Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

Ticks Resist Skin Commensals with Immune Factor of Bacterial Origin.

Ticks transmit a diverse array of microbes to vertebrate hosts, including human pathogens, which has led to a human-centric focus in this vector system. Far less is known about pathogens of ticks themselves. Here, we discover that a toxin in blacklegged ticks (Ixodes scapularis) horizontally acquired from bacteria-called domesticated amidase effector 2 (dae2)-has evolved to kill mammalian skin microbes with remarkable efficiency. Secreted into the saliva and gut of ticks, Dae2 limits skin-associated staphylococci in ticks while feeding. In contrast, Dae2 has no intrinsic ability to kill Borrelia burgdorferi, the tick-borne Lyme disease bacterial pathogen. These findings suggest ticks resist their own pathogens while tolerating symbionts. Thus, just as tick symbionts can be pathogenic to humans, mammalian commensals can be harmful to ticks. Our study underscores how virulence is context-dependent and bolsters the idea that "pathogen" is a status and not an identity.

Structure-Activity Relationship and Molecular Mechanics Reveal the Importance of Ring Entropy in the Biosynthesis and Activity of a Natural Product.

Macrocycles are appealing drug candidates due to their high affinity, specificity, and favorable pharmacological properties. In this study, we explored the effects of chemical modifications to a natural product macrocycle upon its activity, 3D geometry, and conformational entropy. We chose thiocillin as a model system, a thiopeptide in the ribosomally encoded family of natural products that exhibits potent antimicrobial effects against Gram-positive bacteria. Since thiocillin is derived from a genetically encoded peptide scaffold, site-directed mutagenesis allows for rapid generation of analogues. To understand thiocillin's structure-activity relationship, we generated a site-saturation mutagenesis library covering each position along thiocillin's macrocyclic ring. We report the identification of eight unique compounds more potent than wild-type thiocillin, the best having an 8-fold improvement in potency. Computational modeling of thiocillin's macrocyclic structure revealed a striking requirement for a low-entropy macrocycle for activity. The populated ensembles of the active mutants showed a rigid structure with few adoptable conformations while inactive mutants showed a more flexible macrocycle which is unfavorable for binding. This finding highlights the importance of macrocyclization in combination with rigidifying post-translational modifications to achieve high-potency binding.

Asymmetric Hydrogen Bonding Catalysis for the Synthesis of Dihydroquinazoline-Containing Antiviral, Letermovir.

A weak Brønsted acid-catalyzed asymmetric guanidine aza-conjugate addition reaction has been developed. C2-symmetric, dual hydrogen-bond donating bistriflamides are shown to be highly effective in activating α,ß-unsaturated esters toward the intramolecular addition of a pendant guanidinyl nucleophile. Preliminary mechanistic investigation, including density functional theory calculations and kinetics studies, support a conjugate addition pathway as more favorable energetically than an alternative electrocyclization pathway. This methodology has been successfully applied to the synthesis of the 3,4-dihydroquinazoline-containing antiviral, Letermovir, and a series of analogues.

Unusual (+/-)-electrospray ionization induced fragmentation: Structural elucidation of an in-process synthetic intermediate of doravirine (MK-1439) using liquid chromatography/high-resolution tandem mass spectrometry and two-dimensional nuclear magnetic resonance.

