Discovery, structure-activity relationship studies, and crystal structure of nonpeptide inhibitors bound to the Shank3 PDZ domain

Shank is the central scaffolding protein of the postsynaptic density (PSD) protein complex found in cells of the central nervous system. Cellular studies indicate a prominent role of the protein in the organization of the PSD, in the development of neuronal morphology, in neuronal signaling, and in synaptic plasticity, thus linking Shank functions to the molecular basis of learning and memory. Mutations in the Shank gene have been found in several neuronal disorders including mental retardation, typical autism, and Asperger syndrome. Shank is linked to the PSD complex via its PDZ domain that binds to the C‐terminus of guanylate‐kinase‐associated protein (GKAP). Here, small‐molecule inhibitors of Shank3 PDZ domain are developed. A fluorescence polarization assay based on an identified high‐affinity peptide is established, and tetrahydroquinoline carboxylates are identified as inhibitors of this protein–protein interaction. Chemical synthesis via a hetero‐Diels–Alder strategy is employed for hit optimization, and structure–activity relationship studies are performed. Best hits possess K_i values in the 10 μM range, and binding to the PDZ domain is confirmed by ¹H,¹⁵N HSQC NMR experiments. One of the hits crystallizes with the Shank3 PDZ domain. The structure, analyzed at a resolution of 1.85 Å, reveals details of the binding mode. Finally, binding to PDZ domains of PSD‐95, syntrophin, and DVL3 was studied using ¹H,¹⁵N HSQC NMR spectroscopy.     

Small‐Molecule Inhibitors of AF6 PDZ‐Mediated Protein–Protein Interactions

PDZ (PSD‐95, Dlg, ZO‐1) domains are ubiquitous interaction modules that are involved in many cellular signal transduction pathways. Interference with PDZ‐mediated protein–protein interactions has important implications in disease‐related signaling processes. For this reason, PDZ domains have gained attention as potential targets for inhibitor design and, in the long run, drug development. Herein we report the development of small molecules to probe the function of the PDZ domain from human AF6 (ALL1‐fused gene from chromosome 6), which is an essential component of cell–cell junctions. These compounds bind to AF6 PDZ with substantially higher affinity than the peptide (Ile‐Gln‐Ser‐Val‐Glu‐Val) derived from its natural ligand, EphB2. In intact cells, the compounds inhibit the AF6–Bcr interaction and interfere with epidermal growth factor (EGF)‐dependent signaling.     

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein–protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling‐transduction complexes. In recent years, PDZ domains have emerged as novel and exciting drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C‐terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys/Arg residue in the ligand‐binding site of the second PDZ domain of PSD‐95, by employing a semisynthetic approach. We generated six semisynthetic PDZ domains comprising different proteogenic and nonproteogenic amino acids representing subtle changes of the conserved Lys/Arg residue. These were tested with four peptide interaction partners, representing the two different binding modes. The results highlight the role of a positively charged amino acid in the β1–β2 loop of PDZ domains, and show subtle differences for canonical and noncanonical interaction partners, thus providing additional insight into the mechanism of PDZ/ligand interaction.     

Structural Diversity of PDZ–Lipid Interactions

PDZ domains are globular protein modules that are over‐and‐above appreciated for their interaction with short peptide motifs found in the cytosolic tail of membrane receptors, channels, and adhesion molecules. These domains predominate in scaffold molecules that control the assembly and the location of large signaling complexes. Studies have now emerged showing that PDZ domains can also interact with membrane lipids, and in particular with phosphoinositides. Phosphoinositides control various aspects of cell signaling, vesicular trafficking, and cytoskeleton remodeling. When investigated, lipid binding appears to be extremely relevant for PDZ protein functionality. Studies point to more than one mechanism for PDZ domains to associate with lipids. Few studies have been focused on the structural basis of PDZ–phosphoinositide interactions, and the biological consequences of such interactions. Using the current knowledge on syntenin‐1, syntenin‐2, PTP‐Bas, PAR‐3 and PICK1, we recapitulate our understanding of the structural and biochemical aspects of PDZ–lipid interactions and the consequences for peptide interactions.     

Rigidified Clicked Dimeric Ligands for Studying the Dynamics of the PDZ1‐2 Supramodule of PSD‐95

PSD‐95 is a scaffolding protein of the MAGUK protein family, and engages in several vital protein–protein interactions in the brain with its PDZ domains. It has been suggested that PSD‐95 is composed of two supramodules, one of which is the PDZ1‐2 tandem domain. Here we have developed rigidified high‐affinity dimeric ligands that target the PDZ1‐2 supramodule, and established the biophysical parameters of the dynamic PDZ1‐2/ligand interactions. By employing ITC, protein NMR, and stopped‐flow kinetics this study provides a detailed insight into the overall conformational energetics of the interaction between dimeric ligands and tandem PDZ domains. Our findings expand our understanding of the dynamics of PSD‐95 with potential relevance to its biological role in interacting with multivalent receptor complexes and development of novel drugs.     

