Fungicide-Resistant Botrytis in Forest Nurseries May Impact Disease Control in Norway Spruce.

Gray mold, caused by Botrytis spp., is a serious problem in Norway spruce seedling production in forest nurseries. From 2013 to 2019, 125 isolates of Botrytis were obtained from eight forest nurseries in Norway: 53 from Norway spruce seedlings, 16 from indoor air, 52 from indoor surfaces, and four from weeds growing close to seedlings. The majority of isolates were identified as B. cinerea, and over 60% of these were characterized as Botrytis group S. B. pseudocinerea isolates were obtained along with isolates with DNA sequence similarities to B. prunorum. Fungicide resistance was assessed with a mycelial growth assay, and resistance was found for the following: boscalid (8.8%), fenhexamid (33.6%), fludioxonil (17.6%), pyraclostrobin (36.0%), pyrimethanil (13.6%), and thiophanate-methyl (50.4%). Many isolates (38.4%) were resistant to two to six different fungicides. A selection of isolates was analyzed for the presence of known resistance-conferring mutations in the cytb, erg27, mrr1, sdhB, and tubA genes, and mutations leading to G143A, F412S, ΔL497, H272R, and E198A/F200Y were detected, respectively. Detection of fungicide resistance in Botrytis from Norway spruce and forest nursery facilities reinforces the necessity of employing resistance management strategies to improve control and delay development of fungicide resistance in the gray mold pathogens.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Design, Optimization, and Study of Small Molecules That Target Tau Pre-mRNA and Affect Splicing.

Approximately 95% of human genes are alternatively spliced, and aberrant splicing events can cause disease. One pre-mRNA that is alternatively spliced and linked to neurodegenerative diseases is tau (microtubule-associated protein tau), which can cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and can contribute to Alzheimer's disease. Here, we describe the design of structure-specific lead small molecules that directly target tau pre-mRNA from sequence. This was followed by hit expansion and analogue synthesis to further improve upon these initial lead molecules. The emergent compounds were assessed for functional activity in a battery of assays, including binding assays and an assay that mimics molecular recognition of tau pre-mRNA by a U1 small nuclear ribonucleoprotein (snRNP) splicing factor. Compounds that emerged from these studies had enhanced potency and selectivity for the target RNA relative to the initial hits, while also having significantly improved drug-like properties. The compounds are shown to directly target tau pre-mRNA in cells, via chemical cross-linking and isolation by pull-down target profiling, and to rescue disease-relevant splicing of tau pre-mRNA in a variety of cellular systems, including primary neurons. More broadly, this study shows that lead, structure-specific compounds can be designed from sequence and then further optimized for their physicochemical properties while at the same time enhancing their activity.

Synthesis of Pyridopyrazine-1,6-dione γ-Secretase Modulators via Selective 4-Methylimidazole N1-Buchwald Arylation.

An efficient synthesis of pyridopyrazine-1,6-dione γ-secretase modulators (GSMs) is described. Our route features the construction of a crystalline lactone intermediate via a selective palladium-catalyzed 4-methylimidazole N1-arylation using the Buchwald Xantphos Pd G4 precatalyst, which does not require a preactivation step. The weak inorganic base KHCO3 was employed to minimize saponification of a particularly sensitive lactone substrate. Additional key transformations include DABAL-Me3-mediated lactone aminolysis and a mild TBD/ethyl trifluoroacetate mediated lactam ring closure to afford a representative GSM in high yield.

Discovery of indole-derived pyridopyrazine-1,6-dione γ-secretase modulators that target presenilin.

Herein we describe design strategies that led to the discovery of novel pyridopyrazine-1,6-dione γ-secretase modulators (GSMs) incorporating an indole motif as a heterocyclic replacement for a naphthyl moiety that was present in the original lead 9. Tactics involving parallel medicinal chemistry and in situ monomer synthesis to prepare focused libraries are discussed. Optimized indole GSM 29 exhibited good alignment of in vitro potency and physicochemical properties, and moderate reduction of brain Aß42 was achieved in a rat efficacy model when dosed orally at 30mg/kg. Labeling experiments using a clickable, indole-derived GSM photoaffinity probe demonstrated that this series binds to the presenilin N-terminal fragment (PS1-NTF) of the γ-secretase complex.