RATIONALE: During the development of a novel synthetic route to doravirine (1), a human immunodeficiency type 1 virus (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI), an unanticipated reaction intermediate, methyl (Z)-2-(3-chloro-5-cyanophenoxy)-5-(3-(3-chloro-5-cyanophenoxy)-2-oxo-4-(trifluoromethyl)pyridin-1(2H)-yl)-5-ethoxy-3-(trifluoromethyl)pent-2-enoate (2), was isolated. Moreover, an unusual electrospray ionization (ESI)-induced fragmentation was observed for 2. Hence, efforts were made towards the understanding of the structure of 2, which was crucial for the understanding of the reaction mechanism. METHODS: The isolated impurity was fully characterized by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC/HRMS/MS), hydrogen/deuterium (H/D) exchange, and an ensemble of two-dimensional nuclear magnetic resonance (2D-NMR) techniques. Density functional theory (DFT) calculations were also conducted. RESULTS: An unusual ESI-induced fragmentation was observed for intermediate 2, giving an ion for half of the molecule in the positive ion mode, with the other half of the molecule affording an ion in the negative ion mode. CONCLUSIONS: To the best of our knowledge, this unique ESI-induced fragmentation has not been previously reported in the literature. The underlying mechanism was explored and is supported by DFT calculations, which could greatly help the structural characterization of unknown impurities with similar structural features using ESI-MS in the future. Copyright © 2017 John Wiley & Sons, Ltd.

Identifying binding hot spots on protein surfaces by mixed-solvent molecular dynamics: HIV-1 protease as a test case.

Mixed-solvent molecular dynamics (MixMD) simulations use full protein flexibility and competition between water and small organic probes to achieve accurate hot-spot mapping on protein surfaces. In this study, we improved MixMD using human immunodeficiency virus type-1 protease (HIVp) as the test case. We used three probe-water solutions (acetonitrile-water, isopropanol-water, and pyrimidine-water), first at 50% w/w concentration and later at 5% v/v. Paradoxically, better mapping was achieved by using fewer probes; 5% simulations gave a superior signal-to-noise ratio and far fewer spurious hot spots than 50% MixMD. Furthermore, very intense and well-defined probe occupancies were observed in the catalytic site and potential allosteric sites that have been confirmed experimentally. The Eye site, an allosteric site underneath the flap of HIVp, has been confirmed by the presence of a 5-nitroindole fragment in a crystal structure. MixMD also mapped two additional hot spots: the Exo site (between the Gly16-Gly17 and Cys67-Gly68 loops) and the Face site (between Glu21-Ala22 and Val84-Ile85 loops). The Exo site was observed to overlap with crystallographic additives such as acetate and dimethyl sulfoxide that are present in different crystal forms of the protein. Analysis of crystal structures of HIVp in different symmetry groups has shown that some surface sites are common interfaces for crystal contacts, which means that they are surfaces that are relatively easy to desolvate and complement with organic molecules. MixMD should identify these sites; in fact, their occupancy values help establish a solid cut-off where "druggable" sites are required to have higher occupancies than the crystal-packing faces.

Protein flexibility in docking and surface mapping.

Structure-based drug design has become an essential tool for rapid lead discovery and optimization. As available structural information has increased, researchers have become increasingly aware of the importance of protein flexibility for accurate description of the native state. Typical protein-ligand docking efforts still rely on a single rigid receptor, which is an incomplete representation of potential binding conformations of the protein. These rigid docking efforts typically show the best performance rates between 50 and 75%, while fully flexible docking methods can enhance pose prediction up to 80-95%. This review examines the current toolbox for flexible protein-ligand docking and receptor surface mapping. Present limitations and possibilities for future development are discussed.

Parameter choice matters: validating probe parameters for use in mixed-solvent simulations.

Probe mapping is a common approach for identifying potential binding sites in structure-based drug design; however, it typically relies on energy minimizations of probes in the gas phase and a static protein structure. The mixed-solvent molecular dynamics (MixMD) approach was recently developed to account for full protein flexibility and solvation effects in hot-spot mapping. Our first study used only acetonitrile as a probe, and here, we have augmented the set of functional group probes through careful testing and parameter validation. A diverse range of probes are needed in order to map complex binding interactions. A small variation in probe parameters can adversely effect mixed-solvent behavior, which we highlight with isopropanol. We tested 11 solvents to identify six with appropriate behavior in TIP3P water to use as organic probes in the MixMD method. In addition to acetonitrile and isopropanol, we have identified acetone, N-methylacetamide, imidazole, and pyrimidine. These probe solvents will enable MixMD studies to recover hydrogen-bonding sites, hydrophobic pockets, protein-protein interactions, and aromatic hotspots. Also, we show that ternary-solvent systems can be incorporated within a single simulation. Importantly, these binary and ternary solvents do not require artificial repulsion terms like other methods. Within merely 5 ns, layered solvent boxes become evenly mixed for soluble probes. We used radial distribution functions to evaluate solvent behavior, determine adequate mixing, and confirm the absence of phase separation. We recommend that radial distribution functions should be used to assess adequate sampling in all mixed-solvent techniques rather than the current practice of examining the solvent ratios at the edges of the solvent box.

