Signal peptide mimicry primes Sec61 for client-selective inhibition.

Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryogenic electron microscopy structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis.

Structure-based discovery of nonopioid analgesics acting through the α2A-adrenergic receptor.

Because nonopioid analgesics are much sought after, we computationally docked more than 301 million virtual molecules against a validated pain target, the α2A-adrenergic receptor (α2AAR), seeking new α2AAR agonists chemotypes that lack the sedation conferred by known α2AAR drugs, such as dexmedetomidine. We identified 17 ligands with potencies as low as 12 nanomolar, many with partial agonism and preferential Gi and Go signaling. Experimental structures of α2AAR complexed with two of these agonists confirmed the docking predictions and templated further optimization. Several compounds, including the initial docking hit '9087 [mean effective concentration (EC50) of 52 nanomolar] and two analogs, '7075 and PS75 (EC50 4.1 and 4.8 nanomolar), exerted on-target analgesic activity in multiple in vivo pain models without sedation. These newly discovered agonists are interesting as therapeutic leads that lack the liabilities of opioids and the sedation of dexmedetomidine.

Kinetic and structural investigations of novel inhibitors of human epithelial 15-lipoxygenase-2.

Human epithelial 15-lipoxygenase-2 (h15-LOX-2, ALOX15B) is expressed in many tissues and has been implicated in atherosclerosis, cystic fibrosis and ferroptosis. However, there are few reported potent/selective inhibitors that are active ex vivo. In the current work, we report newly discovered molecules that are more potent and structurally distinct from our previous inhibitors, MLS000545091 and MLS000536924 (Jameson et al, PLoS One, 2014, 9, e104094), in that they contain a central imidazole ring, which is substituted at the 1-position with a phenyl moiety and with a benzylthio moiety at the 2-position. The initial three molecules were mixed-type, non-reductive inhibitors, with IC50 values of 0.34 ±â€¯0.05 µM for MLS000327069, 0.53 ±â€¯0.04 µM for MLS000327186 and 0.87 ±â€¯0.06 µM for MLS000327206 and greater than 50-fold selectivity versus h5-LOX, h12-LOX, h15-LOX-1, COX-1 and COX-2. A small set of focused analogs was synthesized to demonstrate the validity of the hits. In addition, a binding model was developed for the three imidazole inhibitors based on computational docking and a co-structure of h15-LOX-2 with MLS000536924. Hydrogen/deuterium exchange (HDX) results indicate a similar binding mode between MLS000536924 and MLS000327069, however, the latter restricts protein motion of helix-α2 more, consistent with its greater potency. Given these results, we designed, docked, and synthesized novel inhibitors of the imidazole scaffold and confirmed our binding mode hypothesis. Importantly, four of the five inhibitors mentioned above are active in an h15-LOX-2/HEK293 cell assay and thus they could be important tool compounds in gaining a better understanding of h15-LOX-2's role in human biology. As such, a suite of similar pharmacophores that target h15-LOX-2 both in vitro and ex vivo are presented in the hope of developing them as therapeutic agents.

Docking and mutagenesis studies lead to improved inhibitor development of ML355 for human platelet 12-lipoxygenase.

Human platelet 12-(S)-Lipoxygenase (12-LOX) is a fatty acid metabolizing oxygenase that plays an important role in platelet activation and cardiometabolic disease. ML355 is a specific 12-LOX inhibitor that has been shown to decrease thrombosis without prolonging hemostasis and protect human pancreatic islets from inflammatory injury. It has an amenable drug-like scaffold with nM potency and encouraging ADME and PK profiles, but its binding mode to the active site of 12-LOX remains unclear. In the current work, we combined computational modeling and experimental mutagenesis to propose a model in which ML355 conforms to the "U" shape of the 12-LOX active site, with the phenyl linker region wrapping around L407. The benzothiazole of ML355 extends into the bottom of the active site cavity, pointing towards residues A417 and V418. However, reducing the active site depth alone did not affect ML355 potency. In order to lower the potency of ML355, the cavity needed to be reduced in both length and width. In addition, H596 appears to position ML355 in the active site through an interaction with the 2-methoxy phenol moiety of ML355. Combined, this binding model suggested that the benzothiazole of ML355 could be enlarged. Therefore, a naphthyl-benzothiazole derivative of ML355, Lox12Slug001, was synthesized and shown to have 7.2-fold greater potency than ML355. This greater potency is proposed to be due to additional van der Waals interactions and pi-pi stacking with F414 and F352. Lox12Slug001 was also shown to be highly selective against 12-LOX relative to the other LOX isozymes and more importantly, it showed activity in rescuing human islets exposed to inflammatory cytokines with comparable potency to ML355. Further studies are currently being pursued to derivatize ML355 in order to optimize the additional space in the active site, while maintaining acceptable drug-like properties.

