Nicholas Barlow

DOTA-Amide Lanthanide Tag for Reliable Generation of Pseudocontact Shifts in Protein NMR Spectra

Structural studies of proteins and protein-ligand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to create paramagnetic centers. In particular, pseudocontact shifts (PCS) generated by paramagnetic lanthanides contain important and unique long-range structure information. Here, we present a high-affinity lanthanide binding tag that can be attached to single cysteine residues of proteins. The new tag has many advantageous features that are not available in this combination from previously published tags: (i) it binds lanthanide ions very tightly, minimizing the generation of nonspecific effects, (ii) it produces PCSs with high reliability as its bulkiness prevents complete motional averaging of PCSs, (iii) it can be attached to single cysteine residues, alleviating the need of detailed prior knowledge of the 3D structure of the target protein, and (iv) it does not display conformational exchange phenomena that would increase the number of signals in the NMR spectrum. The performance of the tag is demonstrated with the N-terminal domain of the E. coli arginine repressor and the A28C mutant of human ubiquitin.

Cathepsin S Causes Inflammatory Pain via Biased Agonism of PAR2 and TRPV4

Background: Proteases trigger inflammation and pain by cleaving protease-activated receptors (PARs) at defined sites. Results: Cathepsin S (Cat-S) cleaved PAR2 at a unique site E56↓T57, leading to Gαs-mediated cAMP accumulation and TRPV4-dependent inflammation and pain. Conclusion: Cat-S is a biased agonist of PAR2- and TRPV4-dependent inflammation and pain. Significance: PARs integrate responses to diverse proteases. Serine proteases such as trypsin and mast cell tryptase cleave protease-activated receptor-2 (PAR2) at R36↓S37 and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR2 at distinct sites are biased agonists that also induce inflammation and pain is unexplored. Cathepsin S (Cat-S) is a lysosomal cysteine protease of antigen-presenting cells that is secreted during inflammation and which retains activity at extracellular pH. We observed that Cat-S cleaved PAR2 at E56↓T57, which removed the canonical tethered ligand and prevented trypsin activation. In HEK and KNRK cell lines and in nociceptive neurons of mouse dorsal root ganglia, Cat-S and a decapeptide mimicking the Cat-S-revealed tethered ligand-stimulated PAR2 coupling to Gαs and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca2+, activate ERK1/2, recruit β-arrestins, or induce PAR2 endocytosis. Cat-S caused PAR2-dependent activation of transient receptor potential vanilloid 4 (TRPV4) in Xenopus laevis oocytes, HEK cells and nociceptive neurons, and stimulated neuronal hyperexcitability by adenylyl cyclase and protein kinase A-dependent mechanisms. Intraplantar injection of Cat-S caused inflammation and hyperalgesia in mice that was attenuated by PAR2 or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR2 antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR2 in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR2 that causes PAR2- and TRPV4-dependent inflammation and pain. They expand the role of PAR2 as a mediator of protease-driven inflammatory pain.

Dynamic binding mode of a Synaptotagmin-1–SNARE complex in solution

Rapid neurotransmitter release depends on the Ca2+ sensor Synaptotagmin-1 (Syt1) and the SNARE complex formed by synaptobrevin, syntaxin-1 and SNAP-25. How Syt1 triggers release has been unclear, partly because elucidating high-resolution structures of Syt1–SNARE complexes has been challenging. An NMR approach based on lanthanide-induced pseudocontact shifts now reveals a dynamic binding mode in which basic residues in the concave side of the Syt1 C2B-domain β-sandwich interact with a polyacidic region of the SNARE complex formed by syntaxin-1 and SNAP-25. The physiological relevance of this dynamic structural model is supported by mutations in basic residues of Syt1 that markedly impair SNARE-complex binding in vitro and Syt1 function in neurons. Mutations with milder effects on binding have correspondingly milder effects on Syt1 function. Our results support a model whereby dynamic interaction facilitates cooperation between Syt1 and the SNAREs in inducing membrane fusion.

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain.

Background: Proteases cleave protease-activated receptor-2 (PAR2), which activates transient receptor potential (TRP) ion channels to cause inflammation and pain. Results: Neutrophil elastase cleaves PAR2, resulting in Gαs-mediated cAMP formation, transient receptor potential vanilloid 4 (TRPV4) activation, and sensitization of nociceptive neurons, inflammation, and pain. Conclusion: Elastase causes PAR2- and TRPV4-mediated inflammation and pain. Significance: PARs and TRP channels mediate responses to diverse proteases. Proteases that cleave protease-activated receptor-2 (PAR2) at Arg36↓Ser37 reveal a tethered ligand that binds to the cleaved receptor. PAR2 activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR2 at non-canonical sites can trigger distinct signaling cascades, the functional importance of the PAR2-biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR2, causes inflammation and pain by activating PAR2 and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR2 at Ala66↓Ser67 and Ser67↓Val68. Elastase stimulated PAR2-dependent cAMP accumulation and ERK1/2 activation, but not Ca2+ mobilization, in KNRK cells. Elastase induced PAR2 coupling to Gαs but not Gαq in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK2) or β-arrestin to PAR2, consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR2-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR2-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR2- and TRPV4-mediated influx of extracellular Ca2+ in mouse nociceptors. Adenylyl cyclase and PKA-mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR2- and TRPV4-mediated mechanisms. Thus, the elastase-biased agonism of PAR2 causes Gαs-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR2 as a mediator of protease-driven inflammation and pain.