Discovery of DNL343: A Potent, Selective, and Brain-Penetrant eIF2B Activator Designed for the Treatment of Neurodegenerative Diseases.

Eukaryotic translation initiation factor 2B (eIF2B) is a key component of the integrated stress response (ISR), which regulates protein synthesis and stress granule formation in response to cellular insult. Modulation of the ISR has been proposed as a therapeutic strategy for treatment of neurodegenerative diseases such as vanishing white matter (VWM) disease and amyotrophic lateral sclerosis (ALS) based on its ability to improve cellular homeostasis and prevent neuronal degeneration. Herein, we report the small-molecule discovery campaign that identified potent, selective, and CNS-penetrant eIF2B activators using both structure- and ligand-based drug design. These discovery efforts culminated in the identification of DNL343, which demonstrated a desirable preclinical drug profile, including a long half-life and high oral bioavailability across preclinical species. DNL343 was progressed into clinical studies and is currently undergoing evaluation in late-stage clinical trials for ALS.

Improving protocols for protein mapping through proper comparison to crystallography data.

Computational approaches to fragment-based drug design (FBDD) can complement experiments and facilitate the identification of potential hot spots along the protein surface. However, the evaluation of computational methods for mapping binding sites frequently focuses upon the ability to reproduce crystallographic coordinates to within a low RMSD threshold. This dependency on the deposited coordinate data overlooks the original electron density from the experiment, thus techniques may be developed based upon subjective-or even erroneous-atomic coordinates. This can become a significant drawback in applications to systems where the location of hot spots is unknown. On the basis of comparison to crystallographic density, we previously showed that mixed-solvent molecular dynamics (MixMD) accurately identifies the active site for HEWL, with acetonitrile as an organic solvent. Here, we concentrated on the influence of protic solvent on simulation and refined the optimal MixMD approach for extrapolation of the method to systems without established sites. Our results establish an accurate approach for comparing simulations to experiment. We have outlined the most efficient strategy for MixMD, based on simulation length and number of runs. The development outlined here makes MixMD a robust method which should prove useful across a broad range of target structures. Lastly, our results with MixMD match experimental data so well that consistency between simulations and density may be a useful way to aid the identification of probes vs waters during the refinement of future multiple solvent crystallographic structures.

CD98hc is a target for brain delivery of biotherapeutics.

Brain exposure of systemically administered biotherapeutics is highly restricted by the blood-brain barrier (BBB). Here, we report the engineering and characterization of a BBB transport vehicle targeting the CD98 heavy chain (CD98hc or SLC3A2) of heterodimeric amino acid transporters (TVCD98hc). The pharmacokinetic and biodistribution properties of a CD98hc antibody transport vehicle (ATVCD98hc) are assessed in humanized CD98hc knock-in mice and cynomolgus monkeys. Compared to most existing BBB platforms targeting the transferrin receptor, peripherally administered ATVCD98hc demonstrates differentiated brain delivery with markedly slower and more prolonged kinetic properties. Specific biodistribution profiles within the brain parenchyma can be modulated by introducing Fc mutations on ATVCD98hc that impact FcγR engagement, changing the valency of CD98hc binding, and by altering the extent of target engagement with Fabs. Our study establishes TVCD98hc as a modular brain delivery platform with favorable kinetic, biodistribution, and safety properties distinct from previously reported BBB platforms.

