Discovery of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic approach for Alzheimer's disease.

Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module strongly linked to Alzheimer's disease (AD) traits and microglia. These proteins are more abundant in Alzheimer's patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) and the cytoplasmic tail of CD44. Inhibiting the MSN-CD44 interaction may help limit AD-associated neuronal damage. Here, we investigated the feasibility of developing inhibitors that target this protein-protein interaction. We have employed structural, mutational, and phage-display studies to examine how CD44 binds to the FERM domain of MSN. Interestingly, we have identified an allosteric site located close to the PIP2 binding pocket that influences CD44 binding. These findings suggest a mechanism in which PIP2 binding to the FERM domain stimulates CD44 binding through an allosteric effect, leading to the formation of a neighboring pocket capable of accommodating a receptor tail. Furthermore, high-throughput screening of a chemical library identified two compounds that disrupt the MSN-CD44 interaction. One compound series was further optimized for biochemical activity, specificity, and solubility. Our results suggest that the FERM domain holds potential as a drug development target. Small molecule preliminary leads generated from this study could serve as a foundation for additional medicinal chemistry efforts with the goal of controlling microglial activity in AD by modifying the MSN-CD44 interaction.

Development of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic strategy for Alzheimer's disease.

Recent genome-wide association studies have revealed genetic risk factors for Alzheimer's disease (AD) that are exclusively expressed in microglia within the brain. A proteomics approach identified moesin (MSN), a FERM (four-point-one ezrin radixin moesin) domain protein, and the receptor CD44 as hub proteins found within a co-expression module strongly linked to AD clinical and pathological traits as well as microglia. The FERM domain of MSN interacts with the phospholipid PIP2 and the cytoplasmic tails of receptors such as CD44. This study explored the feasibility of developing protein-protein interaction inhibitors that target the MSN-CD44 interaction. Structural and mutational analyses revealed that the FERM domain of MSN binds to CD44 by incorporating a beta strand within the F3 lobe. Phage-display studies identified an allosteric site located close to the PIP2 binding site in the FERM domain that affects CD44 binding within the F3 lobe. These findings support a model in which PIP2 binding to the FERM domain stimulates receptor tail binding through an allosteric mechanism that causes the F3 lobe to adopt an open conformation permissive for binding. High-throughput screening of a chemical library identified two compounds that disrupt the MSN-CD44 interaction, and one compound series was further optimized for biochemical activity, specificity, and solubility. The results suggest that the FERM domain holds potential as a drug development target. The small molecule preliminary leads generated from the study could serve as a foundation for additional medicinal chemistry effort with the goal of controlling microglial activity in AD by modifying the MSN-CD44 interaction.

Use of AD Informer Set compounds to explore validity of novel targets in Alzheimer's disease pathology.

Introduction: A chemogenomic set of small molecules with annotated activities and implicated roles in Alzheimer's disease (AD) called the AD Informer Set was recently developed and made available to the AD research community: https://treatad.org/data-tools/ad-informer-set/. Methods: Small subsets of AD Informer Set compounds were selected for AD-relevant profiling. Nine compounds targeting proteins expressed by six AD-implicated genes prioritized for study by Target Enablement to Accelerate Therapy Development for Alzheimer's Disease (TREAT-AD) teams were selected for G-protein coupled receptor (GPCR), amyloid beta (Aß) and tau, and pharmacokinetic (PK) studies. Four non-overlapping compounds were analyzed in microglial cytotoxicity and phagocytosis assays. Results: The nine compounds targeting CAPN2, EPHX2, MDK, MerTK/FLT3, or SYK proteins were profiled in 46 to 47 primary GPCR binding assays. Human induced pluripotent stem cell (iPSC)-derived neurons were treated with the same nine compounds and secretion of Aß peptides (Aß40 and Aß42) as well as levels of phosphophorylated tau (p-tau, Thr231) and total tau (t-tau) peptides measured at two concentrations and two timepoints. Finally, CD1 mice were dosed intravenously to determine preliminary PK and/or brain-specific penetrance values for these compounds. As a final cell-based study, a non-overlapping subset of four compounds was selected based on single-concentration screening for analysis of both cytotoxicity and phagocytosis in murine and human microglia cells. Discussion: We have demonstrated the utility of the AD Informer Set in the validation of novel AD hypotheses using biochemical, cellular (primary and immortalized), and in vivo studies. The selectivity for their primary targets versus essential GPCRs in the brain was established for our compounds. Statistical changes in tau, p-tau, Aß40, and/or Aß42 and blood-brain barrier penetrance were observed, solidifying the utility of specific compounds for AD. Single-concentration phagocytosis results were validated as predictive of dose-response findings. These studies established workflows, validated assays, and illuminated next steps for protein targets and compounds.

AD Informer Set: Chemical tools to facilitate Alzheimer's disease drug discovery.