In Vitro Biosynthetic Pathway Investigations of Neuroprotectin D1 (NPD1) and Protectin DX (PDX) by Human 12-Lipoxygenase, 15-Lipoxygenase-1, and 15-Lipoxygenase-2.

In this paper, human platelet 12-lipoxygenase [h12-LOX (ALOX12)], human reticulocyte 15-lipoxygenase-1 [h15-LOX-1 (ALOX15)], and human epithelial 15-lipoxygenase-2 [h15-LOX-2 (ALOX15B)] were observed to react with docosahexaenoic acid (DHA) and produce 17S-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17S-HpDHA). The kcat/KM values with DHA for h12-LOX, h15-LOX-1, and h15-LOX-2 were 12, 0.35, and 0.43 s-1 µM-1, respectively, which demonstrate h12-LOX as the most efficient of the three. These values are comparable to their counterpart kcat/KM values with arachidonic acid (AA), 14, 0.98, and 0.24 s-1 µM-1, respectively. Comparison of their product profiles with DHA demonstrates that the three LOX isozymes produce 11S-HpDHA, 14S-HpDHA, and 17S-HpDHA, to varying degrees, with 17S-HpDHA being the majority product only for the 15-LOX isozymes. The effective kcat/KM values (kcat/KM × percent product formation) for 17S-HpDHA of the three isozymes indicate that the in vitro value of h12-LOX was 2.8-fold greater than that of h15-LOX-1 and 1.3-fold greater than that of h15-LOX-2. 17S-HpDHA was an effective substrate for h12-LOX and h15-LOX-1, with four products being observed under reducing conditions: protectin DX (PDX), 16S,17S-epoxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid (16S,17S-epoxyDHA), the key intermediate in neuroprotection D1 biosynthesis [NPD1, also known as protectin D1 (PD1)], 11,17S-diHDHA, and 16,17S-diHDHA. However, h15-LOX-2 did not react with 17-HpDHA. With respect to their effective kcat/KM values, h12-LOX was markedly less effective than h15-LOX-1 in reacting with 17S-HpDHA, with a 55-fold lower effective kcat/KM in producing 16S,17S-epoxyDHA and a 27-fold lower effective kcat/KM in generating PDX. This is the first direct demonstration of h15-LOX-1 catalyzing this reaction and reveals an in vitro pathway for PDX and NPD1 intermediate biosynthesis. In addition, epoxide formation from 17S-HpDHA and h15-LOX-1 was negatively affected via allosteric regulation by 17S-HpDHA (Kd = 5.9 µM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (Kd = 2.5 µM), and 17S-hydroxy-13Z,15E,19Z-docosatrienoic acid (17S-HDTA) (Kd = 1.4 µM), suggesting a possible regulatory pathway in reducing epoxide formation. Finally, 17S-HpDHA and PDX inhibited platelet aggregation, with EC50 values of approximately 1 and 3 µM, respectively. The in vitro results presented here may help advise in vivo PDX and NPD1 intermediate (i.e., 16S,17S-epoxyDHA) biosynthetic investigations and support the benefits of DHA rich diets.

Mutagenesis, Hydrogen-Deuterium Exchange, and Molecular Docking Investigations Establish the Dimeric Interface of Human Platelet-Type 12-Lipoxygenase.

It was previously shown that human platelet 12S-lipoxygenase (h12-LOX) exists as a dimer; however, the specific structure is unknown. In this study, we create a model of the dimer through a combination of computational methods, experimental mutagenesis, and hydrogen-deuterium exchange (HDX) investigations. Initially, Leu183 and Leu187 were replaced by negatively charged glutamate residues and neighboring aromatic residues were replaced with alanine residues (F174A/W176A/L183E/L187E/Y191A). This quintuple mutant disrupted both the hydrophobic and π-π interactions, generating an h12-LOX monomer. To refine the determinants for dimer formation further, the L183E/L187E mutant was generated and the equilibrium shifted mostly toward the monomer. We then submitted the predicted monomeric structure to protein-protein docking to create a model of the dimeric complex. A total of nine of the top 10 most energetically favorable docking conformations predict a TOP-to-TOP dimeric arrangement of h12-LOX, with the α-helices containing a Leu-rich region (L172, L183, L187, and L194), corroborating our experimental results showing the importance of these hydrophobic interactions for dimerization. This model was supported by HDX investigations that demonstrated the stabilization of four, non-overlapping peptides within helix α2 of the TOP subdomain for wt-h12-LOX, consistent with the dimer interface. Most importantly, our data reveal that the dimer and monomer of h12-LOX have distinct biochemical properties, suggesting that the structural changes due to dimerization have allosteric effects on active site catalysis and inhibitor binding.

