Use of phosphotyrosine-containing peptides to target SH2 domains: Antagonist peptides of the Crk/CrkL-p130Cas axis.

Protein-protein interactions between SH2 domains and segments of proteins that include a post-translationally phosphorylated tyrosine residue (pY) underpin numerous signal transduction cascades that allow cells to respond to their environment. Dysregulation of the writing, erasing, and reading of these posttranslational modifications is a hallmark of human disease, notably cancer. Elucidating the precise role of the SH2 domain-containing adaptor proteins Crk and CrkL in tumor cell migration and invasion is challenging because there are no specific and potent antagonists available. Crk and CrkL SH2s interact with a region of the docking protein p130Cas containing 15 potential pY-containing tetrapeptide motifs. This chapter summarizes recent efforts toward peptide antagonists for this Crk/CrkL-p130Cas interaction. We describe our protocol for recombinant expression and purification of Crk and CrkL SH2s for functional assays and our procedure to determine the consensus binding motif from the p130Cas sequence. To develop a more potent antagonist, we employ methods often associated with structure-based drug design. Computational docking using Rosetta FlexPepDock, which accounts for peptides having a greater number of conformational degrees of freedom than small organic molecules that typically constitute libraries, provides quantitative docking metrics to prioritize candidate peptides for experimental testing. A battery of biophysical assays, including fluorescence polarization, differential scanning fluorimetry and saturation transfer difference nuclear magnetic resonance spectroscopy, were employed to assess the candidates. In parallel, GST pulldown competition assays characterized protein-protein binding in vitro. Taken together, our methodology yields peptide antagonists of the Crk/CrkL-p130Cas axis that will be used to validate targets, assess druggability, foster in vitro assay development, and potentially serve as lead compounds for therapeutic intervention.

Tuning the redox profile of the 6,6'-biazulenic platform through functionalization along its molecular axis.

The E1/2 potential associated with reduction of the linearly-functionalized 6,6'-biazulenic scaffold is accurately correlated to the combined σp Hammett parameters of the substituents over >600 mV range. X-ray crystallographic analysis of the 2,2'-dichloro-substituted derivative revealed unexpectedly short C-Cl bond distances, along with other metric changes, suggesting a non-trivial cycloheptafulvalene-like structural contribution.

Familial Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes.

Mutations that cause familial Alzheimer's disease (FAD) are found in amyloid precursor protein (APP) and presenilin, the catalytic component of γ-secretase, that together produce amyloid ß-peptide (Aß). Nevertheless, whether Aß is the primary disease driver remains controversial. We report here that FAD mutations disrupt initial proteolytic events in the multistep processing of APP substrate C99 by γ-secretase. Cryoelectron microscopy reveals that a substrate mimetic traps γ-secretase during the transition state, and this structure aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and in cellulo fluorescence microscopy support stabilization of enzyme-substrate complexes by FAD mutations. Neuronal expression of C99 and/or presenilin-1 in Caenorhabditis elegans leads to synaptic loss only with FAD-mutant transgenes. Designed mutations that stabilize the enzyme-substrate complex and block Aß production likewise led to synaptic loss. Collectively, these findings implicate the stalled process-not the products-of γ-secretase cleavage of substrates in FAD pathogenesis.

Vanadyl as a Spectroscopic Probe of Tunable Ligand Donor Strength in Bimetallic Complexes.

Incorporation of secondary metal ions into heterobimetallic complexes has emerged as an attractive strategy for rational tuning of compounds' properties and reactivity, but direct solution-phase spectroscopic interrogation of tuning effects has received less attention than it deserves. Here, we report the assembly and study of a series of heterobimetallic complexes containing the vanadyl ion, [VO]2+, paired with monovalent cations (Cs+, Rb+, K+, Na+, and Li+) and a divalent cation (Ca2+). These complexes, which can be isolated in pure form or generated in situ from a common monometallic vanadyl-containing precursor, enable experimental spectroscopic and electrochemical quantification of the influence of the incorporated cations on the properties of the vanadyl moiety. The data reveal systematic shifts in the V-O stretching frequency, isotropic hyperfine coupling constant for the vanadium center, and V(V)/V(IV) reduction potential in the complexes. These shifts can be interpreted as charge density effects parametrized through the Lewis acidities of the cations, suggesting broad potential for the vanadyl ion to serve as a spectroscopic probe in multimetallic species.