Introduction: The portfolio of novel targets to treat Alzheimer's disease (AD) has been enriched by the Accelerating Medicines Partnership Program for Alzheimer's Disease (AMP AD) program. Methods: Publicly available resources, such as literature and databases, enabled a data-driven effort to identify existing small molecule modulators for many protein products expressed by the genes nominated by AMP AD and suitable positive control compounds to be included in the set. Compounds contained within the set were manually selected and annotated with associated published, predicted, and/or experimental data. Results: We built an annotated set of 171 small molecule modulators targeting 98 unique proteins that have been nominated by AMP AD consortium members as novel targets for the treatment of AD. The majority of compounds included in the set are inhibitors. These small molecules vary in their quality and should be considered chemical tools that can be used in efforts to validate therapeutic hypotheses, but which will require further optimization. A physical copy of the AD Informer Set can be requested on the Target Enablement to Accelerate Therapy Development for Alzheimer's Disease (TREAT-AD) website. Discussion: Small molecules that enable target validation are important tools for the translation of novel hypotheses into viable therapeutic strategies for AD.

Identification of Pyrimidine-Based Lead Compounds for Understudied Kinases Implicated in Driving Neurodegeneration.

The pyrimidine core has been utilized extensively to construct kinase inhibitors, including eight FDA-approved drugs. Because the pyrimidine hinge-binding motif is accommodated by many human kinases, kinome-wide selectivity of resultant molecules can be poor. This liability was seen as an advantage since it is well tolerated by many understudied kinases. We hypothesized that nonexemplified aminopyrimidines bearing side chains from well-annotated pyrimidine-based inhibitors with off-target activity on understudied kinases would provide us with useful inhibitors of these lesser studied kinases. Our strategy paired mixing and matching the side chains from the 2- and 4-positions of the parent compounds with modifications at the 5-position of the pyrimidine core, which is situated near the gatekeeper residue of the binding pocket. Utilizing this approach, we imparted improved kinome-wide selectivity to most members of the resultant library. Importantly, we also identified potent biochemical and cell-active lead compounds for understudied kinases like DRAK1, BMP2K, and MARK3/4.

Dihydroquinolines, Dihydronaphthyridines and Quinolones by Domino Reactions of Morita-Baylis-Hillman Acetates.

An efficient synthetic route to highly substituted dihydroquinolines and dihydronaphthyridines has been developed using a domino reaction of Morita-Baylis-Hillman (MBH) acetates with primary aliphatic and aromatic amines in DMF at 50-90 °C. The MBH substrates incorporate a side chain acetate positioned adjacent to an acrylate or acrylonitrile aza-Michael acceptor as well as an aromatic ring activated toward SNAr ring closure. A control experiment established that the initial reaction was an SN2'-type displacement of the side chain acetate by the amine to generate the alkene product with the added nitrogen nucleophile positioned trans to the SNAr aromatic ring acceptor. Thus, equilibration of the initial alkene geometry is required prior to cyclization. A further double bond migration was observed for several reactions targeting dihydronaphthyridines from substrates with a side chain acrylonitrile moiety. MBH acetates incorporating a 2,5-difluorophenyl moiety were found to have dual reactivity in these annulations. In the absence of O2, the expected dihydroquinolines were formed, while in the presence of O2, quinolones were produced. All of the products were isolated in good to excellent yields (72-93%). Numerous cases (42) are reported, and mechanisms are discussed.

Small Molecule Inhibitors of the Bacterioferritin (BfrB)-Ferredoxin (Bfd) Complex Kill Biofilm-Embedded Pseudomonas aeruginosa Cells.

Bacteria depend on a well-regulated iron homeostasis to survive adverse environments. A key component of the iron homeostasis machinery is the compartmentalization of Fe3+ in bacterioferritin and its subsequent mobilization as Fe2+ to satisfy metabolic requirements. In Pseudomonas aeruginosa Fe3+ is compartmentalized in bacterioferritin (BfrB), and its mobilization to the cytosol requires binding of a ferredoxin (Bfd) to reduce the stored Fe3+ and release the soluble Fe2+. Blocking the BfrB-Bfd complex in P. aeruginosa by deletion of the bfd gene triggers an irreversible accumulation of Fe3+ in BfrB, concomitant cytosolic iron deficiency and significant impairment of biofilm development. Herein we report that small molecules developed to bind BfrB at the Bfd binding site block the BfrB-Bfd complex, inhibit the mobilization of iron from BfrB in P. aeruginosa cells, elicit a bacteriostatic effect on planktonic cells, and are bactericidal to cells embedded in mature biofilms.

Naphthalenes and Quinolines by Domino Reactions of Morita-Baylis-Hillman Acetates.

