José S. Duca

Antibodies to influenza nucleoprotein cross-react with human hypocretin receptor 2.

Similarity between influenza nucleoprotein and hypocretin receptor 2 may trigger vaccine-associated narcolepsy. Immunological mistaken identity New reports of narcolepsy increased after the vaccination campaign against the 2009 A(H1N1) influenza pandemic in some countries but not others. Now Ahmed et al. examine differences between the vaccines used and find a potential mechanistic explanation for the vaccine-specific effect. They found a peptide in influenza nucleopeptide A that shared protein residues with human hypocretin receptor 2, which has been linked to narcolepsy. The vaccine used in unaffected countries contained less influenza nucleoprotein. Indeed, patients with putative vaccine-associated narcolepsy produced antibodies that cross-reacted to both the influenza and the hypocretin receptor 2 epitopes. Although these data do not demonstrate causation, they provide a possible explanation for the association of this particular influenza vaccination with increased reports of narcolepsy. The sleep disorder narcolepsy is linked to the HLA-DQB1*0602 haplotype and dysregulation of the hypocretin ligand-hypocretin receptor pathway. Narcolepsy was associated with Pandemrix vaccination (an adjuvanted, influenza pandemic vaccine) and also with infection by influenza virus during the 2009 A(H1N1) influenza pandemic. In contrast, very few cases were reported after Focetria vaccination (a differently manufactured adjuvanted influenza pandemic vaccine). We hypothesized that differences between these vaccines (which are derived from inactivated influenza viral proteins) explain the association of narcolepsy with Pandemrix-vaccinated subjects. A mimic peptide was identified from a surface-exposed region of influenza nucleoprotein A that shared protein residues in common with a fragment of the first extracellular domain of hypocretin receptor 2. A significant proportion of sera from HLA-DQB1*0602 haplotype–positive narcoleptic Finnish patients with a history of Pandemrix vaccination (vaccine-associated narcolepsy) contained antibodies to hypocretin receptor 2 compared to sera from nonnarcoleptic individuals with either 2009 A(H1N1) pandemic influenza infection or history of Focetria vaccination. Antibodies from vaccine-associated narcolepsy sera cross-reacted with both influenza nucleoprotein and hypocretin receptor 2, which was demonstrated by competitive binding using 21-mer peptide (containing the identified nucleoprotein mimic) and 55-mer recombinant peptide (first extracellular domain of hypocretin receptor 2) on cell lines expressing human hypocretin receptor 2. Mass spectrometry indicated that relative to Pandemrix, Focetria contained 72.7% less influenza nucleoprotein. In accord, no durable antibody responses to nucleoprotein were detected in sera from Focetria-vaccinated nonnarcoleptic subjects. Thus, differences in vaccine nucleoprotein content and respective immune response may explain the narcolepsy association with Pandemrix.

LMX1B Mutations Cause Hereditary FSGS without Extrarenal Involvement

LMX1B encodes a homeodomain-containing transcription factor that is essential during development. Mutations in LMX1B cause nail-patella syndrome, characterized by dysplasia of the patellae, nails, and elbows and FSGS with specific ultrastructural lesions of the glomerular basement membrane (GBM). By linkage analysis and exome sequencing, we unexpectedly identified an LMX1B mutation segregating with disease in a pedigree of five patients with autosomal dominant FSGS but without either extrarenal features or ultrastructural abnormalities of the GBM suggestive of nail-patella-like renal disease. Subsequently, we screened 73 additional unrelated families with FSGS and found mutations involving the same amino acid (R246) in 2 families. An LMX1B in silico homology model suggested that the mutated residue plays an important role in strengthening the interaction between the LMX1B homeodomain and DNA; both identified mutations would be expected to diminish such interactions. In summary, these results suggest that isolated FSGS could result from mutations in genes that are also involved in syndromic forms of FSGS. This highlights the need to include these genes in all diagnostic approaches to FSGS that involve next-generation sequencing.

Toward the Validation of Maternal Embryonic Leucine Zipper Kinase: Discovery, Optimization of Highly Potent and Selective Inhibitors, and Preliminary Biology Insight.

MELK kinase has been implicated in playing an important role in tumorigenesis. Our previous studies suggested that MELK is involved in the regulation of cell cycle and its genetic depletion leads to growth inhibition in a subset of high MELK-expressing basal-like breast cancer cell lines. Herein we describe the discovery and optimization of novel MELK inhibitors 8a and 8b that recapitulate the cellular effects observed by short hairpin ribonucleic acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered a novel fluorine-induced hydrophobic collapse that locked the ligand in its bioactive conformation and led to a 20-fold gain in potency. These novel pharmacological inhibitors achieved high exposure in vivo and were well tolerated, which may allow further in vivo evaluation.

Estimation of Solvation Entropy and Enthalpy via Analysis of Water Oxygen-Hydrogen Correlations.

A statistical-mechanical framework for estimation of solvation entropies and enthalpies is proposed, which is based on the analysis of water as a mixture of correlated water oxygens and water hydrogens. Entropic contributions of increasing order are cast in terms of a Mutual Information Expansion that is evaluated to pairwise interactions. In turn, the enthalpy is computed directly from a distance-based hydrogen bonding energy algorithm. The resulting expressions are employed for grid-based analyses of Molecular Dynamics simulations. In this first assessment of the methodology, we obtained global estimates of the excess entropy and enthalpy of water that are in good agreement with experiment and examined the methods ability to enable detailed elucidation of solvation thermodynamic structures, which can provide valuable knowledge toward molecular design.