Insight into the Spatial Arrangement of the Lysine Tyrosylquinone and Cu2+ in the Active Site of Lysyl Oxidase-like 2.

Lysyl oxidase-2 (LOXL2) is a Cu2+ and lysine tyrosylquinone (LTQ)-dependent amine oxidase that catalyzes the oxidative deamination of peptidyl lysine and hydroxylysine residues to promote crosslinking of extracellular matrix proteins. LTQ is post-translationally derived from Lys653 and Tyr689, but its biogenesis mechanism remains still elusive. A 2.4 Å Zn2+-bound precursor structure lacking LTQ (PDB:5ZE3) has become available, where Lys653 and Tyr689 are 16.6 Å apart, thus a substantial conformational rearrangement is expected to take place for LTQ biogenesis. However, we have recently shown that the overall structures of the precursor (no LTQ) and the mature (LTQ-containing) LOXL2s are very similar and disulfide bonds are conserved. In this study, we aim to gain insights into the spatial arrangement of LTQ and the active site Cu2+ in the mature LOXL2 using a recombinant LOXL2 that is inhibited by 2-hydrazinopyridine (2HP). Comparative UV-vis and resonance Raman spectroscopic studies of the 2HP-inhibited LOXL2 and the corresponding model compounds and an EPR study of the latter support that 2HP-modified LTQ serves as a tridentate ligand to the active site Cu2. We propose that LTQ resides within 2.9 Å of the active site of Cu2+ in the mature LOXL2, and both LTQ and Cu2+ are solvent-exposed.

Cellular Target Deconvolution of Small Molecules Using a Selection-Based Genetic Screening Platform.

Small-molecule drug target identification is an essential and often rate-limiting step in phenotypic drug discovery and remains a major challenge. Here, we report a novel platform for target identification of activators of signaling pathways by leveraging the power of a clustered regularly interspaced short palindromic repeats (CRISPR) knockout library. This platform links the expression of a suicide gene to the small-molecule-activated signaling pathway to create a selection system. With this system, loss-of-function screening using a CRISPR single-guide (sg) RNA library positively enriches cells in which the target has been knocked out. The identities of the drug targets and other essential genes required for the activity of small molecules of interest are then uncovered by sequencing. We tested this platform on BDW568, a newly discovered type-I interferon signaling activator, and identified stimulator of interferon genes (STING) as its target and carboxylesterase 1 (CES1) to be a key metabolizing enzyme required to activate BDW568 for target engagement. The platform we present here can be a general method applicable for target identification for a wide range of small molecules that activate different signaling pathways.

Recognition of single-stranded nucleic acids by small-molecule splicing modulators.

Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.

Do Macrocyclic Peptide Drugs Interact with Bile Salts under Simulated Gastrointestinal Conditions?

