Divide and Conquer. Pocket-Opening Mixed-Solvent Simulations in the Perspective of Docking Virtual Screening Applications for Drug Discovery.

The existence of a druggable binding pocket is a prerequisite for computational drug-target interaction studies including virtual screening. Retrospective studies have shown that extended sampling methods like Markov State Modeling and mixed-solvent simulations can identify cryptic pockets relevant for drug discovery. Here, we apply a combination of mixed-solvent molecular dynamics (MD) and time-structure independent component analysis (TICA) to four retrospective case studies: NPC2, the CECR2 bromodomain, TEM-1, and MCL-1. We compare previous experimental and computational findings to our results. It is shown that the successful identification of cryptic pockets depends on the system and the cosolvent probes. We used alternative TICA internal features such as the unbiased backbone coordinates or backbone dihedrals versus biased interatomic distances. We found that in the case of NPC2, TEM-1, and MCL-1, the use of unbiased features is able to identify cryptic pockets, although in the case of the CECR2 bromodomain, more specific features are required to properly capture a pocket opening. In the perspective of virtual screening applications, it is shown how docking studies with the parent ligands depend critically on the conformational state of the targets.

Plasma ß-secretase1 concentrations correlate with basal forebrain atrophy and neurodegeneration in cognitively healthy individuals at risk for AD.

BACKGROUND: Increased ß-secretase 1 (BACE1) protein concentration, in body fluids, is a candidate biomarker of Alzheimer's disease (AD).We reported that plasma BACE1 protein concentrations are associated with the levels of brain amyloidß (Αß) accumulation in cognitively healthy individuals with subjective memory complaint (SMC). METHODS: In 302 individuals from the same cohort, we investigated the cross-sectional and longitudinal association between plasma BACE1 protein concentrations and AD biomarkers of neurodegeneration (plasma t-tau and Neurofilament light chain (NfL), fluorodeoxyglucose-positron emission tomography (FDG-PET), brain volumes in the basal forebrain [BF], hippocampus, and entorhinal cortex). RESULTS: We report a positive longitudinal correlation of BACE1 with both NfL and t-tau, as well as a correlation between annual BACE1 changes and bi-annual reduction of BF volume. We show a positive association between BACE1 and FDG-PET signal at baseline. CONCLUSIONS: The association between plasma BACE1 protein concentrations and BF atrophy we found in cognitively healthy individuals with SMC corroborates translational studies, suggesting a role of BACE1 in neurodegeneration.

3,3-Difluoro-3,4,5,6-tetrahydropyridin-2-amines: Potent and permeable BACE-1 inhibitors.

Since its discovery in 1999, BACE-1, a membrane anchored aspartyl protease expressed primarily in the CNS, has been the target of numerous medicinal chemistry research programs. These efforts have produced highly potent inhibitors with nanomolar affinity and ever-increasing structural complexity. However, only a handful of these molecules have been able to combine in vitro potency with CNS permeability and progressed to the clinic. Herein, we describe a set of novel piperidine-based inhibitors. This investigation culminated with the identification of 43, a highly potent (IC50: 1.5 nM), permeable BACE-1 inhibitor with a low susceptibility to Pgp-mediatedefflux.

Preparation of 4'-Spirocyclobutyl Nucleoside Analogues as Novel and Versatile Adenosine Scaffolds.

Despite the large variety of modified nucleosides that have been reported, the preparation of constrained 4'-spirocyclic adenosine analogues has received very little attention. We discovered that the [2+2]-cycloaddition of dichloroketene on readily available 4'-exo-methylene furanose sugars efficiently results in the diastereoselective formation of novel 4'-spirocyclobutanones. The reaction mechanism was investigated via density functional theory (DFT) and found to proceed either via a non-synchronous or stepwise reaction sequence, controlled by the stereochemistry at the 3'-position of the sugar substrate. The obtained dichlorocyclobutanones were converted into nucleoside analogues, providing access to a novel class of chiral 4'-spirocyclobutyl adenosine mimetics in eight steps from commercially available sugars. Assessment of the biological activity of designed 4'-spirocyclic adenosine analogues identified potent inhibitors for protein methyltransferase target PRMT5.

Plasma amyloid ß 40/42 ratio predicts cerebral amyloidosis in cognitively normal individuals at risk for Alzheimer's disease.