Collaborating to improve the use of free-energy and other quantitative methods in drug discovery

In May and August, 2016, several pharmaceutical companies convened to discuss and compare experiences with Free Energy Perturbation (FEP). This unusual synchronization of interest was prompted by Schrödinger’s FEP+ implementation and offered the opportunity to share fresh studies with FEP and enable broader discussions on the topic. This article summarizes key conclusions of the meetings, including a path forward of actions for this group to aid the accelerated evaluation, application and development of free energy and related quantitative, structure-based design methods.

Uncoupling the Structure–Activity Relationships of β2 Adrenergic Receptor Ligands from Membrane Binding

Ligand binding to membrane proteins may be significantly influenced by the interaction of ligands with the membrane. In particular, the microscopic ligand concentration within the membrane surface solvation layer may exceed that in bulk solvent, resulting in overestimation of the intrinsic protein-ligand binding contribution to the apparent/measured affinity. Using published binding data for a set of small molecules with the β2 adrenergic receptor, we demonstrate that deconvolution of membrane and protein binding contributions allows for improved structure-activity relationship analysis and structure-based drug design. Molecular dynamics simulations of ligand bound membrane protein complexes were used to validate binding poses, allowing analysis of key interactions and binding site solvation to develop structure-activity relationships of β2 ligand binding. The resulting relationships are consistent with intrinsic binding affinity (corrected for membrane interaction). The successful structure-based design of ligands targeting membrane proteins may require an assessment of membrane affinity to uncouple protein binding from membrane interactions.

Gazing into the crystal ball; the future of computer-aided drug design

Twenty-five years is almost a full career for a scientist, but before looking to the future, we should ask what is really new in the last 25 years, i.e. since 1986? Surprisingly little! Here is a partial but still fairly good list of techniques routinely used by modellers: high throughput docking, high precision docking, free-energy calculations, quantum mechanics, molecular mechanics, distance geometry, molecular dynamics, statistical thermodynamics, conformational searching, scaffold morphing, solvation, QSPR, QSAR, bioavailability predictions, pharmacophores, protein modeling, de novo design, library design, chemical databases and searching, data analysis and visualization, virtual screening, chemometrics, interaction analysis using small molecule and protein x-rays, and FBDD. The majority of these techniques were introduced in the early to mid 1980s, and we think everything on the list except FBDD was introduced by the early 1990s (many techniques have been re-invented since; the collective memory of the literature seems to be under 10 years and falling). The biggest revolution in computational chemistry over the last 25 years was not a new computational technique, but rather the introduction of Beowulf clusters around 2000, which in just a few years increased processing power by about 1009 beyond Moore’s Law for many problems, i.e., it skipped at least a decade. This ‘‘suddenly’’ enabled application of a large number of the techniques from the 1980s to real systems.

A theoretical approach to understanding the fragmentation reaction of halonitrobenzene radical anions

We present a semiempirical AM1 study of the radical anions of o-, m- and p-halonitrobenzenes and some alkyl substituted derivatives in relation to their σ–π orbital isomerism and the energy of their interconversion (ΔEσπ). Halobenzene radical anions are also included for comparison. The results obtained with the RHF/CI(5) formalism account for the differences observed in the fragmentation rate of these radical anions under thermal and photochemical conditions. Based on the calculated ΔEσπ the intramolecular thermal electron transfer from the π system to the σ* C–X bond involved in the fragmentation of the intermediates into an aromatic radical and the anion of the leaving group occurs with considerable energy for the p-, m- and o-chloronitrobenzenes (1a–c) and the p- and m-bromo (2a, b) derivatives. The fragmentation of these radical anions is favoured either from the first or from higher energy excited doublet states. On the other hand, the intramolecular thermal electron transfer is favoured for the p-, m-, o-iodo (3a–c) and o-bromo (2c) derivatives. The results obtained for some alkyl substituted halonitrobenzene radical anions are in agreement with their known experimental fragmentation rates.

Theoretical study on haloaromatic radical anions and their intramolecular electron transfer reactions

An AM1 theoretical study was made on the electronic nature of radical anions of halobenzenes, 2-halopyridines, halobenzonitriles, ortho-, para-, meta-haloacetophenones and related compounds (X = Cl, Br and I). The experimentally determined fragmentation rate of these intermediates is discussed in terms of the possibility of the existence of π* and σ* orbital isomeric radical anions, their energy difference and the probability of an intramolecular electron-transfer reaction from the π* to the σ* system. Good correlation was obtained between the properties determined theoretically and the experimental fragmentation rates for the family of halobenzonitrile and haloacetophenone compounds.Inspection of the potential surface for the intramolecular electron-transfer reaction between both isomers indicates that the carbon–halogen bond elongation and bending angle are the main reaction coordinates for the electron-transfer step which results from an avoided crossing of potential energy surfaces (non-vanishing Hσπ integral).

“Addition” and “Subtraction”: Selectivity Design for Type II Maternal Embryonic Leucine Zipper Kinase Inhibitors

While adding the structural features that are more favored by on-target activity is the more common strategy in selectivity optimization, the opposite strategy of subtracting the structural features that contribute more to off-target activity can also be very effective. Reported here is our successful effort of improving the kinase selectivity of type II maternal embryonic leucine zipper kinase inhibitors by applying these two complementary approaches together, which clearly demonstrates the powerful synergy between them.