Characterization of a putative phosphorylation switch: adaptation of SPOT synthesis to analyze PDZ domain regulation mechanisms

Transient macromolecular complexes are often formed by protein-protein interaction domains (e.g., PDZ, SH2, SH3, WW), which are often regulated (positively or negatively) by phosphorylation. To address the in vitro analysis of PDZ domain regulation by such phosphorylation, we improved the inverted peptide method. This method is based on standard SPOT synthesis, followed by inversion of the peptide under acidic conditions to generate the free C termini necessary for PDZ domain ligand recognition. The benefit of the newly introduced acidic conditions is the preservation of the incorporated phosphate group during peptide synthesis. Furthermore, the improved method is more robust and shows an increased signal-to-noise ratio. As representative examples, we used the AF6, ERBIN, and SNA1 (alpha-1-syntrophin) PDZ domains to analyze the influence of ligand-position-dependent phosphorylation. We could clearly demonstrate severe down-regulation by phosphorylation of the PDZ ligand position -2 (<50 %) and slightly less at position -1 ( approximately 50 %). These results are specific and reproducible for all three PDZ domains. Finally, we confirmed the influence of negative regulation by using the protein kinase BCR as the AF6 PDZ domain ligand. For the first time, this approach allows the SPOT synthesis technique to be used to screen large libraries of phosphorylated peptides in vitro. This should ultimately help in the identification of phosphorylation-dependent regulation mechanisms in vivo.     

NMR-Guided Repositioning of Non-Steroidal Anti-Inflammatory Drugs into Tight Junction Modulators

Bioavailability is a major bottleneck in the clinical application of medium molecular weight therapeutics, including protein and peptide drugs. Paracellular transport of these molecules is hampered by intercellular tight junction (TJ) complexes. Therefore, safe chemical regulators for TJ loosening are desired. Here, we showed a potential application of select non-steroidal anti-inflammatory drugs (NSAIDs) as TJ modulators. Based on our previous observation that diclofenac and flufenamic acid directly bound various PDZ domains with a broad specificity, we applied solution nuclear magnetic resonance techniques to examine the interaction of other NSAIDs and the first PDZ domain (PDZ1) of zonula occludens (ZO)-1, ZO-1(PDZ1). Inhibition of ZO-1(PDZ1) is expected to provide loosening of the epithelial barrier function because the domain plays a crucial role in maintaining TJ integrity. Accordingly, diclofenac and indomethacin were found to decrease the subcellular localization of claudin (CLD)-2 but not occludin and ZO-1 at the apicolateral intercellular compartment of Madin–Darby canine kidney (MDCK) II cells. These NSAIDs exhibited 125–155% improved paracellular efflux of fluorescein isothiocyanate insulin for the Caco-2 cell monolayer. We propose that these NSAIDs can be repurposed as drug absorption enhancers for peptide drugs.     

Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment

By constructing Fv and single-chain Fv (scFv) fragments of antibodies, the variable domains are taken out of their natural context in the Fab fragment, where they are associated with the constant domains of the light (CL) and heavy chain (CH1). As a consequence, all residues of the former variable/constant domain interface become solvent exposed. In an analysis of 30 non-redundant Fab structures it was found that at the former variable/constant domain interface of the Fv fragment the frequency of exposed hydrophobic residues is much higher than in the rest of the Fv fragment surface. We investigated the importance of these residues for different properties such as folding in vivo and in vitro, thermodynamic stability, solubility of the native protein and antigen affinity. The experimental model system was the scFv fragment of the anti-fluorescein antibody 4-4-20, of which only 2% is native when expressed in the periplasm of Escherichia coli. To improve its in vivo folding, a mutagenesis study of three newly exposed interfacial residues in various combinations was carried out. The replacement of one of the residues (V84D in VH) led to a 25-fold increase of the functional periplasmic expression yield of the scFv fragment of the antibody 4-4-20. With the purified scFv fragment it was shown that the thermodynamic stability and the antigen binding constant are not influenced by these mutations, but the rate of the thermally induced aggregation reaction is decreased. Only a minor effect on the solubility of the native protein was observed, demonstrating that the mutations prevent aggregation during folding and not of the native protein. Since the construction of all scFv fragments leads to the exposure of these residues at the former variable/constant domain interface, this strategy should be generally applicable for improving the in vivo folding of scFv fragments and, by analogy, also the in vivo folding of other engineered protein domains.      