Lanthanide Tags for Site-Specific Ligation to an Unnatural Amino Acid and Generation of Pseudocontact Shifts in Proteins

Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structural restraints for structural biology by NMR spectroscopy, but site-specific tagging of proteins with lanthanides remains a challenge, as most of the available lanthanide tags require proteins with single cysteine residues. We show that cyclen-based paramagnetic lanthanide tags can be attached to proteins in a site-specific manner by Cu(I)-catalyzed azide-alkyne cycloaddition to a genetically encoded p-azido-l-phenylalanine residue with a tether that proved sufficiently short and rigid for the observation of PCSs in several proteins. Despite the sterically demanding conditions associated with bulky tags and reactions close to the protein surface, ligation yields consistently above 50% and approaching 100% were obtained with the help of the Cu(I)-stabilizing ligand BTTAA. The yields were high independent of the presence of cysteine residues, thereby avoiding the need for cysteine mutations associated with conventional lanthanide-tagging strategies.

Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief

Therapeutic targeting of the neurokinin 1 receptor in endosomes provides efficacious and prolonged pain relief. Targeting the enemy within endosomes With opioid addiction on the rise, there is a great need for effective nonopioid approaches to treat pain. Jensen et al. examined the function of substance P neurokinin 1 receptor, which plays a role in the transmission of pain signals in the central nervous system. The authors demonstrated that endocytosis of this receptor is required for the transmission of pain signals. Although systemic inhibition of endocytosis would not be feasible in a living organism, the authors discovered another way to take advantage of this information. They conjugated neurokinin 1 receptor antagonists to cholestanol, promoting their incorporation into endosomes, where they successfully inhibited their target to block pain transmission. Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)–coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK1R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK1R in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane–targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and β-arrestin blocked SP-induced NK1R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal–regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK1R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK1R antagonists. These results reveal a critical role for endosomal signaling of the NK1R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.

Partly-PEGylated Poly-L-lysine dendrimers have reduced plasma stability and circulation times compared with fully PEGylated dendrimers

The current study was performed to examine the effect of 50% surface PEGylation on the plasma circulation, biodistribution and metabolism of intravenously administered Generation 4 poly-L-lysine dendrimers in rats. Partial surface PEGylation blocked the rapid and extensive vascular binding previously described for the non-PEGylated dendrimer and slowed metabolism of the core, however the effect was less than that observed for total surface coverage of PEG. Capping the residual surface amines with acetyl groups further improved plasma stability.

Exploring the Chemical Space around 8-Mercaptoguanine as a Route to New Inhibitors of the Folate Biosynthesis Enzyme HPPK

As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg2+-dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (KD ∼13 µM), inhibited the S. aureus enzyme (SaHPPK) (IC50 ∼ 41 µM), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N 9 substitution, or tautomerization arising from N 7 substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N 9-H···Val46 hydrogen bond, combined with the limited space available around the N 9 position. The water-filled pocket under the N 7 position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N 7 (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (KD ∼ 12 µM). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 Å X-ray structure of the SaHPPK/21a complex confirms that extension from the N 7 position towards the Mg2+-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg2+ centres or Mg2+ -binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.

Structure-Based Design and Development of Functionalized Mercaptoguanine Derivatives as Inhibitors of the Folate Biosynthesis Pathway Enzyme 6-Hydroxymethyl-7,8-Dihydropterin Pyrophosphokinase from Staphylococcus Aureus.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), an enzyme from the folate biosynthesis pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin and is a yet-to-be-drugged antimicrobial target. Building on our previous discovery that 8-mercaptoguanine (8MG) is an inhibitor of Staphylococcus aureus HPPK (SaHPPK), we have identified and characterized the binding of an S8-functionalized derivative (3). X-ray structures of both the SaHPPK/3/cofactor analogue ternary and the SaHPPK/cofactor analogue binary complexes have provided insight into cofactor recognition and key residues that move over 30 Å upon binding of 3, whereas NMR measurements reveal a partially plastic ternary complex active site. Synthesis and binding analysis of a set of analogues of 3 have identified an advanced new lead compound (11) displaying >20-fold higher affinity for SaHPPK than 8MG. A number of these exhibited low micromolar affinity for dihydropteroate synthase (DHPS), the adjacent, downstream enzyme to HPPK, and may thus represent promising new leads to bienzyme inhibitors.

Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation.

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.