Simulating asymmetric membranes using P21 periodic boundary conditions.

Molecular dynamics (MD) simulations of symmetric lipid bilayers are now well established, while those of asymmetric ones are considerably less developed. This disjunction arises in part because the surface tensions of leaflets in asymmetric bilayers can differ (unlike those of symmetric ones), and there is no simple way to determine them without assumptions. This chapter describes the use of P21 periodic boundary conditions (PBC), which allow lipids to switch leaflets, to generate asymmetric bilayers under the assumption of equal chemical potentials of lipids in opposing leaflets. A series of examples, ranging from bilayers with one lipid type to those with peptides and proteins, provides a guide for the use of P21 PBC. Critical properties of asymmetric membranes, such as spontaneous curvature, are highly sensitive to differences in the leaflet surface tensions (or differential stress), and equilibration with P21 PBC substantially reduces differential stress of asymmetric bilayers assembled with surface area-based methods. Limitations of the method are discussed. Technically, the nonstandard unit cell is difficult to parallelize and to incorporate restraints. Inherently, the assumption of equal chemical potentials, and therefore the method itself, is not applicable to all target systems. Despite these limitations, it is argued that P21 simulations should be considered when designing equilibration protocols for MD studies of most asymmetric membranes.

Spontaneous curvature generation by peptides in asymmetric bilayers.

Peptides and proteins play crucial roles in membrane remodeling by inducing spontaneous curvature. However, extracting spontaneous curvatures from simulations of asymmetric bilayers is challenging because differential stress (i.e., the difference of the leaflet surface tensions) arising from leaflet area strains can vary substantially among initial conditions. This study investigates peptide-induced spontaneous curvature δc 0 p in asymmetric bilayers consisting of a single lipid type and a peptide confined to one leaflet; δc 0 p is calculated from the Helfrich equation using the first moment of the lateral pressure tensor and an alternative expression using the differential stress. It is shown that differential stress introduced during initial system generation is effectively relaxed by equilibrating using P21 periodic boundary conditions, which allows lipids to switch leaflets across cell boundaries and equalize their chemical potentials across leaflets. This procedure leads to robust estimates of δc 0 p for the systems simulated, and is recommended when equality of chemical potentials between the leaflets is a primary consideration.

Optimization of Orally Bioavailable Antileishmanial 2,4,5-Trisubstituted Benzamides.

Leishmaniasis, a neglected tropical disease caused by Leishmania species parasites, annually affects over 1 million individuals worldwide. Treatment options for leishmaniasis are limited due to high cost, severe adverse effects, poor efficacy, difficulty of use, and emerging drug resistance to all approved therapies. We discovered 2,4,5-trisubstituted benzamides (4) that possess potent antileishmanial activity but poor aqueous solubility. Herein, we disclose our optimization of the physicochemical and metabolic properties of 2,4,5-trisubstituted benzamide that retains potency. Extensive structure-activity and structure-property relationship studies allowed selection of early leads with suitable potency, microsomal stability, and improved solubility for progression. Early lead 79 exhibited an 80% oral bioavailability and potently blocked proliferation of Leishmania in murine models. These benzamide early leads are suitable for development as orally available antileishmanial drugs.

Initiation and evolution of pores formed by influenza fusion peptides probed by lysolipid inclusion.

The fusion peptide (FP) domain is necessary for the fusogenic activity of spike proteins in a variety of enveloped viruses, allowing the virus to infect the host cell, and is the only part of the protein that interacts directly with the target membrane lipid tails during fusion. There are consistent findings of poration by this domain in experimental model membrane systems, and, in certain conditions, the isolated FPs can generate pores. Here, we use molecular dynamics simulations to investigate the specifics of how these FP-induced pores form in membranes with different compositions of lysolipid and POPC. The simulations show that pores form spontaneously at high lysolipid concentrations via hybrid intermediates, where FP aggregates in the cis leaflet tilt to form a funnel-like structure that spans the leaflet and locally reduces the hydrophobic thickness that must be traversed by water to form a pore. By restraining a single FP within an FP aggregate to this tilted conformation, pores can be formed in lower-lysolipid-content membranes, including pure POPC, on the 100-ns timescale, much more rapidly than in unbiased simulations in bilayers with the same composition. The pore formation pathway is similar to the spontaneous formation in high lysolipid concentrations. Depending on the membrane composition, the pores can be metastable (as seen in POPC) or lead to membrane rupture.