INTRODUCTION: Blood-based biomarkers of pathophysiological brain amyloid ß (Aß) accumulation, particularly for preclinical target and large-scale interventions, are warranted to effectively enrich Alzheimer's disease clinical trials and management. METHODS: We investigated whether plasma concentrations of the Aß1-40/Aß1-42 ratio, assessed using the single-molecule array (Simoa) immunoassay, may predict brain Aß positron emission tomography status in a large-scale longitudinal monocentric cohort (N = 276) of older individuals with subjective memory complaints. We performed a hypothesis-driven investigation followed by a no-a-priori hypothesis study using machine learning. RESULTS: The receiver operating characteristic curve and machine learning showed a balanced accuracy of 76.5% and 81%, respectively, for the plasma Aß1-40/Aß1-42 ratio. The accuracy is not affected by the apolipoprotein E (APOE) ε4 allele, sex, or age. DISCUSSION: Our results encourage an independent validation cohort study to confirm the indication that the plasma Aß1-40/Aß1-42 ratio, assessed via Simoa, may improve future standard of care and clinical trial design.

Brain Aß load association and sexual dimorphism of plasma BACE1 concentrations in cognitively normal individuals at risk for AD.

INTRODUCTION: Successful development of effective ß-site amyloid precursor protein cleaving enzyme 1 (BACE1)-targeted therapies for early stages of Alzheimer's disease requires biomarker-guided intervention strategies. METHODS: We investigated whether key biological factors such as sex, apolipoprotein E (APOE ε4) allele, and age affect longitudinal plasma BACE1 concentrations in a large monocenter cohort of individuals at risk for Alzheimer's disease. We explored the relationship between plasma BACE1 concentrations and levels of brain amyloid-ß (Aß) deposition, using positron emission tomography global standard uptake value ratios. RESULTS: Baseline and longitudinal mean concentrations of plasma BACE1 were significantly higher in women than men. We also found a positive significant impact of plasma BACE1 on baseline Aß-positron emission tomography global standard uptake value ratios. DISCUSSION: Our results suggest a sexual dimorphism in BACE1-related upstream mechanisms of brain Aß production and deposition. We argue that plasma BACE1 should be considered in further biomarker validation and qualification studies as well as in BACE1 clinical trials.

Neurogranin as Cerebrospinal Fluid Biomarker for Alzheimer Disease: An Assay Comparison Study.

BACKGROUND: Neurogranin in cerebrospinal fluid (CSF) correlates with cognitive decline and is a potential novel biomarker for Alzheimer disease (AD) dementia. We investigated the analytical and diagnostic performance of 3 commonly used neurogranin assays in the same cohort of patients to improve the interpretability of CSF neurogranin test results. METHODS: The neurogranin Erenna® assay from Washington University, St. Louis, MO (WashU); ELISA from ADx Neurosciences; and ELISA from Gothenburg University, Mölndal, Sweden (UGot), were compared using silver staining and Western blot after gel electrophoresis. Clinical performance of the 3 assays was compared in samples from individuals diagnosed with subjective cognitive decline (n = 22), and in patients with AD (n = 22), frontotemporal dementia (n = 22), dementia with Lewy bodies (n = 22), or vascular dementia (n = 20), adjusted for sex and age. RESULTS: The assays detected different epitopes of neurogranin: the WashU assay detected the N-terminal part of neurogranin (S10-D23) and a C-terminal part (G49-G60), the ADx assay detected C-terminal neurogranin truncated at P75, and the UGot assay detected the C-terminal neurogranin with intact ending (D78). Spearman ρ was 0.95 between ADx and WashU, 0.87 between UGot and WashU, and 0.81 between UGot and ADx. ANCOVA (analysis of covariance) showed group differences for ranked neurogranin concentrations in each assay (all P < 0.05), with specific increases in AD. CONCLUSIONS: Although the 3 assays target different epitopes on neurogranin and have different calibrators, the high correlations and the similar group differences suggest that the different forms of neurogranin in CSF carry similar diagnostic information, at least in the context of neurodegenerative diseases.

Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential.

Neurogranin and tau in cerebrospinal fluid and plasma of patients with acute ischemic stroke.

