Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

Peptide neurotoxins are powerful tools for research, diagnosis, and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high affinity and specificity. This paper describes isolation of toxins for one such orphan target, KcsA, a potassium channel that has been fundamental to delineating the structural basis for ion channel function. A phage-display strategy is presented whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We describe two toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (K_i) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a K_i of 0.5 nM whereas Shaker, Kv1.2, and Kv1.3 channels are blocked over 200-fold less well. HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2, and Kv1.3 channels with K_i of 1–4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys₂₁ and Tyr₂₂, are essential for high-affinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg₂₃, indicating that Lys₂₁ is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of high-affinity toxins for orphan receptors.Venomous animals produce neurotoxic peptides for defense and to capture prey. With potencies in the nanomolar range, the peptides act by modulating the function of target receptors. Toxins isolated from venoms have been used to identify and purify ion channels, to clarify their roles in physiology, to elucidate the structural basis for their function, and, recently, to diagnose and treat disease. Given their utility, it is frustrating that natural toxins cross-react with related receptors (or have no known target) and that most receptors lack a specific, high-affinity toxin. This state of affairs is easily understood; the small amounts of toxins isolated from natural sources makes target identification a challenge and their purpose in the wild does not favor target specificity. Here, we advance our approach to overcoming these problems, that is, creation of expression libraries of toxins allowing cloning based on target binding (1), by seeking a specific, high-affinity ligand for an orphan channel receptor.Our strategy is to start with a known toxin and to design a phage-display library using the genetic database of its predicted homologs, in native and combinatorial fashion, so the encoded peptides share the same structural scaffold. As a proof of concept, we previously addressed a case of inadequate target discrimination by known natural toxins using a library of ∼11,200 peptides designed to share the fold in α-KTx scorpion toxins and a specific ligand for the human voltage-gated potassium channel Kv1.3 was isolated (1). Moka1, composed of domains from three scorpion species, blocks Kv1.3 with nanomolar affinity, allowing it to suppress T-cell-mediated immune responses, and is without unwanted side effects on gastrointestinal motility seen with natural toxins because it does not cross-inhibit Kv1.1 and Kv1.2. Supporting the design premise that encoded peptides are expressed, correctly folded, and accessible on the phage surface in a manner permissive of sorting based on target binding, the determined 3D structure of Moka1 revealed it to be constructed on an α-KTx scaffold. Here, we sought to extend our strategy by testing another scaffold and creation of a library sufficiently large to achieve isolation of peptides specific for a target with no known ligand.KcsA is a prokaryotic channel with high potassium conductance and selectivity (2). The first potassium channel visualized at high resolution (3), KcsA has a single ion conduction pathway on the central axis of symmetry formed by four identical subunits, each with two transmembrane segments and a reentrant pore-forming loop (TM1-P-TM2). The 3D structure of KcsA confirmed explanations for selective ion permeation and conduction pathway gating deduced in the period before crystallization and its continued interrogation has been key to delineating the mechanistic bases for channel function (4–6). Although described 20 y ago (7), KcsA remains an orphan target so that studies with peptide toxins have required production of mutant channels with multiple mutations in the pore domain (8) or chimeras such as Kv1.3-KcsA, where the entire KcsA pore domain is replaced by the one in Kv1.3 (9).To isolate toxins for KcsA, a peptide library was designed with ∼1,562,750 variants via combinatorial permutation of sequences related to the sea anemone type I (SAK1) toxin ShK. Phage sorting was performed on purified, wild-type KcsA channels. Peptides expressed on the enriched phage were synthesized and studied by surface plasmon resonance (SPR) to characterize their binding to purified KcsA and by voltage-clamp electrophysiology to assess channel blockade. Hui1, a novel and specific inhibitor of KcsA, HmK, a natural and promiscuous blocker, and Hui1 mutants were evaluated to identify toxin segments and residues responsible for specificity and affinity and to discern the mechanism of channel inhibition. The 3D structure of Hui1 determined by NMR, the 1:1 stoichiometry of KcsA inhibition via a pore-directed mechanism, and the role of two, canonical “dyad” residues (Lys₂₁ and Tyr₂₂) in high-affinity binding all met expectations for a SAK1-type toxin. In contrast, the influence of permeant trans ions (those traversing the channel after entering from the opposite side of the membrane) on dissociation of Hui1 from its external binding site indicated that Arg₂₃, a residue with a side chain too bulky to fit snugly into the potassium conduction pore (10), was responsible for the voltage dependence of block rather than Lys₂₁. This unexpected role for Hui1-Arg₂₃ could reflect a new SAK1 binding orientation for the novel toxin; however, some models have located the ShK dyad Lys in the outer pore vestibule of Kv1.3 (11) rather than in the narrow portion of the conduction pathway (12). We posit that Hui1 binds and blocks like some, and perhaps most, natural SAK1 toxins.     

