Inducers of the endothelial cell barrier identified through chemogenomic screening in genome-edited hPSC-endothelial cells.

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.

Homologous ligands accommodated by discrete conformations of a buried cavity.

Conformational change in protein-ligand complexes is widely modeled, but the protein accommodation expected on binding a congeneric series of ligands has received less attention. Given their use in medicinal chemistry, there are surprisingly few substantial series of congeneric ligand complexes in the Protein Data Bank (PDB). Here we determine the structures of eight alkyl benzenes, in single-methylene increases from benzene to n-hexylbenzene, bound to an enclosed cavity in T4 lysozyme. The volume of the apo cavity suffices to accommodate benzene but, even with toluene, larger cavity conformations become observable in the electron density, and over the series two other major conformations are observed. These involve discrete changes in main-chain conformation, expanding the site; few continuous changes in the site are observed. In most structures, two discrete protein conformations are observed simultaneously, and energetic considerations suggest that these conformations are low in energy relative to the ground state. An analysis of 121 lysozyme cavity structures in the PDB finds that these three conformations dominate the previously determined structures, largely modeled in a single conformation. An investigation of the few congeneric series in the PDB suggests that discrete changes are common adaptations to a series of growing ligands. The discrete, but relatively few, conformational states observed here, and their energetic accessibility, may have implications for anticipating protein conformational change in ligand design.

Covalent docking of large libraries for the discovery of chemical probes.

Chemical probes that form a covalent bond with a protein target often show enhanced selectivity, potency and utility for biological studies. Despite these advantages, protein-reactive compounds are usually avoided in high-throughput screening campaigns. Here we describe a general method (DOCKovalent) for screening large virtual libraries of electrophilic small molecules. We apply this method prospectively to discover reversible covalent fragments that target distinct protein nucleophiles, including the catalytic serine of AmpC ß-lactamase and noncatalytic cysteines in RSK2, MSK1 and JAK3 kinases. We identify submicromolar to low-nanomolar hits with high ligand efficiency, cellular activity and selectivity, including what are to our knowledge the first reported reversible covalent inhibitors of JAK3. Crystal structures of inhibitor complexes with AmpC and RSK2 confirm the docking predictions and guide further optimization. As covalent virtual screening may have broad utility for the rapid discovery of chemical probes, we have made the method freely available through an automated web server (http://covalent.docking.org/).

Increasing chemical space coverage by combining empirical and computational fragment screens.

Most libraries for fragment-based drug discovery are restricted to 1,000-10,000 compounds, but over 500,000 fragments are commercially available and potentially accessible by virtual screening. Whether this larger set would increase chemotype coverage, and whether a computational screen can pragmatically prioritize them, is debated. To investigate this question, a 1281-fragment library was screened by nuclear magnetic resonance (NMR) against AmpC ß-lactamase, and hits were confirmed by surface plasmon resonance (SPR). Nine hits with novel chemotypes were confirmed biochemically with KI values from 0.2 to low mM. We also computationally docked 290,000 purchasable fragments with chemotypes unrepresented in the empirical library, finding 10 that had KI values from 0.03 to low mM. Though less novel than those discovered by NMR, the docking-derived fragments filled chemotype holes from the empirical library. Crystal structures of nine of the fragments in complex with AmpC ß-lactamase revealed new binding sites and explained the relatively high affinity of the docking-derived fragments. The existence of chemotype holes is likely a general feature of fragment libraries, as calculation suggests that to represent the fragment substructures of even known biogenic molecules would demand a library of minimally over 32,000 fragments. Combining computational and empirical fragment screens enables the discovery of unexpected chemotypes, here by the NMR screen, while capturing chemotypes missing from the empirical library and tailored to the target, with little extra cost in resources.

Development and binding mode assessment of N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-l-γ-glutamyl-D-glutamic acid (BGC 945), a novel thymidylate synthase inhibitor that targets tumor cells.

N-[4-[2-Propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-l-γ-glutamyl-d-glutamic acid 1 (BGC 945, now known as ONX 0801), is a small molecule thymidylate synthase (TS) inhibitor discovered at the Institute of Cancer Research in London. It is licensed by Onyx Pharmaceuticals and is in phase 1 clinical studies. It is a novel antifolate drug resembling TS inhibitors plevitrexed and raltitrexed that combines enzymatic inhibition of thymidylate synthase with α-folate receptor-mediated targeting of tumor cells. Thus, it has potential for efficacy with lower toxicity due to selective intracellular accumulation through α-folate receptor (α-FR) transport. The α-FR, a cell-surface receptor glycoprotein, which is overexpressed mainly in ovarian and lung cancer tumors, has an affinity for 1 similar to that for its natural ligand, folic acid. This study describes a novel synthesis of 1, an X-ray crystal structure of its complex with Escherichia coli TS and 2'-deoxyuridine-5'-monophosphate, and a model for a similar complex with human TS.