Peptide drugs face several barriers to oral delivery, including enzymatic degradation in the gastrointestinal tract and low membrane permeability. Importantly, the direct interaction between various biorelevant colloids (i.e., bile salt micelles and bile salt-phospholipid mixed micelles) present in the aqueous gastrointestinal environment and peptide drug molecules has not been studied. In this work, we systematically characterized interactions between a water-soluble model peptide drug, octreotide, and a range of physiologically relevant bile salts in solution. Octreotide membrane flux in pure bile salt solutions and commercially available biorelevant media, i.e., fasted state simulated intestinal fluid (FaSSIF) and fed state simulated intestinal fluid (FeSSIF), was evaluated using a side-by-side diffusion cell equipped with a cellulose dialysis membrane. All seven micellar bile salt solutions as well as FaSSIF and FeSSIF decreased octreotide membrane flux, and dihydroxy bile salts were found to have a much larger effect than trihydroxy bile salts. An inverse relationship between octreotide membrane flux and pancreatic enzymatic stability was also observed; bile salt micelles and bile salt-phospholipid mixed micelles provided a protective effect toward enzymatic degradation and prolonged octreotide half-life in vitro. Diffusion ordered nuclear magnetic resonance (DOSY NMR) spectroscopy and dynamic light scattering (DLS) were used as complementary experimental techniques to confirm peptide-micelle interactions in solution. Experiments were also performed using desmopressin as a second model peptide drug; desmopressin interacted with bile salts in solution, albeit to a lower extent relative to octreotide. The findings described herein demonstrate that amphiphilic, water-soluble peptide drugs do interact with bile salts and phospholipids in solution, with an effect on peptide membrane flux and enzymatic stability. Correspondingly, oral peptide drug absorption and bioavailability may be impacted.

The 15-Amino Acid Repeat Region of Adenomatous Polyposis Coli Is Intrinsically Disordered and Retains Conformational Flexibility upon Binding ß-Catenin.

The tumor suppressor Adenomatous polyposis coli (APC) is a large, multidomain protein with many identified cellular functions. The best characterized role of APC is to scaffold a protein complex that negatively regulates Wnt signaling via ß-catenin destruction. This destruction is mediated by ß-catenin binding to centrally located 15- and 20-amino acid repeat regions of APC. More than 80% of cancers of the colon and rectum present with an APC mutation. Most carcinomas with mutant APC express a truncated APC protein that retains the ∼200-amino acid long' 15-amino acid repeat region'. This study demonstrates that the 15-amino acid repeat region of APC is intrinsically disordered. We investigated the backbone dynamics in the presence of ß-catenin and predicted residues that may contribute to transient secondary features. This study reveals that the 15-amino acid region of APC retains flexibility upon binding ß-catenin and that APC does not have a single, observable "highest-affinity" binding site for ß-catenin. This flexibility potentially allows ß-catenin to be more readily captured by APC and then remain accessible to other elements of the destruction complex for subsequent processing.

Cobalt-Catalyzed Selective Unsymmetrical Dioxidation of gem-Difluoroalkenes.

gem-Difluoroalkenes represent valuable synthetic handles for organofluorine chemistry; however, most reactions of this substructure proceed through reactive intermediates prone to eliminate a fluorine atom and generate monofluorinated products. Taking advantage of the distinct reactivity of gem-difluoroalkenes, we present a cobalt-catalyzed regioselective unsymmetrical dioxygenation of gem-difluoroalkenes using phenols and molecular oxygen, which retains both fluorine atoms and provides ß-phenoxy-ß,ß-difluorobenzyl alcohols. Mechanistic studies suggest that the reaction operates through a radical chain process initiated by Co(II)/O2/phenol and quenched by the Co-based catalyst. This mechanism enables the retention of both fluorine atoms, which contrasts most transition-metal-catalyzed reactions of gem-difluoroalkenes that typically involve defluorination.

Design of Substrate Transmembrane Mimetics as Structural Probes for γ-Secretase.

γ-Secretase is a membrane-embedded aspartyl protease complex central in biology and medicine. How this enzyme recognizes transmembrane substrates and catalyzes hydrolysis in the lipid bilayer is unclear. Inhibitors that mimic the entire substrate transmembrane domain and engage the active site should provide important tools for structural biology, yielding insight into substrate gating and trapping the protease in the active state. Here, we report transmembrane peptidomimetic inhibitors of the γ-secretase complex that contain an N-terminal helical peptide region that engages a substrate docking exosite and a C-terminal transition-state analog moiety targeted to the active site. Both regions are required for stoichiometric inhibition of γ-secretase. Moreover, enzyme inhibition kinetics and photoaffinity probe displacement experiments demonstrate that both the docking exosite and the active site are engaged by the bipartite inhibitors. The solution conformations of these potent transmembrane-mimetic inhibitors are similar to those of bound natural substrates, suggesting these probes are preorganized for high-affinity binding and should allow visualization of the active γ-secretase complex, poised for intramembrane proteolysis, by cryo-electron microscopy.