In vitro activities and targets of three cephem antibiotics against Haemophilus influenzae.

The antimicrobial activities of cefixime, cefpodoxime, and ceftibuten were determined with 18 ampicillin-susceptible (Amps), 13 ampicillin-resistant beta-lactamase-producing (AmprBLP), and 7 ampicillin-resistant non-beta-lactamase-producing (AmprNBLP) strains of Haemophilus influenzae. An effect of inoculum density on apparent MIC, the bactericidal activity of these agents, and the targets of the three cephems were determined. The MICs of cefixime, cefpodoxime, and ceftibuten for 90% of the Amps and AmprBLP isolates were 0.04, 0.08, and 0.08 microgram/ml, respectively. In contrast, the MICs for 90% of the AmprNBLP strains were 0.96, 1.92, and 7.68 micrograms/ml. No significant inoculum effect was observed for any group of strains comparing inocula of 10(3) and 10(5) CFU, whereas only the AmprNBLP isolates showed a marked effect at an inoculum of 10(6) CFU. Although bactericidal levels were achieved for the Amps and AmprBLP strains, tolerance to cefixime and ceftibuten was observed. The bactericidal activity for the AmprNBLP strains was limited, with cefixime showing the highest activity of the three cephems. Penicillin-binding proteins 2, 4, and 5 revealed high affinity, with 50% inhibitory concentration levels below the MIC for all three cephems, suggesting that these are important targets of these agents in H. influenzae. We conclude that the cephems are highly active in vitro against Amps and AmprBLP strains of H. influenzae, but less so against AmprNBLP isolates.     

In vitro flow based systems to study platelet function and thrombus formation: Recommendations for standardization: Communication from the SSC on Biorheology of the ISTH

Experimental videomicroscopic in vitro assays of thrombus formation based on blood perfusion are instrumental in a wide range of basic studies in thrombosis, screening for hereditary or acquired plateletrelated pathologies, and assessing the effectiveness of novel anti‐platelet therapies. Here, we discuss application of the broadly used “in vitro thrombosis model”: a frequently used assay to study the formation of 3D aggregates under flow, which involves perfusing anticoagulated whole blood over fibrillar collagen in a flow geometry of rectangular cross‐section, such as glass microcapillaries or parallel‐plate flow chambers. Major advantaged of this assay are simplicity and ability to reproduce the four main stages of platelet thrombus formation, i.e. platelet tethering, adhesion, activation and aggregation under a wide range of hemodynamic conditions. On the other hand, these devices represent, at best, simple reductive models of thrombosis. We also describe how blood flow assays can be used to study various aspects of platelet function on adhesive proteins and discuss the relevance of such flow models. Finally, we propose recommendations for standardization related to the use of this assay that cover collagen source, coating methods, micropatterning, sample composition, anticoagulation, choice of flow device, hemodynamic conditions, quantification challenges, variability, pre‐analytical conditions and other issues.     

Heparanase-neutralizing antibodies attenuate lymphoma tumor growth and metastasis