Fragment-guided design of subnanomolar ß-lactamase inhibitors active in vivo.

Fragment-based design was used to guide derivatization of a lead series of ß-lactamase inhibitors that had heretofore resisted optimization for in vivo activity. X-ray structures of fragments overlaid with the lead suggested new, unanticipated functionality and points of attachment. Synthesis of three derivatives improved affinity over 20-fold and improved efficacy in cell culture. Crystal structures were consistent with the fragment-based design, enabling further optimization to a K(i) of 50 pM, a 500-fold improvement that required the synthesis of only six derivatives. One of these, compound 5, was tested in mice. Whereas cefotaxime alone failed to cure mice infected with ß-lactamase-expressing Escherichia coli, 65% were cleared of infection when treated with a cefotaxime:5 combination. Fragment complexes offer a path around design hurdles, even for advanced molecules; the series described here may provide leads to overcome ß-lactamase-based resistance, a key clinical challenge.

Quality control of disulfide bond formation in pilus subunits by the chaperone FimC.

Type 1 pili from uropathogenic Escherichia coli are filamentous, noncovalent protein complexes mediating bacterial adhesion to the host tissue. All structural pilus subunits are homologous proteins sharing an invariant disulfide bridge. Here we show that disulfide bond formation in the unfolded subunits, catalyzed by the periplasmic oxidoreductase DsbA, is required for subunit recognition by the assembly chaperone FimC and for FimC-catalyzed subunit folding. FimC thus guarantees quantitative disulfide bond formation in each of the up to 3,000 subunits of the pilus. The X-ray structure of the complex between FimC and the main pilus subunit FimA and the kinetics of FimC-catalyzed FimA folding indicate that FimC accelerates folding of pilus subunits by lowering their topological complexity. The kinetic data, together with the measured in vivo concentrations of DsbA and FimC, predict an in vivo half-life of 2 s for oxidative folding of FimA in the periplasm.

Design, synthesis, crystal structures, and antimicrobial activity of sulfonamide boronic acids as ß-lactamase inhibitors.

We investigated a series of sulfonamide boronic acids that resulted from the merging of two unrelated AmpC ß-lactamase inhibitor series. The new boronic acids differed in the replacement of the canonical carboxamide, found in all penicillin and cephalosporin antibiotics, with a sulfonamide. Surprisingly, these sulfonamides had a highly distinct structure-activity relationship from the previously explored carboxamides, high ligand efficiencies (up to 0.91), and K(i) values down to 25 nM and up to 23 times better for smaller analogues. Conversely, K(i) values were 10-20 times worse for larger molecules than in the carboxamide congener series. X-ray crystal structures (1.6-1.8 Å) of AmpC with three of the new sulfonamides suggest that this altered structure-activity relationship results from the different geometry and polarity of the sulfonamide versus the carboxamide. The most potent inhibitor reversed ß-lactamase-mediated resistance to third generation cephalosporins, lowering their minimum inhibitory concentrations up to 32-fold in cell culture.

Complementarity between a docking and a high-throughput screen in discovering new cruzain inhibitors.

Virtual and high-throughput screens (HTS) should have complementary strengths and weaknesses, but studies that prospectively and comprehensively compare them are rare. We undertook a parallel docking and HTS screen of 197861 compounds against cruzain, a thiol protease target for Chagas disease, looking for reversible, competitive inhibitors. On workup, 99% of the hits were eliminated as false positives, yielding 146 well-behaved, competitive ligands. These fell into five chemotypes: two were prioritized by scoring among the top 0.1% of the docking-ranked library, two were prioritized by behavior in the HTS and by clustering, and one chemotype was prioritized by both approaches. Determination of an inhibitor/cruzain crystal structure and comparison of the high-scoring docking hits to experiment illuminated the origins of docking false-negatives and false-positives. Prioritizing molecules that are both predicted by docking and are HTS-active yields well-behaved molecules, relatively unobscured by the false-positives to which both techniques are individually prone.

Anguinomycins and derivatives: total syntheses, modeling, and biological evaluation of the inhibition of nucleocytoplasmic transport.

The preparation of the polyketide natural products anguinomycin C and D is reported based on key steps such as Negishi stereoinversion cross coupling, Jacobsen Cr(III)-catalyzed Hetero Diels-Alder reaction, Evans B-mediated syn-aldol chemistry, and B-alkyl Suzuki-Miyaura cross coupling. The configuration of both natural products was established as (5R,10R,16R,18S,19R,20S). Biological evaluation demonstrated that these natural products are inhibitors of the nuclear export receptor CRM1, leading to shutdown of CRM1-mediated nuclear protein export at concentrations above 10 nM. Analogues of anguinomycin and leptomycin B (LMB) have been prepared, and the simple alpha,beta-unsaturated lactone analogue 4 with a truncated polyketide chain retains most of the biological activity (inhibition above 25 nM). The structural basis for this inhibition has been demonstrated by modeling the transport inhibitors into X-ray crystal structures, thus highlighting key points for successful and strong biological action of anguinomycin and LMB.