Planar aggregation of the influenza viral fusion peptide alters membrane structure and hydration, promoting poration.

To infect, enveloped viruses employ spike protein, spearheaded by its amphipathic fusion peptide (FP), that upon activation extends out from the viral surface to embed into the target cellular membrane. Here we report that synthesized influenza virus FPs are membrane active, generating pores in giant unilamellar vesicles (GUV), and thus potentially explain both influenza virus' hemolytic activity and the liposome poration seen in cryo-electron tomography. Experimentally, FPs are heterogeneously distributed on the GUV at the time of poration. Consistent with this heterogeneous distribution, molecular dynamics (MD) simulations of asymmetric bilayers with different numbers of FPs in one leaflet show FP aggregation. At the center of FP aggregates, a profound change in the membrane structure results in thinning, higher water permeability, and curvature. Ultimately, a hybrid bilayer nanodomain forms with one lipidic leaflet and one peptidic leaflet. Membrane elastic theory predicts a reduced barrier to water pore formation when even a dimer of FPs thins the membrane as above, and the FPs of that dimer tilt, to continue the leaflet bending initiated by the hydrophobic mismatch between the FP dimer and the surrounding lipid.

Identification of Small Molecule Inhibitors against Staphylococcus aureus Dihydroorotase via HTS.

Drug-resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)-an integral step in the de novo pyrimidine biosynthesis pathway. This study sets forth a high-throughput screening (HTS) of 3000 fragment compounds by a colorimetry-based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca-asp determined the best compound to be a competitive inhibitor with a KD value of 11 µM, which is 10-fold tighter than Ca-asp. Preliminary structure-activity relationship (SAR) provides the foundation for further structure-based antimicrobial inhibitor design against S. aureus.

Amino-Substituted 3-Aryl- and 3-Heteroarylquinolines as Potential Antileishmanial Agents.

Leishmaniasis, a disease caused by protozoa of the Leishmania species, afflicts roughly 12 million individuals worldwide. Most existing drugs for leishmaniasis are toxic, expensive, difficult to administer, and subject to drug resistance. We report a new class of antileishmanial leads, the 3-arylquinolines, that potently block proliferation of the intramacrophage amastigote form of Leishmania parasites with good selectivity relative to the host macrophages. Early lead 34 was rapidly acting and possessed good potency against L. mexicana (EC50 = 120 nM), 30-fold selectivity for the parasite relative to the macrophage (EC50 = 3.7 µM), and also blocked proliferation of Leishmania donovani parasites resistant to antimonial drugs. Finally, another early lead, 27, which exhibited reasonable in vivo tolerability, impaired disease progression during the dosing period in a murine model of cutaneous leishmaniasis. These results suggest that the arylquinolines provide a fruitful departure point for the development of new antileishmanial drugs.

Antimalarial activity of 2,6-dibenzylidenecyclohexanone derivatives.

Malaria remains one of the deadliest infectious diseases worldwide and continues to infect hundreds of millions of individuals each year. Here we report the discovery and derivatization of a series of 2,6-dibenzylidenecyclohexanones targeting the chloroquine-sensitive 3D7 strain of Plasmodium falciparum . While the initial lead compound displayed significant toxicity in a human cell proliferation assay, we were able to identify a derivative with no detectable toxicity and sub-micromolar potency.

Improvement of Oral Bioavailability of Pyrazolo-Pyridone Inhibitors of the Interaction of DCN1/2 and UBE2M.