利用DNA改组筛选高活性抗凝血酶Ⅲ

目的　利用DNA改组技术大幅度提高抗凝血酶Ⅲ (AT -Ⅲ )的生物活性。方法　采用RQ1DNaseⅠ不完全酶解ST -Ⅲ基因 ,产生长度为 5 0～ 10 0bp的随机片段 ,用低熔点胶回收。无引物PCR从小片段组装成大片段 ,通过有引物PCR得到正确大小的片段。线性化的质粒通过电转转入毕赤酵母GS115细胞。结果　获得了 5株高活性的克隆。结论　探索了在毕赤酶母进行DNA改组和筛选的方法和路线 ,成功进行了AT Ⅲ的改组 ,并为其它分子的改组提供借鉴。     

Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388

Fragments of human thrombomodulin (TM) have been expressed inlarge quantities in the Pichia pastoris yeast expression systemand purified to homogeneity. Fermentation of P.pastoris resultedin yields of 170 mg/1 TM. Purification to homogeneity resultedin an overall 10% yield, so that quantities of –20 mgpurified fragments can be readily obtained. Smaller fragmentsof TM, such as the individual fourth or fifth domains, werenot active, nor were equimolar mixtures of the two domains.These results demonstrate that the fourth and fifth epidermalgrowth factor (EGF)–like domains together comprise thesmallest active fragment of TM. The fragment containing thefourth and fifth EGFlike domains (TMEGF(4–5)] had 10%the specific activity of rabbit TM. Comparison of the M388Lmutant TMEGF(4–5) fragment with the same mutant TMEGF(4–5–6)fragment showed that the fragment with the sixth domain hada 10–fold better Km value for thrombin than the fragmentthat did not contain the sixth domain; this factor completelyaccounts for the higher specific activity of the fragments containingthe sixth domain. Comparison of the wild–type and M388Lmutants showed that the M388L mutation resulted in a 2–foldincrease in k_cat for the activation of protein C by the thrombin–TMfragment complex, completely accounting for the 2–foldincrease in specific activity of these mutant fragments.     

Development of Fragment‐Based n‐FABS NMR Screening Applied to the Membrane Enzyme FAAH

Despite the recognized importance of membrane proteins as pharmaceutical targets, the reliable identification of fragment hits that are able to bind these proteins is still a major challenge. Among different ¹⁹F NMR spectroscopic methods, n‐fluorine atoms for biochemical screening (n‐FABS) is a highly sensitive technique that has been used efficiently for fragment screening, but its application for membrane enzymes has not been reported yet. Herein, we present the first successful application of n‐FABS to the discovery of novel fragment hits, targeting the membrane‐bound enzyme fatty acid amide hydrolase (FAAH), using a library of fluorinated fragments generated based on the different local environment of fluorine concept. The use of the recombinant fusion protein MBP‐FAAH and the design of compound 11 as a suitable novel fluorinated substrate analogue allowed n‐FABS screening to be efficiently performed using a very small amount of enzyme. Notably, we have identified 19 novel fragment hits that inhibit FAAH with a median effective concentration (IC₅₀) in the low mM –μM range. To the best of our knowledge, these results represent the first application of a ¹⁹F NMR fragment‐based functional assay to a membrane protein.     

Bivalent peptides as models for multimeric targets of PDZ domains

PDZ domains are among the most common modules in eukaryotic, including human, genomes. They are found exclusively in large, multidomain cytosolic proteins--often with other domains that belong to a variety of families--and are involved in a plethora of physiological and pathophysiological events. PDZ domains mediate protein-protein interactions by binding to solvent-exposed and extended C-terminal short fragments of membrane-associated proteins, such as receptors and ion channels. Most of what is known about the mechanisms of target binding by PDZ domains is inferred from studies that involve isolated recombinant PDZ domains and short synthetic peptides that represent the targets. These binary systems constitute an obvious oversimplification and disregard factors such as noncanonical modes of binding and enhanced affinity due to multimeric interactions mediated by clusters and oligomers of PDZ-domain-containing proteins. We have tested whether the interaction between a dimeric form of PDZ domain that mimics a functional dimeric guanine nucleotide exchange factor, PDZ-RhoGEF (PDZ-containing RhoA-specific guanine nucleotide exchange factor) or LARG (leukemia-associated RhoA specific guanine nucleotide exchange factor), and a bivalent peptide that mimics the dimer of the plexin B receptor, could enhance the interaction between the two moieties. Peptide dimerization was achieved by cross-linking the N-terminal ends of peptides attached to Wang resin with poly(ethylene glycol) spacers (30-45 Angstroms in length). The interaction of dimeric PDZ domains with dimeric peptides resulted in an up to 20-fold increase in affinity compared to the simple binary system. This is consistent with the notion that multimerization of both receptors and PDZ-containing proteins might constitute an important regulatory mechanism.     