Discovery of Potent and Selective Dual Leucine Zipper Kinase/Leucine Zipper-Bearing Kinase Inhibitors with Neuroprotective Properties in In Vitro and In Vivo Models of Amyotrophic Lateral Sclerosis.

Dual leucine zipper kinase (DLK) and leucine zipper-bearing kinase (LZK) are regulators of neuronal degeneration and axon growth. Therefore, there is a considerable interest in developing DLK/LZK inhibitors for neurodegenerative diseases. Herein, we use ligand- and structure-based drug design approaches for identifying novel amino-pyrazine inhibitors of DLK/LZK. DN-1289 (14), a potent and selective dual DLK/LZK inhibitor, demonstrated excellent in vivo plasma half-life across species and is anticipated to freely penetrate the central nervous system with no brain impairment based on in vivo rodent pharmacokinetic studies and human in vitro transporter data. Proximal target engagement and disease relevant pathway biomarkers were also favorably regulated in an in vivo model of amyotrophic lateral sclerosis.

A chemoenzymatic strategy for site-selective functionalization of native peptides and proteins.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

Binding to the open conformation of HIV-1 protease.

A recent crystal structure of HIV-1 protease (HIVp) was the first to experimentally observe a ligand targeting an open-flap conformation. Researchers studying a symmetric pyrrolidine inhibitor found that two ligands cocrystallized with the protease, forcing an unusual configuration and unique crystallographic contacts. One molecule is centered in the traditional binding site (α pose) and the other binds between the flaps (ß pose). The ligands stack against each other in a region termed the "eye" site. Ligands bound to the eye site should prevent flap closure, but it is unclear if the pyrrolidine inhibitors or the crystal packing are causing the open state. Molecular dynamics simulations were used to examine the solution-state behavior of three possible binding modes: the ternary complex of HIVp+αß and the binary complexes, HIVp+α and HIVp+ß. We show that HIVp+α is the most stable of the three states. During conformational sampling, α takes an asymmetric binding pose, with one naphthyl ring occupying the eye site and the other reoriented down to occupy positions seen with traditional inhibitors. This finding supports previous studies that reveal a requirement for asymmetric binding at the eye site. In fact, if the α pose is modified to splay both naphthyl rings across the binding site like traditional inhibitors, one ring consistently flips to occupy the eye site. Our simulations reveal that interactions to the eye site encourage a conformationally restrained state, and understanding those contacts may aid the design of ligands to specifically target alternate conformations of the protease.

Full protein flexibility is essential for proper hot-spot mapping.

A traditional technique for structure-based drug design (SBDD) is mapping of protein surfaces with probe molecules to identify "hot spots" where key functional groups can best complement the receptor. Common methods, such as minimization of probes or calculation of grids, use a fixed protein structure in the gas phase, ignoring both protein flexibility and proper competition between the probes and water. As a result, the potential surface is quite rugged, and many spurious local minima are identified. In this work, we compared rigid and fully flexible proteins in mixed-solvent molecular dynamics, which allows for flexibility and full solvent effects. We were surprised to find that the large number of local minima are still found when a protein's conformational sampling is restricted; the dynamic averaging of probes and competition with water do not smooth the potential surface as one might expect. Only when a protein is allowed to be fully flexible in the simulation are the proper minima located and the spurious ones eliminated. Our results indicate that inclusion of full protein flexibility is critical to accurate hot-spot mapping for SBDD.

CSAR benchmark exercise of 2010: selection of the protein-ligand complexes.