The cullin-RING ubiquitin ligases (CRLs) are ubiquitin E3 enzymes that play a key role in controlling proteasomal degradation and are activated by neddylation. We previously reported inhibitors that target CRL activation by disrupting the interaction of defective in cullin neddylation 1 (DCN1), a CRL neddylation co-E3, and UBE2M, a neddylation E2. Our first-generation inhibitors possessed poor oral bioavailability and fairly rapid clearance that hindered the study of acute inhibition of DCN-controlled CRL activity in vivo. Herein, we report studies to improve the pharmacokinetic performance of the pyrazolo-pyridone inhibitors. The current best inhibitor, 40, inhibits the interaction of DCN1 and UBE2M, blocks NEDD8 transfer in biochemical assays, thermally stabilizes cellular DCN1, and inhibits anchorage-independent growth in a DCN1 amplified squamous cell carcinoma cell line. Additionally, we demonstrate that a single oral 50 mg/kg dose sustains plasma exposures above the biochemical IC90 for 24 h in mice.

Identification of Plasmodium falciparum heat shock 90 inhibitors via molecular docking.

A virtual screen was performed to identify anti-malarial compounds targeting Plasmodium falciparum heat shock 90 protein by applying a series of drug-like and commercial availability filters to compounds in the ZINC database, resulting in a virtual library of more than 13 million candidates. The goal of the virtual screen was to identify novel compounds which could serve as a starting point for the development of antimalarials with a mode of action different from anything currently used in the clinic. The screen targeted the ATP binding pocket of the highly conserved Plasmodium heat shock 90 protein, as this protein is critical to the survival of the parasite and has several significant structural differences from the human homolog. The top twelve compounds from the virtual screen were tested in vitro, with all twelve showing no antiproliferative activity against the human fibroblast cell line and three compounds exhibiting single digit or better micromolar antiproliferative activity against the chloroquine-sensitive P. falciparum 3D7 strain.

Evaluation of 1,1-cyclopropylidene as a thioether isostere in the 4-thio-thienopyrimidine (TTP) series of antimalarials.

The 4-(heteroarylthio)thieno[2,3-d]pyrimidine (TTP) series of antimalarials, represented by 1 and 17, potently inhibit proliferation of the 3D7 strain of P. falciparum (EC50 70-100 nM), but suffer from oxidative metabolism. The 1,1-cyclopropylidene isosteres 6 and 16 were designed to obviate this drawback. They were prepared by a short route that features a combined Peterson methylenation / cyclopropanation transformation of, e. g., ketone 7. Isosteres 6 and 16 possess significantly attenuated antimalarial potency relative to parents 1 and 17. This outcome can be rationalized based on the increased out-of-plane steric demands of the latter two. In support of this hypothesis, the relatively flat ketone 7 retains some of the potency of 1, even though it appears to be a comparatively inferior mimic with respect to electronics and bond lengths and angles. We also demonstrate crystallographically and computationally an apparent increase in the strength of the intramolecular sulfur hole interaction of 1 upon protonation.

Surveillance of spotted fever rickettsioses at Army installations in the U.S. Central and Atlantic regions, 2012-2018.

Spotted fever rickettsioses (SFR) are emerging in the Atlantic and Central regions of the U.S., though cases have been reported across the contiguous U.S. Military populations may be at increased risk for SFR because of residence in these regions and frequent field training in tick habitats. Surveillance for Rocky Mountain spotted fever in the Army began in 1998 and was expanded to include all SFR in 2017. Between 2016 and 2017, the rate of active component cases reported from Army installations in the Atlantic and Central regions of the U.S. increased nearly five-fold from 2016 (0.55 per 100,000 person-years [p-yrs]) to 2017 (2.65 per 100,000 p-yrs). The majority of SFR cases were reported from Fort Leonard Wood, MO, and Fort Bragg, NC. Most reported cases had no documented symptoms consistent with SFR and could not be confirmed as "cases" by standard case-defining methods. SFR surveillance and control efforts in military populations can be improved by better adherence to guidelines for SFR diagnosis and through the use of available advanced laboratory techniques.