NMR Studies of a MnIII-hydroxo Adduct Reveal an Equilibrium between MnIII-hydroxo and µ-Oxodimanganese(III,III) Species.

The solution properties of MnIII-hydroxo and MnIII-methoxy complexes featuring N5 amide-containing ligands were investigated using 1H NMR spectroscopy. The 1H NMR spectrum for one of these complexes, the previously reported [MnIII(OH)(dpaq)](OTf) (dpaq = 2-[bis(pyridin-2-ylmethyl)]amino- N-quinolin-8-yl-acetamidate) shows hyperfine-shifted signals, as expected for this S = 2 MnIII-hydroxo adduct. However, the 1H NMR spectrum of [MnIII(OH)(dpaq)](OTf) also shows a large number of proton resonances in the diamagnetic region, suggesting the presence of multiple species in CD3CN solution. The majority of the signals in the diamagnetic region disappear when a small amount of water is added to a CH3CN solution of [MnIII(OH)(dpaq)](OTf). Electronic absorption and Mn K-edge X-ray absorption experiments support the formulation of this diamagnetic species as the µ-oxodimanganese(III,III) complex [MnIII2(µ-O)(dpaq)2)]2+. On the basis of these observations, we propose that the dissolution of [MnIII(OH)(dpaq)](OTf) in CD3CN results in the formation of mononuclear MnIII-hydroxo and dinuclear µ-oxodimanganese(III,III) species that are in equilibrium. The addition of a small amount of water is sufficient to shift this equilibrium in favor of the MnIII-hydroxo adduct. Surprisingly, electronic absorption experiments show that the conversion of [MnIII2(µ-O)(dpaq)2)]2+ to [MnIII(OH)(dpaq)]+ by added water is relatively slow. Because this dimer to monomer conversion is slower than TEMPOH oxidation by [MnIII(OH)(dpaq)]+, the previously observed TEMPOH oxidation rates for [MnIII(OH)(dpaq)]+ reflected both processes. Here, we report the bona fide TEMPOH oxidation rate for [MnIII(OH)(dpaq)]+, which is significantly faster than previously reported. 1H NMR spectra are also reported for the related [MnIII(OMe)(dpaq)]+ and [MnIII(OH)(dpaq2Me)]+ complexes. These spectra only show hyperfine-shifted signals, suggesting the presence of only mononuclear MnIII-methoxy and MnIII-hydroxo species in solution. Measurements of T1 relaxation times and proton peak integrations for [MnIII(OMe)(dpaq)]+ provide preliminary assignments for 1H NMR resonances.

Mitochondrial Dysfunction Triggers Synaptic Deficits via Activation of p38 MAP Kinase Signaling in Differentiated Alzheimer's Disease Trans-Mitochondrial Cybrid Cells.