Structure and functional complementation of engineered fragments from yeast phosphoglycerate kinase

Previous studies have shown that, although the isolated structuraldomains of yeast phosphoglycerate kinase recover a quasi-nativestructure in vitro as well as in vivo, they do not reassociatenor generate a functional enzyme. The aim of this work was firstto study the folding of complementary fragments different fromstructural domains and second to determine the requirementsfor their reassociation and functional complementation. Themethod used for producing rigorously defined fragments consistsof the introduction of a unique cysteinyl residue in the proteinfollowed by a specific cleavage by 5'5'-dithiobis(2-nitrobenzoate)/potassiumcyanide at this residue. Two pairs of complementary fragmentswere thus obtained, 1–96/97–415 and 1–248/249–415.The structure and stabilities of the different fragments werestudied. The short fragments, i.e. 1–96 and 249–415were found to contain some secondary structure, but to havea low stability. Each large fragment has a high structural contentand a stability close to that of the corresponding domain. Incontrast to that observed with the isolated domains, a weakbut significant complementation was observed for the two pairsof fragments; the pair of fragments 1–248/249–415recovered 8% of the activity of the native enzyme upon complementation.An independent refolding of the complementary fragments beforereassociation decreased the yield of complementation for thepair of fragments 1–96/97–415, but did not affectthe complementation for the other pair (1–248/249–415).From the present data and previous work on the isolated domains,it appears that the correct folding of the isolated fragmentsis not a prerequisite for their complementation.     

Directed evolution of PDZ variants to generate high-affinity detection reagents

High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific 'beta1-beta3' interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.     

Off-Pathway-Sensitive Protein-Splicing Screening Based on a Toxin/Antitoxin System

Protein-splicing domains are frequently used engineering tools that find application in the in vivo and in vitro ligation of protein domains. Directed evolution is among the most promising technologies used to advance this technology. However, the available screening systems for protein-splicing activity are associated with bottlenecks such as the selection of pseudo-positive clones arising from off-pathway reaction products or fragment complementation. Herein, we report a stringent screening method for protein-splicing activity in cis and trans, that exclusively selects productively splicing domains. By fusing splicing domains to an intrinsically disordered region of the antidote from the Escherichia coli CcdA/CcdB type II toxin/antitoxin system, we linked protein splicing to cell survival. The screen allows selecting novel cis- and trans-splicing inteins catalyzing productive highly efficient protein splicing, for example, from directed-evolution approaches or the natural intein sequence space.     

Efficient Screening of Target-Specific Selected Compounds in Mixtures by 19F NMR Binding Assay with Predicted 19F NMR Chemical Shifts

Ligand-based ¹⁹F NMR screening is a highly effective and well-established hit-finding approach. The high sensitivity to protein binding makes it particularly suitable for fragment screening. Different criteria can be considered for generating fluorinated fragment libraries. One common strategy is to assemble a large, diverse, well-designed and characterized fragment library which is screened in mixtures, generated based on experimental ¹⁹F NMR chemical shifts. Here, we introduce a complementary knowledge-based ¹⁹F NMR screening approach, named ¹⁹Focused screening, enabling the efficient screening of putative active molecules selected by computational hit finding methodologies, in mixtures assembled and on-the-fly deconvoluted based on predicted ¹⁹F NMR chemical shifts. In this study, we developed a novel approach, named LEFshift, for ¹⁹F NMR chemical shift prediction using rooted topological fluorine torsion fingerprints in combination with a random forest machine learning method. A demonstration of this approach to a real test case is reported.     

