ABSTRACT
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.
Subject(s)
Amyotrophic Lateral Sclerosis , Leukoencephalopathies , Neurodegenerative Diseases , Humans , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/metabolism , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/metabolism , Mutation , Eukaryotic Initiation Factor-2B/genetics , Eukaryotic Initiation Factor-2B/metabolism , Brain/metabolism , Leukoencephalopathies/metabolismABSTRACT
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.
Subject(s)
Blood-Brain Barrier , Brain , Animals , Mice , Tissue Distribution , Antibodies , Engineering , Macaca fascicularisABSTRACT
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.
Subject(s)
Amyotrophic Lateral Sclerosis , Neurodegenerative Diseases , Humans , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/metabolism , Leucine Zippers , MAP Kinase Kinase Kinases , Central Nervous System/metabolism , Brain/metabolism , Neurodegenerative Diseases/metabolismABSTRACT
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.
Subject(s)
Insulin , Penicillin Amidase , Peptides , Protein Engineering , Amino Acid Sequence , Humans , Insulin/analogs & derivatives , Insulin/biosynthesis , Lysine/chemistry , Penicillin Amidase/chemistry , Penicillin Amidase/genetics , Peptides/chemistry , Peptides/genetics , Protein Engineering/methodsABSTRACT
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.
Subject(s)
Biological Products/therapeutic use , Brain/metabolism , Lysosomal Storage Diseases/therapy , Progranulins/therapeutic use , Animals , Bone Morphogenetic Proteins/metabolism , Endosomes/metabolism , Female , Frontotemporal Dementia/blood , Frontotemporal Dementia/cerebrospinal fluid , Gliosis/complications , Gliosis/pathology , Humans , Induced Pluripotent Stem Cells/metabolism , Inflammation/pathology , Lipid Metabolism , Lipofuscin/metabolism , Lysosomes/metabolism , Macrophages/metabolism , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/metabolism , Nerve Degeneration/pathology , Phenotype , Progranulins/deficiency , Progranulins/metabolism , Receptors, Immunologic/metabolism , Receptors, Transferrin/metabolism , Tissue DistributionABSTRACT
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.
Subject(s)
Bacteria/metabolism , Immunologic Factors/metabolism , Ixodes/physiology , Skin/microbiology , Symbiosis , Animals , Anti-Bacterial Agents/pharmacology , Biocatalysis , Cell Wall/metabolism , Feeding Behavior , Female , Gastrointestinal Tract/metabolism , Host-Pathogen Interactions , Mice , Models, Molecular , Peptidoglycan/metabolism , Phylogeny , Saliva/metabolism , Salivary Glands/metabolism , Staphylococcus epidermidis/physiology , Structural Homology, Protein , Substrate Specificity , Up-RegulationABSTRACT
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.
ABSTRACT
Directed evolution experiments designed to improve the activity of a biocatalyst have increased in sophistication from the early days of completely random mutagenesis. Sequence-based and structure-based methods have been developed to identify "hotspot" positions that when randomized provide a higher frequency of beneficial mutations that improve activity. These focused mutagenesis methods reduce library sizes and therefore reduce screening burden, accelerating the rate of finding improved enzymes. Looking for further acceleration in finding improved enzymes, we investigated whether two existing methods, one sequence-based (Protein GPS) and one structure-based (using Bioluminate and MOE), were sufficiently predictive to provide not just the hotspot position, but also the amino acid substitution that improved activity at that position. By limiting the libraries to variants that contained only specific amino acid substitutions, library sizes were kept to less than 100 variants. For an initial round of ATA-117 R-selective transaminase evolution, we found that the methods used produced libraries where 9% and 18% of the amino acid substitutions chosen were amino acids that improved reaction performance in lysates. The ability to create combinations of mutations as part of the initial design was confounded by the relatively large number of predicted mutations that were inactivating (30% and 45% for the sequence-based and structure-based methods, respectively). Despite this, combining several mutations identified within a given method produced variant lysates 7- and 9-fold more active than the wild-type lysate, highlighting the capability of mutations chosen this way to generate large advances in activity in addition to the reductions in screening.