Loss of synapse and synaptic dysfunction contribute importantly to cognitive impairment in Alzheimer's disease (AD). Mitochondrial dysfunction and oxidative stress are early pathological features in AD-affected brain. However, the effect of AD mitochondria on synaptogenesis remains to be determined. Using human trans-mitochondrial "cybrid" (cytoplasmic hybrid) neuronal cells whose mitochondria were transferred from platelets of patients with sporadic AD or age-matched non-AD subjects with relatively normal cognition, we provide the first evidence of mitochondrial dysfunction compromises synaptic development and formation of synapse in AD cybrid cells in response to chemical-induced neuronal differentiation. Compared to non-AD control cybrids, AD cybrid cells showed synaptic loss which was evidenced by a significant reduction in expression of two synaptic marker proteins: synaptophysin (presynaptic marker) and postsynaptic density protein-95, and neuronal proteins (MAP-2 and NeuN) upon neuronal differentiation. In parallel, AD-mediated synaptic deficits correlate to mitochondrial dysfunction and oxidative stress as well as activation of p38 MAP kinase. Notably, inhibition of p38 MAP kinase by pharmacological specific p38 inhibitor significantly increased synaptic density, improved mitochondrial function, and reduced oxidative stress. These results suggest that activation of p38 MAP kinase signaling pathway contributes to AD-mediated impairment in neurogenesis, possibly by inhibiting the neuronal differentiation. Our results provide new insight into the crosstalk of dysfunctional AD mitochondria to synaptic formation and maturation via activation of p38 MAP kinase. Therefore, blockade of p38 MAP kinase signal transduction could be a potential therapeutic strategy for AD by alleviating loss of synapses.

Human oncoprotein Musashi-2 N-terminal RNA recognition motif backbone assignment and identification of RNA-binding pocket.

RNA-binding protein Musashi-2 (MSI2) is a key regulator in stem cells, it is over-expressed in a variety of cancers and its higher expression is associated with poor prognosis. Like Musashi-1, it contains two N-terminal RRMs (RNA-recognition Motifs, also called RBDs (RNA-binding Domains)), RRM1 and RRM2, which mediate the binding to their target mRNAs. Previous studies have obtained the three-dimensional structures of the RBDs of Musashi-1 and the RBD1:RNA complex. Here we show the binding of MSI2-RRM1 to a 15nt Numb RNA in Fluorescence Polarization assay and time resolved Fluorescence Resonance Energy Transfer assay. Using nuclear magnetic resonance (NMR) spectroscopy we assigned the backbone resonances of MSI2-RRM1, and characterized the direct interaction of RRM1 to Numb RNA r(GUAGU). Our NMR titration and structure modeling studies showed that MSI2-RRM1 and MSI1-RBD1 have similar RNA binding events and binding pockets. This work adds significant information to MSI2-RRM1 structure and RNA binding pocket, and contributes to the development of MSI2 specific and MSI1/MSI2 dual inhibitors.

F1F0 ATP Synthase-Cyclophilin D Interaction Contributes to Diabetes-Induced Synaptic Dysfunction and Cognitive Decline.

Mitochondrial abnormalities are well known to cause cognitive decline. However, the underlying molecular basis of mitochondria-associated neuronal and synaptic dysfunction in the diabetic brain remains unclear. Here, using a mitochondrial single-channel patch clamp and cyclophilin D (CypD)-deficient mice (Ppif -/-) with streptozotocin-induced diabetes, we observed an increase in the probability of Ca2+-induced mitochondrial permeability transition pore (mPTP) opening in brain mitochondria of diabetic mice, which was further confirmed by mitochondrial swelling and cytochrome c release induced by Ca2+ overload. Diabetes-induced elevation of CypD triggers enhancement of F1F0 ATP synthase-CypD interaction, which in turn leads to mPTP opening. Indeed, in patients with diabetes, brain cypD protein levels were increased. Notably, blockade of the F1F0 ATP synthase-CypD interaction by CypD ablation protected against diabetes-induced mPTP opening, ATP synthesis deficits, oxidative stress, and mitochondria dysfunction. Furthermore, the absence of CypD alleviated deficits in synaptic plasticity, learning, and memory in diabetic mice. Thus, blockade of ATP synthase interaction with CypD provides a promising new target for therapeutic intervention in diabetic encephalopathy.