Synthesis and Structure-Activity Relationship of Dual-Stage Antimalarial Pyrazolo[3,4-b]pyridines.

Malaria remains one of the most deadly infectious diseases, causing hundreds of thousands of deaths each year, primarily in young children and pregnant mothers. Here, we report the discovery and derivatization of a series of pyrazolo[3,4-b]pyridines targeting Plasmodium falciparum, the deadliest species of the malaria parasite. Hit compounds in this series display sub-micromolar in vitro activity against the intraerythrocytic stage of the parasite as well as little to no toxicity against the human fibroblast BJ and liver HepG2 cell lines. In addition, our hit compounds show good activity against the liver stage of the parasite but little activity against the gametocyte stage. Parasitological profiles, including rate of killing, docking, and molecular dynamics studies, suggest that our compounds may target the Qo binding site of cytochrome bc1.

Antimalarial activity of tetrahydro-ß-carbolines targeting the ATP binding pocket of the Plasmodium falciparum heat shock 90 protein.

A series of tetrahydro-ß-carboline derivatives of a lead compound known to target the heat shock 90 protein of Plasmodium falciparum were synthesized and assayed for both potency against the parasite and toxicity against a human cell line. Using a rationalized structure based design strategy, a new lead compound with a potency two orders of magnitude greater than the original lead compound was found. Additional modeling of this new lead compound suggests multiple avenues to further increase potency against this target, potentially paving the path for a therapeutic with a mode of action different than any current clinical treatment.

Lipopolysaccharide Simulations Are Sensitive to Phosphate Charge and Ion Parameterization.

The high proportion of lipopolysaccharide (LPS) molecules in the outer membrane of Gram-negative bacteria makes it a highly effective barrier to small molecules, antibiotic drugs, and other antimicrobial agents. Given this vital role in protecting bacteria from potentially hostile environments, simulations of LPS bilayers and outer membrane systems represent a critical tool for understanding the mechanisms of bacterial resistance and the development of new antibiotic compounds that circumvent these defenses. The basis of these simulations is parameterizations of LPS, which have been developed for all major molecular dynamics force fields. However, these parameterizations differ in both the protonation state of LPS and how LPS membranes behave in the presence of various ion species. To address these discrepancies and understand the effects of phosphate charge on bilayer properties, simulations were performed for multiple distinct LPS chemotypes with different ion parameterizations in both protonated or deprotonated lipid A states. These simulations show that bilayer properties, such as the area per lipid and inter-lipid hydrogen bonding, are highly influenced by the choice of phosphate group charges, cation type, and ion parameterization, with protonated LPS and monovalent cations with modified nonbonded parameters providing the best match to the experiments. Additionally, alchemical free energy simulations were performed to determine theoretical pKa values for LPS and subsequently validated by 31P solid-state nuclear magnetic resonance experiments. Results from these complementary computational and experimental studies demonstrate that the protonated state dominates at physiological pH, contrary to the deprotonated form modeled by many LPS force fields. Overall, these results highlight the sensitivity of LPS simulations to phosphate charge and ion parameters while offering recommendations for how existing models should be updated for consistency between force fields as well as to best match experiments.

Gastric emptying of milk in infants and children up to 5 years of age: normative data and influencing factors.