BACKGROUND: While neurogranin has no value as plasma biomarker for Alzheimer's disease, it may be a potential blood biomarker for traumatic brain injury. This evokes the question whether there are changes in neurogranin levels in blood in other conditions of brain injury, such as acute ischemic stroke (AIS). METHODS: We therefore explored neurogranin in paired cerebrospinal fluid (CSF)/plasma samples of AIS patients (n = 50) from a well-described prospective study. In parallel, we investigated another neuronal protein, i.e. tau, which has already been suggested as potential AIS biomarker in CSF and blood. ELISA as well as Single Molecule Array (Simoa) technology were used for the biochemical analyses. Statistical analyses included Shapiro-Wilk testing, Mann-Whitney analyses and Pearson's correlation analysis. RESULTS: In contrast to tau, of which high levels in both CSF and plasma were related to stroke characteristics like severity and long-term outcome, plasma neurogranin levels were only correlated with infarct volume. Likewise, CSF neurogranin levels were significantly higher in patients with an infarct volume > 5 mL than in patients with smaller infarct volumes. Finally, neurogranin and tau were significantly correlated in CSF, whereas a weaker relationship was observed in plasma. CONCLUSIONS: These findings indicate that although plasma and CSF neurogranin may reflect the volume of acute cerebral ischemia, this synaptic protein is less likely to be a potential AIS biomarker. Levels of tau correlated with severity and outcome of stroke in both plasma and CSF, in the present study as well as previous reports, confirming the potential of tau as an AIS biomarker.

C-terminal neurogranin is increased in cerebrospinal fluid but unchanged in plasma in Alzheimer's disease.

INTRODUCTION: Biomarkers monitoring synaptic degeneration/loss would be valuable for Alzheimer's disease (AD) diagnosis. Postsynaptic protein neurogranin may be a promising cerebrospinal fluid (CSF) biomarker but has not yet been evaluated as a plasma biomarker. METHODS: Using an in-house designed prototype enzyme-linked immunosorbent assay (ELISA) targeting neurogranin C-terminally, we studied neurogranin in paired CSF/plasma samples of controls (n = 29) versus patients experiencing MCI, or dementia, due to AD (in total n = 59). RESULTS: CSF neurogranin was increased in AD and positively correlated with CSF tau, whereas there was a negative relationship between CSF neurogranin (and tau) and CSF Aß1-42/Aß1-40. No differences were detected in plasma neurogranin between controls and AD. Also, there was no correlation between CSF and plasma neurogranin, excluding confounding effects of the latter. DISCUSSION: This study strengthens the potential of neurogranin as an AD CSF biomarker, which now needs validation in larger studies. As tools, straightforward immunoassays can be used, as demonstrated by the described ELISA.

Exploring the Severity Strata of Disease Activity and Repigmentation in Vitiligo Based on Validated Physician Global Assessment (PGA) Scores.

Background/Objectives: There is currently no guidance on how to interpret the global degrees of activity (worsening) and repigmentation (improvement) in vitiligo. Stratification into global degrees can be completed for static evaluations (e.g., visible disease activity signs) and dynamic assessments (e.g., evolution over time). For the latter, the Vitiligo Disease Activity Score (VDAS15&60) and Vitiligo Disease Improvement Score (VDIS15&60) were recently validated. Methods: In the current study, a Physician Global Assessment (PGA) for disease activity (worsening) and repigmentation (improvement) was evaluated for validity (construct) and reliability (inter- and intrarater) based on a photo set of 66 patients. Subsequently, the PGA activity (worsening) and repigmentation (improvement) were used to stratify the Vitiligo Extent Score plus (VESplus), VDAS15&60 or VDIS15&60 into three global categories (slightly, moderately and much worse/improved), based on ROC analysis. Results: For the VESplus, cut-off values for the categories 'slightly, moderately and much worse' were >0.3%, >27.71% and >128.75% BSA (relative changes in the affected total BSA), respectively. For the categories 'slightly, moderately and much improved', they were >0%, >4.87% and >36.88% BSA (relative changes in the affected total BSA), respectively. The optimal cut-off values of the number of active (VDAS15) body areas were >0 areas for slightly worse, >2 areas for moderately worse and >7 for much worse. For VDIS15, the cut-off values for slightly improved and moderately improved were >0 and >1. For VDAS60 and VDIS60, the cut-off points were >0.5, >3, >9.5 and >0.5 and >1.5, respectively. The results should be interpreted with caution in patients with extensive vitiligo due to the rather limited disease extent of the included patient population (VESplus (median: 3.2%)). Conclusions: This research will aid in the development of more detailed international definitions.

Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance.

The development of HIV integrase (IN) strand transfer inhibitors (INSTIs) and our understanding of viral resistance to these molecules have been hampered by a paucity of available structural data. We recently reported cocrystal structures of the prototype foamy virus (PFV) intasome with raltegravir and elvitegravir, establishing the general INSTI binding mode. We now present an expanded set of cocrystal structures containing PFV intasomes complexed with first- and second-generation INSTIs at resolutions of up to 2.5 Å. Importantly, the improved resolution allowed us to refine the complete coordination spheres of the catalytic metal cations within the INSTI-bound intasome active site. We show that like the Q148H/G140S and N155H HIV-1 IN variants, the analogous S217H and N224H PFV INs display reduced sensitivity to raltegravir in vitro. Crystal structures of the mutant PFV intasomes in INSTI-free and -bound forms revealed that the amino acid substitutions necessitate considerable conformational rearrangements within the IN active site to accommodate an INSTI, thus explaining their adverse effects on raltegravir antiviral activity. Furthermore, our structures predict physical proximity and an interaction between HIV-1 IN mutant residues His148 and Ser/Ala140, rationalizing the coevolution of Q148H and G140S/A mutations in drug-resistant viral strains.

Macrocyclic Carbon-Linked Pyrazoles As Novel Inhibitors of MCL-1.

Myeloid cell leukemia-1 (MCL-1) is a member of the antiapoptotic BCL-2 proteins family and a key regulator of mitochondrial homeostasis. Overexpression of MCL-1 is found in many cancer cells and contributes to tumor progression, which makes it an attractive therapeutic target. Pursuing our previous study of macrocyclic indoles for the inhibition of MCL-1, we report herein the impact of both pyrazole and indole isomerism on the potency and overall properties of this family of compounds. We demonstrated that the incorporation of a fluorine atom on the naphthalene moiety was a necessary step to improve cellular potency and that, combined with the introduction of various side chains on the pyrazole, it enhanced solubility significantly. This exploration culminated in the discovery of compounds (Ra)-10 and (Ra)-15, possessing remarkable cellular potency and properties.

Discovery of an Oral, Beyond-Rule-of-Five Mcl-1 Protein-Protein Interaction Modulator with the Potential of Treating Hematological Malignancies.

Avoidance of apoptosis is critical for the development and sustained growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 family of proteins which is overexpressed in many cancers. Upregulation of Mcl-1 in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Therefore, pharmacological inhibition of Mcl-1 is regarded as an attractive approach to treating relapsed or refractory malignancies. Herein, we disclose the design, synthesis, optimization, and early preclinical evaluation of a potent and selective small-molecule inhibitor of Mcl-1. Our exploratory design tactics focused on structural modifications which improve the potency and physicochemical properties of the inhibitor while minimizing the risk of functional cardiotoxicity. Despite being in the "non-Lipinski" beyond-Rule-of-Five property space, the developed compound benefits from exquisite oral bioavailability in vivo and induces potent pharmacodynamic inhibition of Mcl-1 in a mouse xenograft model.

Modulating physicochemical properties of tetrahydropyridine-2-amine BACE1 inhibitors with electron-withdrawing groups: A systematic study.

A common challenge for medicinal chemists is to reduce the pKa of strongly basic groups' conjugate acids into a range that preserves the desired effects, usually potency and/or solubility, but avoids undesired effects like high volume of distribution (Vd), limited membrane permeation, and off-target binding to, notably, the hERG channel and monoamine receptors. We faced this challenge with a 3,4,5,6-tetrahydropyridine-2-amine scaffold harboring an amidine, a key structural component of potential inhibitors of BACE1, the rate-limiting enzyme in the production of Aß species that make up amyloid plaques in Alzheimer's disease. In our endeavor to balance potency with desirable properties to achieve brain penetration, we introduced a diverse set of groups in beta position of the amidine that modulate logD, PSA and pKa. Given the synthetic challenge to prepare these highly functionalized warheads, we first developed a design flow including predicted physicochemical parameters which allowed us to select only the most promising candidates for synthesis. For this we evaluated a set of commercial packages to predict physicochemical properties, which can guide medicinal chemists in their endeavors to modulate pKa values of amidine and amine bases.