Heparanase is an endoglycosidase that cleaves heparan sulfate side chains of proteoglycans, resulting in disassembly of the extracellular matrix underlying endothelial and epithelial cells and associating with enhanced cell invasion and metastasis. Heparanase expression is induced in carcinomas and sarcomas, often associating with enhanced tumor metastasis and poor prognosis. In contrast, the function of heparanase in hematological malignancies (except myeloma) was not investigated in depth. Here, we provide evidence that heparanase is expressed by human follicular and diffused non-Hodgkin''s B-lymphomas, and that heparanase inhibitors restrain the growth of tumor xenografts produced by lymphoma cell lines. Furthermore, we describe, for the first time to our knowledge, the development and characterization of heparanase-neutralizing monoclonal antibodies that inhibit cell invasion and tumor metastasis, the hallmark of heparanase activity. Using luciferase-labeled Raji lymphoma cells, we show that the heparanase-neutralizing monoclonal antibodies profoundly inhibit tumor load in the mouse bones, associating with reduced cell proliferation and angiogenesis. Notably, we found that Raji cells lack intrinsic heparanase activity, but tumor xenografts produced by this cell line exhibit typical heparanase activity, likely contributed by host cells composing the tumor microenvironment. Thus, the neutralizing monoclonal antibodies attenuate lymphoma growth by targeting heparanase in the tumor microenvironment.Heparanase is an endo-β-d-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, releasing saccharide products with appreciable size (4–7 kDa) and biological potency. Enzymatic degradation of HS leads to disassembly of the extracellular matrix (ECM) and correlates with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of HS cleavage and remodeling of the ECM and basement membrane underlying epithelial and endothelial cells (1, 2). Heparanase expression is induced in human cancer, most often associating with reduced patients’ survival postoperation, increased tumor metastasis, and higher vessel density (3–5). In addition, heparanase up-regulation is associated with tumors larger in size (3, 5). Likewise, heparanase over-expression enhanced (6, 7), whereas local delivery of anti-heparanase siRNA inhibited (8), the growth of tumor xenografts. These results imply that heparanase function is not limited to tumor metastasis but is engaged in progression of the primary lesion, thus critically supporting the intimate involvement of heparanase in tumor progression and encouraging the development of heparanase inhibitors as anticancer therapeutics (9–12). As a consequence, heparanase inhibitors are currently evaluated in phase 1 clinical trials (13).Heparanase activity is similarly implicated in the progression of multiple myeloma (14–16), but its significance in other hematologic malignancies has not yet been characterized. Lymphomas are a heterogeneous group of cancers that arise from developing lymphocytes and produce tumors predominantly in lymphoid structures (i.e., bone marrow), but also in extranodal tissues. Collectively, lymphomas constitute the fifth most common cancer in North America, with more than 90% of the patients being affected by lymphomas of B-cell origin (17). Despite overall improvements in outcomes of lymphoma, ∼30–40% of patients have disease that is either refractory or relapses after standard therapy (18). Therefore, a better understanding of the molecular pathobiology of lymphomas is needed for the development of new therapeutic approaches. Here, we provide evidence that heparanase is expressed by B-lymphomas and that heparanase inhibitors restrain tumor growth. Furthermore, we describe the development of novel heparanase-neutralizing monoclonal antibodies (mAbs) that attenuate lymphoma growth by targeting heparanase in the tumor microenvironment.     

Non-Hodgkin lymphomas in pregnancy: Tackling therapeutic quandaries

Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) often present with systemic symptoms such as fatigue, shortness of breath and night sweats, mimicking pregnancy-related features which may result in delayed disease diagnosis. Furthermore, the wish to avoid investigational imaging, aiming to protect the fetus from radiation exposure, may lead to a further delay, which does not often result in significant changes in HL clinical nature and patient outcome. In contrast, a more aggressive behavior (i.e., advanced disease stage and reproductive organ involvement) of most NHL types diagnosed in pregnancy may require urgent therapeutic intervention to prevent disease progression.     

Recently Graduated Medical Students Lack Exposure to and Comfort with Chronic Liver Diseases

Digestive Diseases and Sciences - The prevalence of chronic liver disease (CLD) is rising, but it remains unclear if medical school curricula are emphasizing CLD to reflect its growing...     

Genetic diversity of penicillin G-resistant Neisseria meningitidis from Spain.

Genotypic and phenotypic diversity among 16 penicillin G-resistant (Penr) isolates of Neisseria meningitidis recovered from human blood or cerebrospinal fluid in Spain was compared with that among 12 penicillin-susceptible (Pens) isolates by the use of multilocus enzyme electrophoresis, serotyping, auxotroph testing in chemically defined media, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of penicillin-binding proteins (PBPs). Thirteen distinctive multilocus enzyme genotypes (electrophoretic types [ETs] ) were identified among the 28 isolates. There was slightly less genetic diversity among the eight ETs of Penr isolates (H = 0.385) than among the eight ETs of Pens isolates (H = 0.431). Cluster analysis demonstrated two distinctive complexes of ETs and one ET that was not closely related to either complex. The possibility of a singular clonal origin of penicillin G-resistant isolates was excluded by the observations that resistance occurred in isolates of each of the two distantly related complexes of ETs, that three of the four ETs represented by multiple isolates included both susceptible and resistant strains, and that serotypes and growth requirements were not associated with the resistance phenotype. The 28 isolates showed a relatively homogeneous pattern of four PBPs, with apparently reduced penicillin G binding by PBP 3 of the Penr isolates.