Subject(s)
Directed Molecular Evolution , Mutagenesis/genetics , Mutation/geneticsABSTRACT
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.
Subject(s)
Acetates/chemical synthesis , Antiviral Agents/chemical synthesis , Quantum Theory , Quinazolines/chemistry , Acetates/chemistry , Acetates/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Catalysis , Cyclization , Hydrogen Bonding , Molecular Structure , Quinazolines/chemical synthesis , Quinazolines/pharmacologyABSTRACT
Significant catalyst loading reduction and increased reaction robustness have been achieved for a Pd-catalyzed asymmetric intramolecular C-N coupling through comprehensive mechanistic studies. Detailed kinetic, spectroscopic, and crystallographic analyses revealed that the mono-oxidation of the bis-phosphine ligand is critical for a successful transformation. 31P NMR studies provided an understanding of the inefficient activation of the Pd(OAc)2/(R,R)-QuinoxP* pre-catalyst to form the active bis-phosphine mono-oxide-Pd(0) catalyst with competitive formation of a less active (R,R)-QuinoxP*·PdBr2 complex. Based on these detailed mechanistic studies, a new series of bis-phosphine mono-oxides (BPMO)-ligated Pd(ii) pre-catalysts have been rationally developed that allow for reliable and complete catalyst activation which should have general utility in academic and industrial settings.
ABSTRACT
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.
Subject(s)
Biological Products , Peptides/chemistry , Biological Products/chemistry , Biological Products/pharmacology , Macrocyclic Compounds/chemistry , Molecular Conformation , Molecular Dynamics Simulation , Peptides/genetics , Peptides/pharmacology , Structure-Activity RelationshipABSTRACT
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.
ABSTRACT
An unusual in-source fragmentation pattern observed for 14 doubly quaternized cinchona alkaloid-based phase-transfer catalysts (PTC) was studied using (+)-ESI high resolution mass spectrometry. Loss of the substituted benzyl cation (R1 or R2) was found to be the major product ion [M2+ - R1+ or R2+]+ in MS spectra of all PTC compounds. A Hofmann elimination product ion [M - H]+ was also observed. Only a small amount of the doubly charged M2+ ions were observed in the MS spectra, likely due to strong Columbic repulsion between the two quaternary ammonium cations in the gas phase. The positive voltage in the MS inlet but not the ESI probe was found to induce this extensive fragmentation for all PTC diboromo-salts. Compound 1 was used as an example to illustrate the proposed in-source fragmentation mechanism. The mechanism of formation of the Hofmann elimination product ion [M - H]+ was further investigated using HRMS/MS, H/D exchange, and DFT calculations. The proposed formation of 2b as the major Hofmann elimination product ion was supported both by HRMS/MS and DFT calculations. Formation of product ion 2b through a concerted unimolecular Ei elimination pathway is proposed rather than a bimolecular E2 elimination pathway for common solution Hofmann eliminations. Graphical Abstract á .
ABSTRACT
Natural product and synthetic macrocycles are chemically and topologically diverse. An efficient, accurate, and general method for generating macrocycle conformations would enable structure-based design of macrocycle drugs or host-guest complexes. Computational sampling also provides insight into transiently populated states, complementing crystallographic and NMR data. Here, we report a new algorithm, BRIKARD, that addresses this challenge through computational algebraic geometry and inverse kinematics together with local energy minimization. BRIKARD is demonstrated on 67 diverse macrocycles with structural data, encompassing various ring topologies. We find this approach enumerates diverse structures with macrocyclic RMSD < 1.0 Å to the experimental conformation for 85% of our data set in contrast to success rates of 67-75% with other approaches, while for the subset of 21 more challenging compounds in the data set, these rates are 57% and 10-29%, respectively. Because the algorithm can be efficiently run in parallel on many processors, exhaustive conformational sampling of complex cycles can be obtained in minutes rather than hours: with a 40 processor implementation protocol, BRIKARD samples the conformational diversity of a potential energy landscape in a median of 1.3 minutes of wallclock time, much faster than 3.1-10.3 hours necessary with current programs. By rigorously testing BRIKARD on a broad range of scaffolds with highly complex ring systems, we push the frontiers of macrocycle sampling to encompass multiring compounds, including those with more than 50 ring atoms and up to seven interlaced flexible rings.