A Brain-Penetrant and Bioavailable Pyrazolopiperazine BACE1 Inhibitor Elicits Sustained Reduction of Amyloid ß In Vivo.

We recently disclosed a set of heteroaryl-fused piperazine inhibitors of BACE1 that combined nanomolar potency with good intrinsic permeability and low Pgp-mediated efflux. Herein we describe further work on two prototypes of this family of inhibitors aimed at modulating their basicity and reducing binding to the human ether-a-go-go-related gene (hERG) channel. This effort has led to the identification of compound 36, a highly potent (hAß42 cell IC50 = 1.3 nM), cardiovascularly safe, and orally bioavailable compound that elicited sustained Aß42 reduction in mouse and dog animal models.

1a/1b subtype profiling of nonnucleoside polymerase inhibitors of hepatitis C virus.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.

Development and implementation of an enterprise-wide predictive model for early absorption, distribution, metabolism and excretion properties.

Background: Accurate prediction of absorption, distribution, metabolism and excretion (ADME) properties can facilitate the identification of promising drug candidates. Methodology & Results: The authors present the Janssen generic Target Product Profile (gTPP) model, which predicts 18 early ADME properties, employs a graph convolutional neural network algorithm and was trained on between 1000-10,000 internal data points per predicted parameter. gTPP demonstrated stronger predictive power than pretrained commercial ADME models and automatic model builders. Through a novel logging method, the authors report gTPP usage for more than 200 Janssen drug discovery scientists. Conclusion: The investigators successfully enabled the rapid and systematic implementation of predictive ML tools across a drug discovery pipeline in all therapeutic areas. This experience provides useful guidance for other large-scale AI/ML deployment efforts.

The ß-Secretase BACE1 in Alzheimer's Disease.

BACE1 (beta-site amyloid precursor protein cleaving enzyme 1) was initially cloned and characterized in 1999. It is required for the generation of all monomeric forms of amyloid-ß (Aß), including Aß42, which aggregates into bioactive conformational species and likely initiates toxicity in Alzheimer's disease (AD). BACE1 concentrations and rates of activity are increased in AD brains and body fluids, thereby supporting the hypothesis that BACE1 plays a critical role in AD pathophysiology. Therefore, BACE1 is a prime drug target for slowing down Aß production in early AD. Besides the amyloidogenic pathway, BACE1 has other substrates that may be important for synaptic plasticity and synaptic homeostasis. Indeed, germline and adult conditional BACE1 knockout mice display complex neurological phenotypes. Despite BACE1 inhibitor clinical trials conducted so far being discontinued for futility or safety reasons, BACE1 remains a well-validated therapeutic target for AD. A safe and efficacious compound with high substrate selectivity as well as a more accurate dose regimen, patient population, and disease stage may yet be found. Further research should focus on the role of Aß and BACE1 in physiological processes and key pathophysiological mechanisms of AD. The functions of BACE1 and the homologue BACE2, as well as the biology of Aß in neurons and glia, deserve further investigation. Cellular and molecular studies of BACE1 and BACE2 knockout mice coupled with biomarker-based human research will help elucidate the biological functions of these important enzymes and identify their substrates and downstream effects. Such studies will have critical implications for BACE1 inhibition as a therapeutic approach for AD.

JNJ-67569762, A 2-Aminotetrahydropyridine-Based Selective BACE1 Inhibitor Targeting the S3 Pocket: From Discovery to Clinical Candidate.

The discovery of a novel 2-aminotetrahydropyridine class of BACE1 inhibitors is described. Their pKa and lipophilicity were modulated by a pending sulfonyl group, while good permeability and brain penetration were achieved via intramolecular hydrogen bonding. BACE1 selectivity over BACE2 was achieved in the S3 pocket by a novel bicyclic ring system. An optimization addressing reactive metabolite formation, cardiovascular safety, and CNS toxicity is described, leading to the clinical candidate JNJ-67569762 (12), which gave robust dose-dependent BACE1-mediated amyloid ß lowering without showing BACE2-dependent hair depigmentation in preclinical models. We show that 12 has a favorable projected human dose and PK and hence presented us with an opportunity to test a highly selective BACE1 inhibitor in humans. However, 12 was found to have a QT effect upon repeat dosing in dogs and its development was halted in favor of other selective leads, which will be reported in the future.