Increased Electron Paramagnetic Resonance Signal Correlates with Mitochondrial Dysfunction and Oxidative Stress in an Alzheimer's disease Mouse Brain.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized clinically by cognitive decline and memory loss. The pathological features are amyloid-ß peptide (Aß) plaques and intracellular neurofibrillary tangles. Many studies have suggested that oxidative damage induced by reactive oxygen species (ROS) is an important mechanism for AD progression. Our recent study demonstrated that oxidative stress could further impair mitochondrial function. In the present study, we adopted a transgenic mouse model of AD (mAPP, overexpressing AßPP/Aß in neurons) and performed redox measurements using in vivo electron paramagnetic resonance (EPR) imaging with methoxycarbamyl-proxyl (MCP) as a redox-sensitive probe for studying oxidative stress in an early stage of pathology in a transgenic AD mouse model. Through assessing oxidative stress, mitochondrial function and cognitive behaviors of mAPP mice at the age of 8-9 months, we found that oxidative stress and mitochondrial dysfunction appeared in the early onset of AD. Increased ROS levels were associated with defects of mitochondrial and cognitive dysfunction. Notably, the in vivo EPR method offers a unique way of assessing tissue oxidative stress in living animals under noninvasive conditions, and thus holds a potential for early diagnosis and monitoring the progression of AD.

Combining hard and soft magnetism into a single core-shell nanoparticle to achieve both hyperthermia and image contrast.

BACKGROUND: A biocompatible core/shell structured magnetic nanoparticles (MNPs) was developed to mediate simultaneous cancer therapy and imaging. METHODS & RESULTS: A 22-nm MNP was first synthesized via magnetically coupling hard (FePt) and soft (Fe3O4) materials to produce high relative energy transfer. Colloidal stability of the FePt@Fe3O4 MNPs was achieved through surface modification with silane-polyethylene glycol (PEG). Intravenous administration of PEG-MNPs into tumor-bearing mice resulted in a sustained particle accumulation in the tumor region, and the tumor burden of treated mice was a third that of the mice in control groups 2 weeks after a local hyperthermia treatment. In vivo magnetic resonance imaging exhibited enhanced T2 contrast in the tumor region. CONCLUSION: This work has demonstrated the feasibility of cancer theranostics with PEG-MNPs.

Dual Catalytic Decarboxylative Allylations of α-Amino Acids and Their Divergent Mechanisms.

The room temperature radical decarboxylative allylation of N-protected α-amino acids and esters has been accomplished via a combination of palladium and photoredox catalysis to provide homoallylic amines. Mechanistic investigations revealed that the stability of the α-amino radical, which is formed by decarboxylation, dictates the predominant reaction pathway between competing mechanisms.

Constrained TRPV1 agonists synthesized via silver-mediated intramolecular azo-methine ylide cycloaddition of α-iminoamides.

As part of an effort to identify agonists of TRPV1, a peripheral sensory nerve ion channel, high throughput screening of the NIH Small Molecule Repository (SMR) collection identified MLS002174161, a pentacyclic benzodiazepine. A synthesis effort was initiated that ultimately afforded racemic seco analogs 12 of the SMR compound via a silver mediated intramolecular [3+2] cycloaddition of an azo-methine ylide generated from α-iminoamides 11. The cycloaddition set four contiguous stereocenters and, in some cases, also spontaneously afforded imides 13 from 12. The synthesis of compounds 12, the features that facilitated the conversion of 12-13, and their partial agonist activity against TRPV1 are discussed.

Copper-mediated deoxygenative trifluoromethylation of benzylic xanthates: generation of a C-CF(3) bond from an O-based electrophile.

The conversion of an alcohol-based functional group, into a trifluoromethyl analogue is a desirable transformation. However, few methods are capable of converting O-based electrophiles into trifluoromethanes. The copper-mediated trifluoromethylation of benzylic xanthates using Umemoto's reagent as the source of CF3 to form C-CF3 bonds is described. The method is compatible with an array of benzylic xanthates bearing useful functional groups. A preliminary mechanistic investigation suggests that the C-CF3 bond forms by reaction of the substrate with in situ generated CuCF3 and CuOTf. Further evidence suggests that the reaction could proceed via a radical cation intermediate.