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.

Twisted Intramolecular Charge-Transfer State Addition to Electron-Poor Olefins.

When electron-rich arylpyrrolinium salts are irradiated with ultraviolet light in the presence of Michael acceptors, the pyrrolinyl and aryl fragments add to the activated and polarized double bond in a regioselective manner, forming two C-C bonds and fragmenting the substrate. In this paper, we present a model for this intriguing reaction, supported by spectroscopy and computational analyses, and provide evidence for rectifying previously misassigned structures. We postulate that the photochemical reaction is inefficient because the reaction between the twisted intramolecular charge-transfer state and the olefin competes with fluorescence from this state upon photon absorption. We also discuss the practical advantages of performing this photochemical reaction in a continuous flow setup. Additionally, we explore several subsequent reactions that allow us to further modify the products of the photochemical step, ultimately leading to the creation of new chemical structures.

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.

Histidine Protonation and Conformational Switching in Diphtheria Toxin Translocation Domain.

Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for the delivery of the catalytic domain into the cell. Here, we use a combination of computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, and fluorescence spectroscopy along with the X-ray crystallography) approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H223Q/H257Q double mutant. This replacement suppresses the acid-induced transition, resulting in the retention of a more stable protein structure in solutions at pH 5.5 and, consequently, in reduced membrane-disrupting activity. Here, for the first time, we report the pKa values of the histidine residues of the T-domain, measured by NMR-monitored pH titrations. Most peaks in the histidine side chain spectral region are titrated with pKas ranging from 6.2 to 6.8. However, the two most up-field peaks display little change down to pH 6, which is a limiting pH for this protein in solution at concentrations required for NMR. These peaks are absent in the double mutant, suggesting they belong to H223 and H257. The constant-pH simulations indicate that for the T-domain in solution, the pKa values for histidine residues range from 3.0 to 6.5, with those most difficult to protonate being H251 and H257. Taken together, our experimental and computational data demonstrate that previously suggested cooperative protonation of all six histidines in the T-domain does not occur.

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.

Isolation and Identification of 3,4-Seco-Solanidine-3,4-dioic acid (SSDA) as a Urinary Biomarker of Cytochrome P450 2D6 (CYP2D6) Activity.

Cytochrome P450 2D6 (CYP2D6), is responsible for the metabolism and elimination of approximately 25% of clinically used drugs, including antidepressants and antipsychotics, and its activity varies considerably on a population basis primary due to genetic variation. CYP2D6 phenotype can be assessed in vivo following administration of an exogenous probe compound, such as dextromethorphan or debrisoquine, but use of a biomarker that does not require administration of an exogenous compound (i.e., drug) has considerable appeal for assessing CYP2D6 activity in vulnerable populations, such as children. The goal of this study was to isolate, purify and identify an "endogenous" urinary biomarker (M1; m/z 444.3102) of CYP2D6 activity reported previously. Several chromatographic separation techniques (reverse phase HPLC, cation exchange and analytical reverse phase UPLC) were used to isolate and purify 96 µg of M1 from 40 L of urine. Subsequently, 1D and 2D NMR, and functional group modification reactions were used to elucidate its structure. Analysis of mass spectrometry and NMR data revealed M1 to have similar spectroscopic features to the nitrogen-containing steroidal alkaloid, solanidine. 2D NMR characterization by HMBC, COSY, TOCSY, and HSQC-TOCSY proved to be invaluable in the structural elucidation of M1; derivatization of M1 revealed the presence of two carboxylic acid moieties. M1 was determined to be a steroidal alkaloid with a solanidine backbone that had undergone C-C bond scission to yield 3,4-seco-solanidine-3,4-dioic acid (SSDA). SSDA may have value as a dietary biomarker of CYP2D6 activity in populations where potato consumption is common. Significance Statement Endogenous biomarkers of processes involved in drug disposition and response may allow improved individualization of drug treatment, especially in vulnerable populations, such as children. Given that several CYP2D6 substrates are commonly used in pediatrics and the ubiquitous nature of potato consumption in western diets, SSDA has considerable appeal as non-invasive biomarker of CYP2D6 activity to guide treatment with CYP2D6 substrates in children and adults.

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.