A major goal in drug design is the improvement of computational methods for docking and scoring. The Community Structure Activity Resource (CSAR) aims to collect available data from industry and academia which may be used for this purpose ( www.csardock.org ). Also, CSAR is charged with organizing community-wide exercises based on the collected data. The first of these exercises was aimed to gauge the overall state of docking and scoring, using a large and diverse data set of protein-ligand complexes. Participants were asked to calculate the affinity of the complexes as provided and then recalculate with changes which may improve their specific method. This first data set was selected from existing PDB entries which had binding data (K(d) or K(i)) in Binding MOAD, augmented with entries from PDB bind. The final data set contains 343 diverse protein-ligand complexes and spans 14 pK(d). Sixteen proteins have three or more complexes in the data set, from which a user could start an inspection of congeneric series. Inherent experimental error limits the possible correlation between scores and measured affinity; Pearson R is limited to ~ 0.91 (Pearson R2 0.83) when fitting to the data set without over parameterizing. Pearson R is limited to ~ 0.83(Pearson R2 ~ 0.70) when scoring the data set with a method trained on outside data [corrected]. The details of how the data set was initially selected, and the process by which it matured to better fit the needs of the community are presented. Many groups generously participated in improving the data set, and this underscores the value of a supportive, collaborative effort in moving our field forward.

Clarifying allosteric control of flap conformations in the 1TW7 crystal structure of HIV-1 protease.

The 1TW7 crystal structure of HIV-1 protease shows the flaps placed wider and more open than what is seen in other examples of the semi-open, apo form. It has been proposed that this might be experimental evidence of allosteric control, because crystal packing creates contacts to the "elbow region" of the protease, which may cause deformation of the flaps. Recent dynamics simulations have shown that the conformation seen in 1TW7 relaxes into the typical semi-open conformation in the absence of the crystal contacts, definitively showing that the crystal contacts cause the deformation (Layten et al., J Am Chem Soc 2006;128:13360-13361). However, this does not prove or disprove allosteric modulation at the elbow. In this study, we have conducted additional simulations, supplemented with experimental testing, to further probe the possibility of 1TW7 providing an example of allosteric control of the flap region. We show that the contacts are unstable and do not restrict the conformational sampling of the flaps. The deformation seen in the 1TW7 crystal structure is simply opportunistic crystal packing and not allosteric control.

Ramachandran-type plots for glycosidic linkages: Examples from molecular dynamic simulations using the Glycam06 force field.

The goals of this article are to (1) provide further validation of the Glycam06 force field, specifically for its use in implicit solvent molecular dynamic (MD) simulations, and (2) to present the extension of G.N. Ramachandran's idea of plotting amino acid phi and psi angles to the glycosidic phi, psi, and omega angles formed between carbohydrates. As in traditional Ramachandran plots, these carbohydrate Ramachandran-type (carb-Rama) plots reveal the coupling between the glycosidic angles by displaying the allowed and disallowed conformational space. Considering two-bond glycosidic linkages, there are 18 possible conformational regions that can be defined by (alpha, phi, psi) and (beta, phi, psi), whereas for three-bond linkages, there are 54 possible regions that can be defined by (alpha, phi, psi, omega) and (beta, phi, psi, omega). Illustrating these ideas are molecular dynamic simulations on an implicitly hydrated oligosaccharide (700 ns) and its eight constituent disaccharides (50 ns/disaccharide). For each linkage, we compare and contrast the oligosaccharide and respective disaccharide carb-Rama plots, validate the simulations and the Glycam06 force field through comparison to experimental data, and discuss the general trends observed in the plots.

Combining traditional 2D and modern physical organic-derived descriptors to predict enhanced enantioselectivity for the key aza-Michael conjugate addition in the synthesis of Prevymis™ (letermovir).

Quantitative structure-activity relationships have an extensive history for optimizing drug candidates, yet they have only recently been applied in reaction development. In this report, the predictive power of multivariate parameterization has been explored toward the optimization of a catalyst promoting an aza-Michael conjugate addition for the asymmetric synthesis of letermovir. A hybrid approach combining 2D QSAR and modern 3D physical organic parameters performed better than either approach in isolation. Using these predictive models, a series of new catalysts were identified, which catalyzed the reaction to provide the desired product in improved enantioselectivity relative to the parent catalyst.