BACKGROUND: Gastric emptying scintigraphy is widely used in infants and children, but there is a lack of age-specific normative data. OBJECTIVE: The objectives of this retrospective study were: 1) to establish a range of gastric emptying of milk or formula as a surrogate for normal gastric emptying in infants and young children ≤5 years of age, and 2) to investigate the effects of patient age, feeding volume, feeding route and gastroesophageal reflux on gastric emptying. MATERIALS AND METHODS: The reports of 5,136 gastric emptying studies of children ≤5 years of age performed at Children's National Medical Center from January 1990 to August 2012 were reviewed. Demographic data, 1-h and 3-h gastric emptying values and gastroesophageal reflux status of all patients were stored in a database. Using stringent inclusion and exclusion criteria, the studies of patients as similar to healthy children as possible were selected for this study. RESULTS: The study group included 2,273 children (57% male) ages 0-59 months (median: 4.6 months). The median 1-h gastric emptying was 43% (interquartile range [IQR] 34-54%). The median 3-h gastric emptying was 91% (IQR 79-98%). Sixty-one percent of patients with 1-h gastric emptying value of <50% had 3-h gastric emptying ≥80%. Gastric emptying was significantly faster in children ≤6 months as compared with all older age groups. In each age group, the median gastric emptying decreased with increasing feeding volume. Gastric emptying was significantly faster in patients fed via combined nasogastric tube and oral routes as compared with those fed exclusively orally. There was no significant difference in gastric emptying of children with and without gastroesophageal reflux. CONCLUSION: Although there are statistically significant differences in gastric emptying based on age, volume and route of feeding, the data suggest that overall normal liquid gastric emptying in infants and children ≤5 years of age is ≥80% at 3 h. One-hour emptying measurements are not reliable for detecting delayed gastric emptying.

Salmonella Membrane Structural Remodeling Increases Resistance to Antimicrobial Peptide LL-37.

Gram-negative bacteria are protected from their environment by an outer membrane that is primarily composed of lipopolysaccharides (LPSs). Under stress, pathogenic serotypes of Salmonella enterica remodel their LPSs through the PhoPQ two-component regulatory system that increases resistance to both conventional antibiotics and antimicrobial peptides (AMPs). Acquired resistance to AMPs is contrary to the established narrative that AMPs circumvent bacterial resistance by targeting the general chemical properties of membrane lipids. However, the specific mechanisms underlying AMP resistance remain elusive. Here we report a 2-fold increase in bacteriostatic concentrations of human AMP LL-37 for S. enterica with modified LPSs. LPSs with and without chemical modifications were isolated and investigated by Langmuir films coupled with grazing-incidence X-ray diffraction (GIXD) and specular X-ray reflectivity (XR). The initial interactions between LL-37 and LPS bilayers were probed using all-atom molecular dynamics simulations. These simulations suggest that initial association is nonspecific to the type of LPS and governed by hydrogen bonding to the LPS outer carbohydrates. GIXD experiments indicate that the interactions of the peptide with monolayers reduce the number of crystalline domains but greatly increase the typical domain size in both LPS isoforms. Electron densities derived from XR experiments corroborate the bacteriostatic values found in vitro and indicate that peptide intercalation is reduced by LPS modification. We hypothesize that defects at the liquid-ordered boundary facilitate LL-37 intercalation into the outer membrane, whereas PhoPQ-mediated LPS modification protects against this process by having innately increased crystallinity. Since induced ordering has been observed with other AMPs and drugs, LPS modification may represent a general mechanism by which Gram-negative bacteria protect against host innate immunity.

Identification and design of novel small molecule inhibitors against MERS-CoV papain-like protease via high-throughput screening and molecular modeling.

The development of new therapeutic agents against the coronavirus causing Middle East Respiratory Syndrome (MERS) is a continuing imperative. The initial MERS-CoV epidemic was contained entirely through public health measures, but episodic cases continue, as there are currently no therapeutic agents effective in the treatment of MERS-CoV, although multiple strategies have been proposed. In this study, we screened 30,000 compounds from three different compound libraries against one of the essential proteases, the papain-like protease (PLpro), using a fluorescence-based enzymatic assay followed by surface plasmon resonance (SPR) direct binding analysis for hit confirmation. Mode of inhibition assays and competition SPR studies revealed two compounds to be competitive inhibitors. To improve upon the inhibitory activity of the best hit compounds, a small fragment library consisting of 352 fragments was screened in the presence of each hit compound, resulting in one fragment that enhanced the IC50 value of the best hit compound by 3-fold. Molecular docking and MM/PBSA binding energy calculations were used to predict potential binding sites, providing insight for design and synthesis of next-generation compounds.