scale electronics applications involving metallic and semiconducting electrodes.

Steering electrons on moving pathways.

Electron transfer (ET) reactions provide a nexus among chemistry, biochemistry, and physics. These reactions underpin the "power plants" and "power grids" of bioenergetics, and they challenge us to understand how evolution manipulates structure to control ET kinetics. Ball-and-stick models for the machinery of electron transfer, however, fail to capture the rich electronic and nuclear dynamics of ET molecules: these static representations disguise, for example, the range of thermally accessible molecular conformations. The influence of structural fluctuations on electron-transfer kinetics is amplified by the exponential decay of electron tunneling probabilities with distance, as well as the delicate interference among coupling pathways. Fluctuations in the surrounding medium can also switch transport between coherent and incoherent ET mechanisms--and may gate ET so that its kinetics is limited by conformational interconversion times, rather than by the intrinsic ET time scale. Moreover, preparation of a charge-polarized donor state or of a donor state with linear or angular momentum can have profound dynamical and kinetic consequences. In this Account, we establish a vocabulary to describe how the conformational ensemble and the prepared donor state influence ET kinetics in macromolecules. This framework is helping to unravel the richness of functional biological ET pathways, which have evolved within fluctuating macromolecular structures. The conceptual framework for describing nonadiabatic ET seems disarmingly simple: compute the ensemble-averaged (mean-squared) donor-acceptor (DA) tunneling interaction, , and the Franck-Condon weighted density of states, rho(FC), to describe the rate, (2pi/variant Planck's over 2pi)rho(FC). Modern descriptions of the thermally averaged electronic coupling and of the Franck-Condon factor establish a useful predictive framework in biology, chemistry, and nanoscience. Describing the influence of geometric and energetic fluctuations on ET allows us to address a rich array of mechanistic and kinetic puzzles. How strongly is a protein's fold imprinted on the ET kinetics, and might thermal fluctuations "wash out" signatures of structure? What is the influence of thermal fluctuations on ET kinetics beyond averaging of the tunneling barrier structure? Do electronic coupling mechanisms change as donor and acceptor reposition in a protein, and what are the consequences for the ET kinetics? Do fluctuations access minority species that dominate tunneling? Can energy exchanges between the electron and bridge vibrations generate vibronic signatures that label some of the D-to-A pathways traversed by the electron, thus eliminating unmarked pathways that would otherwise contribute to the DA coupling (as in other "which way" or double-slit experiments)? Might medium fluctuations drive tunneling-hopping mechanistic transitions? How does the donor-state preparation, in particular, its polarization toward the acceptor and its momentum characteristics (which may introduce complex rather than pure real relationships among donor orbital amplitudes), influence the electronic dynamics? In this Account, we describe our recent studies that address puzzling questions of how conformational distributions, excited-state polarization, and electronic and nuclear dynamical effects influence ET in macromolecules. Indeed, conformational and dynamical effects arise in all transport regimes, including the tunneling, resonant transport, and hopping regimes. Importantly, these effects can induce switching among ET mechanisms.

Immune protection is dependent on the gut microbiome in a lethal mouse gammaherpesviral infection.

Immunopathogenesis in systemic viral infections can induce a septic state with leaky capillary syndrome, disseminated coagulopathy, and high mortality with limited treatment options. Murine gammaherpesvirus-68 (MHV-68) intraperitoneal infection is a gammaherpesvirus model for producing severe vasculitis, colitis and lethal hemorrhagic pneumonia in interferon gamma receptor-deficient (IFNγR-/-) mice. In prior work, treatment with myxomavirus-derived Serp-1 or a derivative peptide S-7 (G305TTASSDTAITLIPR319) induced immune protection, reduced disease severity and improved survival after MHV-68 infection. Here, we investigate the gut bacterial microbiome in MHV-68 infection. Antibiotic suppression markedly accelerated MHV-68 pathology causing pulmonary consolidation and hemorrhage, increased mortality and specific modification of gut microbiota. Serp-1 and S-7 reduced pulmonary pathology and detectable MHV-68 with increased CD3 and CD8 cells. Treatment efficacy was lost after antibiotic treatments with associated specific changes in the gut bacterial microbiota. In summary, transkingdom host-virus-microbiome interactions in gammaherpesvirus infection influences gammaherpesviral infection severity and reduces immune modulating therapeutic efficacy.

Role of nucleobase energetics and nucleobase interactions in single-stranded peptide nucleic acid charge transfer.

Self-assembled monolayers of single-stranded (ss) peptide nucleic acids (PNAs) containing seven nucleotides (TTTXTTT), a C-terminus cysteine, and an N-terminus ferrocene redox group were formed on gold electrodes. The PNA monomer group (X) was selected to be either cytosine (C), thymine (T), adenine (A), guanine (G), or a methyl group (Bk). The charge transfer rate through the oligonucleotides was found to correlate with the oxidation potential of X. Kinetic measurements and computational studies of the ss-PNA fragments show that a nucleobase mediated charge transport mechanism in the deep tunneling superexchange regime can explain the observed dependence of the kinetics of charge transfer on the PNA sequence. Theoretical analysis suggests that the charge transport is dominantly hole-mediated and takes place through the filled bridge orbitals. The strongest contribution to conductance comes from the highest filled orbitals (HOMO, HOMO-1, and HOMO-2) of individual bases, with a rapid drop off in contributions from lower lying filled orbitals. Our studies further suggest that the linear correlation observed between the experimental charge transfer rates and the oxidation potential of base X arises from weak average interbase couplings and similar stacking geometries for the four TTTXTTT systems.

Phospholipid-Based Prodrugs for Colon-Targeted Drug Delivery: Experimental Study and In-Silico Simulations.

In ulcerative colitis (UC), the inflammation is localized in the colon, and one of the successful strategies for colon-targeting drug delivery is the prodrug approach. In this work, we present a novel phospholipid (PL)-based prodrug approach, as a tool for colonic drug targeting in UC. We aim to use the phospholipase A2 (PLA2), an enzyme that is overexpressed in the inflamed colonic tissues of UC patients, as the PL-prodrug activating enzyme, to accomplish the liberation of the parent drug from the prodrug complex at the specific diseased tissue(s). Different linker lengths between the PL and the drug moiety can dictate the rate of activation by PLA2, and subsequently determine the amount of free drugs at the site of action. The feasibility of this approach was studied with newly synthesized PL-Fmoc (fluorenylmethyloxycarbonyl) conjugates, using Fmoc as a model compound for testing our hypothesis. In vitro incubation with bee venom PLA2 demonstrated that a 7-carbon linker between the PL and Fmoc has higher activation rate than a 5-carbon linker. 4-fold higher colonic expression of PLA2 was demonstrated in colonic mucosa of colitis-induced rats when compared to healthy animals, validating our hypothesis of a colitis-targeting prodrug approach. Next, a novel molecular dynamics (MD) simulation was developed for PL-based prodrugs containing clinically relevant drugs. PL-methotrexate conjugate with 6-carbon linker showed the highest extent of PLA2-mediated activation, whereas shorter linkers were activated to a lower extent. In conclusion, this work demonstrates that for carefully designed PL-drug conjugates, PLA2 overexpression in inflamed colonic tissues can be used as prodrug-activating enzyme and drug targeting strategy, including insights into the activation mechanisms in a PLA2 binding site.