An efficient synthetic route to highly functionalized naphthalenes and quinolines has been developed using domino reactions between Morita-Baylis-Hillman (MBH) acetates and active methylene compounds (AMCs) promoted by anhydrous K2CO3 in dry N,N-dimethylformamide (DMF) at 23 °C. The substrates incorporate allylic acetates positioned adjacent to a Michael acceptor as well as an aromatic ring activated toward a SNAr ring closure. A control experiment indicated that the initial reaction was an SN2'-type displacement of a side chain acetoxy by the AMC anion to afford the alkene product bearing the added nucleophile trans to the SNAr aromatic ring acceptor. Thus, equilibration of the alkene geometry of the initial product was required prior to cyclization. Products were isolated in good to excellent yields. Numerous cases (24) are reported, and several mechanistic possibilities are discussed.

4H-Benzo[d][1,3]oxazin-4-ones and Dihydro Analogs from Substituted Anthranilic Acids and Orthoesters.

A one-pot route to 2-alkyl and 2-aryl-4H-benzo[d][1,3]oxazin-4-ones (also known as 4H-3,1-benzoxazin-4-ones) has been developed and studied. The method involves the reaction of aryl-substituted anthranilic acids with orthoesters in ethanol catalyzed by acetic acid. Additionally, we have also investigated the reaction under microwave conditions. Not all of the substrates were successful in yielding the target heterocycles as some of the reactions failed to undergo the final elimination. This process led to the isolation of (±)-2-alkyl/aryl-2-ethoxy-1,2-dihydro-4H-benzo[d][1,3]oxazin-4-ones. The formation of the dihydro analogs correlated with the electron density on the aromatic ring: Electron-donating groups favored the 4H- benzo[d][1,3]oxazin-4-ones, while electron-withdrawing groups tended to favor the dihydro product. Substituting a pyridine ring for the benzene ring in the substrate acid suppressed the reaction.

Quinazolin-4(3H)-ones and 5,6-Dihydropyrimidin-4(3H)-ones from ß-Aminoamides and Orthoesters.

Quinazolin-4(3H)-ones have been prepared in one step from 2-aminobenzamides and orthoesters in the presence of acetic acid. Simple 2-aminobenzamides were easily converted to the heterocycles by refluxing in absolute ethanol with 1.5 equivalents of the orthoester and 2 equivalents of acetic acid for 12⁻24 h. Ring-substituted and hindered 2-aminobenzamides as well as cases incorporating an additional basic nitrogen required pressure tube conditions with 3 equivalents each of the orthoester and acetic acid in ethanol at 110 °C for 12⁻72 h. The reaction was tolerant towards functionality on the benzamide and a range of structures was accessible. Workup involved removal of the solvent under vacuum and either recrystallization from ethanol or trituration with ether-pentane. Several 5,6-dihydropyrimidin-4(3H)-ones were also prepared from 3-amino-2,2-dimethylpropionamide. All products were characterized by melting point, FT-IR, ¹H-NMR, 13C-NMR, and HRMS.

Syntheses of 1-Aryl-5-nitro-1H-indazoles and a General One-Pot Route to 1-Aryl-1H-indazoles.

An efficient route to substituted 1-aryl-1H-indazoles has been developed and optimized. The method involved the preparation of arylhydrazones from acetophenone or benzaldehyde substituted by fluorine at C2 and nitro at C5, followed by deprotonation and nucleophilic aromatic substitution (SNAr) ring closure in 45-90%. Modification of this procedure to a one-pot domino process was successful in the acetophenone series (73-96%), while the benzaldehyde series (63-73%) required a step-wise addition of reagents. A general one-pot protocol for 1-aryl-1H-indazole formation without the limiting substitution patterns required for the SNAr cyclization has also been achieved in 62-78% yields. A selection of 1-aryl-1H-indazoles was prepared in high yield by a procedure that requires only a single laboratory operation.

Synthesis and biological activity of fused tetracyclic Pyrrolo[2,1-c][1,4]benzodiazepines.

Cancer remains the second major cause of death in the world. Thus, there is a pressing need to identify potential synthetic route for the development of novel anticancer agents which will serve as lead compounds to effectively combat this life-threatening epidemic. Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) have sparked a great interest as lead compounds because of their cancerostatic and anti-infective properties. The twisted molecular structure of PBD analogs provides both helical and chiral elements. In an effort to expand novel PBDs that interact with the key exocyclic amino group of the DNA-guanine base, we hypothesized that construction of a fused cyclic active system, would likely serve as an electrophilic site when compared to traditional electrophilic C11-N10 imine group. To examine our theory, we report herein the synthesis and cell viability/cytotoxicity of a series of PBD analogs using NCI-60 cell lines screening. Thus, compounds 1-13 were synthesized and fully characterized. The selected PBDs were found to have marginal inhibition of growth, up to 30%, for certain cell lines.