ABSTRACT
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.
Subject(s)
HIV Protease/chemistry , HIV-1/enzymology , Molecular Dynamics Simulation , 2-Propanol/chemistry , Acetonitriles/chemistry , Humans , Water/chemistryABSTRACT
OBJECTIVE: To determine the cause and course of a novel syndrome with progressive encephalopathy and brain atrophy in children. METHODS: Clinical whole-exome sequencing was performed for global developmental delay and intellectual disability; some patients also had spastic paraparesis and evidence of clinical regression. Six patients were identified with de novo missense mutations in the kinesin gene KIF1A. The predicted functional disruption of these mutations was assessed in silico to compare the calculated conformational flexibility and estimated efficiency of ATP binding to kinesin motor domains of wild-type (WT) versus mutant alleles. Additionally, an in vitro microtubule gliding assay was performed to assess the effects of de novo dominant, inherited recessive, and polymorphic variants on KIF1A motor function. RESULTS: All six subjects had severe developmental delay, hypotonia, and varying degrees of hyperreflexia and spastic paraparesis. Microcephaly, cortical visual impairment, optic neuropathy, peripheral neuropathy, ataxia, epilepsy, and movement disorders were also observed. All six patients had a degenerative neurologic course with progressive cerebral and cerebellar atrophy seen on sequential magnetic resonance imaging scans. Computational modeling of mutant protein structures when compared to WT kinesin showed substantial differences in conformational flexibility and ATP-binding efficiency. The de novo KIF1A mutants were nonmotile in the microtubule gliding assay. INTERPRETATION: De novo mutations in KIF1A cause a degenerative neurologic syndrome with brain atrophy. Computational and in vitro assays differentiate the severity of dominant de novo heterozygous versus inherited recessive KIF1A mutations. The profound effect de novo mutations have on axonal transport is likely related to the cause of progressive neurologic impairment in these patients.
ABSTRACT
Many cyclic peptide natural products are larger and structurally more complex than conventional small molecule drugs. Although some molecules in this class are known to possess favorable pharmacokinetic properties, there have been few reports on the membrane permeabilities of cyclic peptide natural products. Here, we present the passive membrane permeabilities of 39 cyclic peptide natural products, and interpret the results using a computational permeability prediction algorithm based on their known or calculated 3D conformations. We found that the permeabilities of these compounds, measured in a parallel artificial membrane permeability assay, spanned a wide range and demonstrated the important influence of conformation on membrane permeability. These results will aid in the development of these compounds as a viable drug paradigm.
Subject(s)
Biological Products/chemistry , Biological Products/pharmacology , Cell Membrane Permeability/drug effects , Membranes, Artificial , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Cyclosporine/chemistry , Cyclosporine/pharmacology , HumansABSTRACT
Cyclic peptide natural products contain a variety of conserved, nonproteinogenic structural elements such as d-amino acids and amide N-methylation. In addition, many cyclic peptides incorporate γ-amino acids and other elements derived from polyketide synthases. We hypothesized that the position and orientation of these extended backbone elements impact the ADME properties of these hybrid molecules, especially their ability to cross cell membranes and avoid metabolic degradation. Here we report the synthesis of cyclic hexapeptide diastereomers containing γ-amino acids (e.g., statines) and systematically investigate their structure-permeability relationships. These compounds were much more water-soluble and, in many cases, were both more membrane permeable and more stable to liver microsomes than a similar non-statine-containing derivative. Permeability correlated well with the extent of intramolecular hydrogen bonding observed in the solution structures determined in the low-dielectric solvent CDCl3, and one compound showed an oral bioavailability of 21% in rat. Thus, the incorporation of γ-amino acids offers a route to increase backbone diversity and improve ADME properties in cyclic peptide scaffolds.