Infinite kinetic stability against dissociation of supramolecular protein complexes through donor strand complementation.

Adhesive type 1 pili from uropathogenic Escherichia coli strains are heat and denaturant resistant, filamentous protein complexes. Individual pilus subunits associate through "donor strand complementation," whereby the incomplete immunoglobulin-like fold of each subunit is completed by the N-terminal extension of a neighboring subunit. We show that antiparallel donor strand insertion generally causes nonequilibrium behavior in protein folding and extreme activation energy barriers for dissociation of subunit-subunit complexes. We identify the most kinetically stable, noncovalent protein complex known to date. The complex between the pilus subunit FimG and the donor strand peptide of the subunit FimF shows an extrapolated dissociation half-life of 3 x 10(9) years. The 15 residue peptide forms ideal intermolecular beta sheet H-bonds with FimG over 10 residues, and its hydrophobic side chains strongly interact with the hydrophobic core of FimG. The results show that kinetic stability and nonequilibrium behavior in protein folding confers infinite stability against dissociation in extracellular protein complexes.

Crystal structure of the ternary FimC-FimF(t)-FimD(N) complex indicates conserved pilus chaperone-subunit complex recognition by the usher FimD.

Type 1 pili, anchored to the outer membrane protein FimD, enable uropathogenic Escherichia coli to attach to host cells. During pilus biogenesis, the N-terminal periplasmic domain of FimD (FimD(N)) binds complexes between the chaperone FimC and pilus subunits via its partly disordered N-terminal segment, as recently shown for the FimC-FimH(P)-FimD(N) ternary complex. We report the structure of a new ternary complex (FimC-FimF(t)-FimD(N)) with the subunit FimF(t) instead of FimH(p). FimD(N) recognizes FimC-FimF(t) and FimC-FimH(P) very similarly, predominantly through hydrophobic interactions. The conserved binding mode at a "hot spot" on the chaperone surface could guide the design of pilus assembly inhibitors.

Evidence for a novel domain of bacterial outer membrane ushers.

Many pathogenic bacteria possess adhesive surface organelles (called pili), anchored to their outer membrane, which mediate the first step of infection by binding to host tissue. Pilus biogenesis occurs via the "chaperone-usher" pathway: the usher, a large outer membrane protein, binds complexes of a periplasmic chaperone with pilus subunits, unloads the subunits from the chaperone, and assembles them into the pilus, which is extruded into the extracellular space. Ushers comprise an N-terminal periplasmic domain, a large transmembrane beta-barrel central domain, and a C-terminal periplasmic domain. Since structural data are available only for the N-terminal domain, we performed an in-depth bioinformatic analysis of bacterial ushers. Our analysis led us to the conclusion that the transmembrane beta-barrel region of ushers contains a so far unrecognized soluble domain, the "middle domain", which possesses a beta-sandwich fold. Two other bacterial beta-sandwich domains, the TT0351 protein from Thermus thermophilus and the carbohydrate binding module CBM36 from Paenibacillus polymyxa, are possible distant relatives of the usher "middle domain". Several mutations reported to abolish in vivo pilus formation cluster in this region, underlining its functional importance.

Structural and functional insights into the assembly of type 1 pili from Escherichia coli.

Type 1 pili are filamentous protein complexes that are anchored to the outer membrane of uropathogenic Escherichia coli and mediate bacterial adhesion to the surface of urinary epithelium cells. We review here the current status of structural and functional studies on the assembly of type 1 pili.

Structural basis of chaperone-subunit complex recognition by the type 1 pilus assembly platform FimD.

Adhesive type 1 pili from uropathogenic Escherichia coli are filamentous protein complexes that are attached to the assembly platform FimD in the outer membrane. During pilus assembly, FimD binds complexes between the chaperone FimC and type 1 pilus subunits in the periplasm and mediates subunit translocation to the cell surface. Here we report nuclear magnetic resonance and X-ray protein structures of the N-terminal substrate recognition domain of FimD (FimD(N)) before and after binding of a chaperone-subunit complex. FimD(N) consists of a flexible N-terminal segment of 24 residues, a structured core with a novel fold, and a C-terminal hinge segment. In the ternary complex, residues 1-24 of FimD(N) specifically interact with both FimC and the subunit, acting as a sensor for loaded FimC molecules. Together with in vivo complementation studies, we show how this mechanism enables recognition and discrimination of different chaperone-subunit complexes by bacterial pilus assembly platforms.