The position of the structurally autonomous kringle 2 domain influences the functional features of tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) is composed of structurallyautonomous domains. From the N-terminus of t-PA, a finger-likedomain (F), an epidermal growth factor-like domain (G), twokringle domains (Kl and K2) and a serine protease domain (P)can be discerned. The K2 domain of t-PA is known to be involvedin lysine binding, fibrin binding and fibrin-dependent plasminogenactivation. To study the functional autonomy of the K2 domainin t-PA we constructed, with the aid of a cassette t-PA gene[Rehberg et al. (1989) Protein Engng, 2,371–377], mutantt-PA genes coding for four molecules (FGK1K2P, FGK2K1P, GK1K2Pand GK2K1P) in which the K2 domain was placed in two differentpositions in t-PA. The DNAs of wild-type t-PA and the t-PA variantswere expressed in Chinese hamster ovary cells and the recombinantproteins were purified by affinity chromatography.All moleculeswere expressed in their single-chain form and could be convertedto their two-chain form. With these molecules, lysine binding,fibrin binding and fibrin-dependent plasminogen activation werestudied. All variants showed affinity for lysyl-Sepharose andaminohexyl-Sepharose. Reversal of the K domains (FGK2K1P versusFGK2K1P and GK1K2P versus GK2K1P) resulted in a 23–47%weaker interaction to both lysyl-Sepharose and aminohexyl-Sepharose.Deleting the F domain (FGK1K2P versus GK1K2P and FGK2K1P versusGK2K1P) resulted in a 20–70% improvement of the interactionslysyl-Sepharose and aminohexyl-Sepharose. All variants boundto a forming fibrin clot. Reversal of the K domains (FGK1K2Pversus FGK2K1P) reduced fibrin binding. In the presence of thelysine analogue -amino caproic acid, only FGK1K2P bound to fibrin.All variants activated plasminogen. In the absence of fibrinogenCNBr fragments (mimic of fibrin), the reversal of the K domain(FGK2K1P) resulted in a 2-fold improved plasminogen activation.In the presence of a fibrin mimic, the plasminogen activationsof the F domain deletion analogues GK1K2P and GK2K1P were foundto be decreased 2- to 4-fold. From these results we concludedthat the function of t-PA in lysine binding, fibrin bindingand fibrin-dependent plasminogen activation is dependent onthe correct spatial orientation of the K2 domain within thet-PA molecule     

Small‐Molecule Inhibitors That Target Protein–Protein Interactions in the RAD51 Family of Recombinases

The development of small molecules that inhibit protein–protein interactions continues to be a challenge in chemical biology and drug discovery. Herein we report the development of indole‐based fragments that bind in a shallow surface pocket of a humanised surrogate of RAD51. RAD51 is an ATP‐dependent recombinase that plays a key role in the repair of double‐strand DNA breaks. It both self‐associates, forming filament structures with DNA, and interacts with the BRCA2 protein through a common “FxxA” tetrapeptide motif. We elaborated previously identified fragment hits that target the FxxA motif site and developed small‐molecule inhibitors that are approximately 500‐fold more potent than the initial fragments. The lead compounds were shown to compete with the BRCA2‐derived Ac‐FHTA‐NH₂ peptide and the self‐association peptide of RAD51, but they had no effect on ATP binding. This study is the first reported elaboration of small‐molecular‐weight fragments against this challenging target.     

Submicron periodical poling by local switching in ion sliced lithium niobate thin films with a dielectric layer

Lithium niobate LiNbO₃ (LN) on insulator wafers (LNOI) representing a submicron-thick LN film bonded to a SiO₂ layer deposited on a thick LN substrate have been manufactured recently by ion slicing. Today, LNOI is one of the most promising materials for fabrication of various integrated optical devices, including waveguides, electro-optical modulators, and wavelength convertors, due to their high drop in refractive index at the interface between the film and the SiO₂ layer. Creation of the stable domain structures with submicron periods in LNOI will improve the properties of nonlinear optical devices. Periodically poled LN with periods below 300 nm can be used to implement an optical parametric oscillator in the backward configuration. We have studied experimentally the ways to create the stripe domains and periodical domain structures in LNOI with a dielectric layer under the film by local switching. The identified scenarios of the domain evolution were attributed to the ineffective screening of depolarization field. We have shown that stripe domains and periodic domain structures can be produced by scanning with a biased tip only at temperatures above 80 °C. Periodical stable domain structures with periods down to 300 nm have been created.     

Quinoline-Conjugated Ruthenacarboranes: Toward Hybrid Drugs with a Dual Mode of Action

The role of autophagy in cancer is often complex, ranging from tumor-promoting to -suppressing effects. In this study, two novel hybrid molecules were designed, containing a ruthenacarborane fragment conjugated with a known modulator of autophagy, namely a quinoline derivative. The complex closo-[3-(η⁶-p-cymene)-1-(quinolin-8-yl-acetate)-3,1,2-RuC₂B₉H₁₀] ( 4 ) showed a dual mode of action against the LN229 (human glioblastoma) cell line, where it inhibited tumor-promoting autophagy, and strongly inhibited cell proliferation, de facto blocking cellular division. These results, together with the tendency to spontaneously form nanoparticles in aqueous solution, make complex 4 a very promising drug candidate for further studies in vivo, for the treatment of autophagy-prone glioblastomas.