γ-Secretase modulator (GSM) photoaffinity probes reveal distinct allosteric binding sites on presenilin.

γ-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic ß-amyloid peptides (i.e. Aß42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule γ-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of Aß42 while not blocking the processing of γ-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active site-directed GSI L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active γ-secretase) but not full-length PS1 (inactive γ-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the γ-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of γ-secretase and the modulatory binding site of certain GSMs.

Enantioselective total syntheses of citrinadins A and B. Stereochemical revision of their assigned structures.

The concise, enantioselective total syntheses of (-)-citrinadin A and (+)-citrinadin B in a total of only 20 and 21 steps, respectively, from commercially available starting materials are described. Our strategy, which minimizes refunctionalization and protection/deprotection operations, features the highly diastereoselective, vinylogous Mannich addition of a dienolate to a chiral pyridinium salt to set the first chiral center. The absolute stereochemistry of this key center was then relayed by a sequence of substrate-controlled reactions, including a highly stereoselective epoxidation/ring opening sequence and an oxidative rearrangement of an indole to furnish a spirooxindole to establish the remaining stereocenters in the pentacyclic core of the citrinadins. An early stage intermediate in the synthesis of (-)-citrinadin A was deoxygenated to generate a dehydroxy compound that was elaborated into (+)-citrinadin B by a sequence of reaction identical to those used to prepare (-)-citrinadin A. These concise syntheses of (-)-citrinadin A and (+)-citrinadin B led to a revision of their stereochemical structures.

Discovery of Clinical Candidate PF-06648671: A Potent γ-Secretase Modulator for the Treatment of Alzheimer's Disease.

Herein, we describe the design and synthesis of γ-secretase modulator (GSM) clinical candidate PF-06648671 (22) for the treatment of Alzheimer's disease. A key component of the design involved a 2,5-cis-tetrahydrofuran (THF) linker to impart conformational rigidity and lock the compound into a putative bioactive conformation. This effort was guided using a pharmacophore model since crystallographic information was not available for the membrane-bound γ-secretase protein complex at the time of this work. PF-06648671 achieved excellent alignment of whole cell in vitro potency (Aß42 IC50 = 9.8 nM) and absorption, distribution, metabolism, and excretion (ADME) parameters. This resulted in favorable in vivo pharmacokinetic (PK) profile in preclinical species, and PF-06648671 achieved a human PK profile suitable for once-a-day dosing. Furthermore, PF-06648671 was found to have favorable brain availability in rodent, which translated into excellent central exposure in human and robust reduction of amyloid ß (Aß) 42 in cerebrospinal fluid (CSF).

Cerebrospinal fluid amyloid-ß (Aß) as an effect biomarker for brain Aß lowering verified by quantitative preclinical analyses.

Reducing the generation of amyloid-ß (Aß) in the brain via inhibition of ß-secretase or inhibition/modulation of γ-secretase has been pursued as a potential disease-modifying treatment for Alzheimer's disease. For the discovery and development of ß-secretase inhibitors (BACEi), γ-secretase inhibitors (GSI), and γ-secretase modulators (GSM), Aß in cerebrospinal fluid (CSF) has been presumed to be an effect biomarker for Aß lowering in the brain. However, this presumption is challenged by the lack of quantitative understanding of the relationship between brain and CSF Aß lowering. In this study, we strived to elucidate how the intrinsic pharmacokinetic (PK)/pharmacodynamic (PD) relationship for CSF Aß lowering is related to that for brain Aß through quantitative modeling of preclinical data for numerous BACEi, GSI, and GSM across multiple species. Our results indicate that the intrinsic PK/PD relationship in CSF is predictive of that in brain, at least in the postulated pharmacologically relevant range, with excellent consistency across mechanisms and species. As such, the validity of CSF Aß as an effect biomarker for brain Aß lowering is confirmed preclinically. Meanwhile, we have been able to reproduce the dose-dependent separation between brain and CSF effect profiles using simulations. We further discuss the implications of our findings to drug discovery and development with regard to preclinical PK/PD characterization and clinical prediction of Aß lowering in the brain.

Development of clickable active site-directed photoaffinity probes for γ-secretase.

We have developed clickable active site-directed photoaffinity probes for γ-secretase which incorporate a photoreactive benzophenone group and an alkyne handle for subsequent click chemistry mediated conjugation with azide-linked reporter tags for visualization (e.g., TAMRA-azide) or enrichment (e.g., biotin-azide) of labeled proteins. Specifically, we synthesized clickable analogs of L646 (2) and L505 (3) and validated specific labeling to presenilin-1N-terminal fragment (PS1-NTF), the active site aspartyl protease component within the γ-secretase complex. Additionally, we were able to identify signal peptide peptidase (SPP) by Western blot analysis. Furthermore, we analyzed the photo-labeled proteins in an unbiased fashion by click chemistry with TAMRA-azide followed by in-gel fluorescence detection. This approach expands the utility of γ-secretase inhibitor (GSI) photoaffinity probes in that labeled proteins can be tagged with any number of azide-linked reporters groups using a single clickable photoaffinity probe for target pull down and/or fluorescent imaging applications.

Design and synthesis of dihydrobenzofuran amides as orally bioavailable, centrally active γ-secretase modulators.

We report the discovery and optimization of a novel series of dihydrobenzofuran amides as γ-secretase modulators (GSMs). Strategies for aligning in vitro potency with drug-like physicochemical properties and good microsomal stability while avoiding P-gp mediated efflux are discussed. Lead compounds such as 35 and 43 have moderate to good in vitro potency and excellent selectivity against Notch. Good oral bioavailability was achieved as well as robust brain Aß42 lowering activity at 100 mg/kg po dose.

Design, synthesis, and pharmacological evaluation of azetedine and pyrrolidine derivatives as dual norepinephrine reuptake inhibitors and 5-HT(1A) partial agonists.

Compounds with combined norepinephrine reuptake inhibitor (NRI) and serotonin 1A (5-HT(1A)) partial agonist pharmacology may offer a new therapeutic approach for treating symptoms of neuropsychiatric disorders including ADHD, depression, and anxiety. Herein we describe the design and optimization of novel chemical matter that exhibits favorable dual NRI and 5-HT(1A) partial agonist activity. Lead compounds in this series were found to be devoid of activity at the dopamine transporter and were shown to be brain penetrant with high receptor occupancy.

Photoaffinity Labeling and Quantitative Chemical Proteomics Identify LXRß as the Functional Target of Enhancers of Astrocytic apoE.

Utilizing a phenotypic screen, we identified chemical matter that increased astrocytic apoE secretion in vitro. We designed a clickable photoaffinity probe based on a pyrrolidine lead compound and carried out probe-based quantitative chemical proteomics in human astrocytoma CCF-STTG1 cells to identify liver x receptor ß (LXRß) as the target. Binding of the small molecule ligand stabilized LXRß, as shown by cellular thermal shift assay (CETSA). In addition, we identified a probe-modified peptide by mass spectrometry and proposed a model where the photoaffinity probe is bound in the ligand-binding pocket of LXRß. Taken together, our findings demonstrated that the lead chemical matter bound directly to LXRß, and our results highlight the power of chemical proteomic approaches to identify the target of a phenotypic screening hit. Additionally, the LXR photoaffinity probe and lead compound described herein may serve as valuable tools to further evaluate the LXR pathway.

Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro , and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion.

Despite the important role of apolipoprotein E (apoE) secretion from astrocytes in brain lipid metabolism and the strong association of apoE4, one of the human apoE isoforms, with sporadic and late onset forms of Alzheimer's disease (AD) little is known about the regulation of astrocytic apoE. Utilizing annotated chemical libraries and a phenotypic screening strategy that measured apoE secretion from a human astrocytoma cell line, inhibition of pan class I histone deacetylases (HDACs) was identified as a mechanism to increase apoE secretion. Knocking down select HDAC family members alone or in combination revealed that inhibition of the class I HDAC family was responsible for enhancing apoE secretion. Knocking down LXRα and LXRß genes revealed that the increase in astrocytic apoE in response to HDAC inhibition occurred via an LXR-independent pathway. Collectively, these data suggest that pan class I HDAC inhibition is a novel pathway for regulating astrocytic apoE secretion.

Concise, stereoselective approach to the spirooxindole ring system of citrinadin A.

The spirooxindole ring system of citrinadin A has been synthesized with excellent control over the absolute stereochemistry at the spirocenter. The key step involves a novel diastereoselective DMDO-mediated oxidative rearrangement employing an 8-phenylmenthol chiral auxiliary on the indole nitrogen.

Trifluoromethyl Oxetanes: Synthesis and Evaluation as a tert-Butyl Isostere.

The synthesis of a new trifluoromethyl oxetane was developed using a Corey-Chaykovsky epoxidation/ring-expansion reaction of trifluoromethyl ketones. The reaction was shown to proceed under mild conditions and displays a broad substrate scope. The trifluoromethyl oxetane was also evaluated as a tert-butyl isostere in the context of the γ-secretase modulator (GSM) program. We demonstrate that the trifluoromethyl oxetane-containing GSM has decreased lipophilicity, improved lipophilic efficiency (LipE) and metabolic stability relative to the corresponding tert-butyl GSM analogue, thus highlighting several benefits of trifluoromethyl oxetane as a more polar tert-butyl isostere.

Structural and Chemical Biology of Presenilin Complexes.

The presenilin proteins are the catalytic subunits of a tetrameric complex containing presenilin 1 or 2, anterior pharynx defective 1 (APH1), nicastrin, and PEN-2. Other components such as TMP21 may exist in a subset of specialized complexes. The presenilin complex is the founding member of a unique class of aspartyl proteases that catalyze the γ, ɛ, ζ site cleavage of the transmembrane domains of Type I membrane proteins including amyloid precursor protein (APP) and Notch. Here, we detail the structural and chemical biology of this unusual enzyme. Taken together, these studies suggest that the complex exists in several conformations, and subtle long-range (allosteric) shifts in the conformation of the complex underpin substrate access to the catalytic site and the mechanism of action for allosteric inhibitors and modulators. Understanding the mechanics of these shifts will facilitate the design of γ-secretase modulator (GSM) compounds that modulate the relative efficiency of γ, ɛ, ζ site cleavage and/or substrate specificity.

Discovery of cyclopropyl chromane-derived pyridopyrazine-1,6-dione γ-secretase modulators with robust central efficacy.

Herein we describe the discovery of a novel series of cyclopropyl chromane-derived pyridopyrazine-1,6-dione γ-secretase modulators for the treatment of Alzheimer's disease (AD). Using ligand-based design tactics such as conformational analysis and molecular modeling, a cyclopropyl chromane unit was identified as a suitable heterocyclic replacement for a naphthyl moiety that was present in the preliminary lead 4. The optimized lead molecule 44 achieved good central exposure resulting in robust and sustained reduction of brain amyloid-ß42 (Aß42) when dosed orally at 10 mg kg-1 in a rat time-course study. Application of the unpaced isolated heart Langendorff model enabled efficient differentiation of compounds with respect to cardiovascular safety, highlighting how minor structural changes can greatly impact the safety profile within a series of compounds.