Synthesis and Screening of α-Xylosides in Human Glioblastoma Cells.

Glycosaminoglycans (GAGs) such as heparan sulfate and chondroitin sulfate decorate all mammalian cell surfaces. These mucopolysaccharides act as coreceptors for extracellular ligands, regulating cell signaling, growth, proliferation, and adhesion. In glioblastoma, the most common type of primary malignant brain tumor, dysregulated GAG biosynthesis results in altered chain length, sulfation patterns, and the ratio of contributing monosaccharides. These events contribute to the loss of normal cellular function, initiating and sustaining malignant growth. Disruption of the aberrant cell surface GAGs with small molecule inhibitors of GAG biosynthetic enzymes is a potential therapeutic approach to blocking the rogue signaling and proliferation in glioma, including glioblastoma. Previously, 4-azido-xylose-α-UDP sugar inhibited both xylosyltransferase (XYLT-1) and ß-1,4-galactosyltransferase-7 (ß-GALT-7)-the first and second enzymes of GAG biosynthesis-when microinjected into a cell. In another study, 4-deoxy-4-fluoro-ß-xylosides inhibited ß-GALT-7 at 1 mM concentration in vitro. In this work, we seek to solve the enduring problem of drug delivery to human glioma cells at low concentrations. We developed a library of hydrophobic, presumed prodrugs 4-deoxy-4-fluoro-2,3-dibenzoyl-(α- or ß-) xylosides and their corresponding hydrophilic inhibitors of XYLT-1 and ß-GALT-7 enzymes. The prodrugs were designed to be activatable by carboxylesterase enzymes overexpressed in glioblastoma. Using a colorimetric MTT assay in human glioblastoma cell lines, we identified a prodrug-drug pair (4-nitrophenyl-α-xylosides) as lead drug candidates. The candidates arrest U251 cell growth at an IC50 = 380 nM (prodrug), 122 µM (drug), and U87 cells at IC50 = 10.57 µM (prodrug). Molecular docking studies were consistent with preferred binding of the α- versus ß-nitro xyloside conformer to XYLT-1 and ß-GALT-7 enzymes.

Role of Human 15-Lipoxygenase-2 in the Biosynthesis of the Lipoxin Intermediate, 5S,15S-diHpETE, Implicated with the Altered Positional Specificity of Human 15-Lipoxygenase-1.

The oxylipins, 5S,12S-dihydroxy-6E,8Z,10E,14Z-eicosatetraenoic acid (5S,12S-diHETE) and 5S,15S-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5S,15S-diHETE), have been identified in cell exudates and have chemotactic activity toward eosinophils and neutrophils. Their biosynthesis has been proposed to occur by sequential oxidations of arachidonic acid (AA) by lipoxygenase enzymes, specifically through oxidation of AA by h5-LOX followed by h12-LOX, h15-LOX-1, or h15-LOX-2. In this work, h15-LOX-1 demonstrates altered positional specificity when reacting with 5S-HETE, producing 90% 5S,12S-diHETE, instead of 5S,15S-diHETE, with kinetics 5-fold greater than that of h12-LOX. This is consistent with previous work in which h15-LOX-1 reacts with 7S-HDHA, producing the noncanonical, DHA-derived, specialized pro-resolving mediator, 7S,14S-diHDHA. It is also determined that oxygenation of 5S-HETE by h15-LOX-2 produces 5S,15S-diHETE and its biosynthetic kcat/KM flux is 2-fold greater than that of h15-LOX-1, suggesting that h15-LOX-2 may have a greater role in lipoxin biosynthesis than previously thought. In addition, it is shown that oxygenation of 12S-HETE and 15S-HETE by h5-LOX is kinetically slow, suggesting that the first step in the in vitro biosynthesis of both 5S,12S-diHETE and 5S,15S-diHETE is the production of 5S-HETE.

15-Lipoxygenase-1 biosynthesis of 7S,14S-diHDHA implicates 15-lipoxygenase-2 in biosynthesis of resolvin D5.

The two oxylipins 7S,14S-dihydroxydocosahexaenoic acid (diHDHA) and 7S,17S-diHDHA [resolvin D5 (RvD5)] have been found in macrophages and infectious inflammatory exudates and are believed to function as specialized pro-resolving mediators (SPMs). Their biosynthesis is thought to proceed through sequential oxidations of DHA by lipoxygenase (LOX) enzymes, specifically, by human 5-LOX (h5-LOX) first to 7(S)-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-DHA (7S-HDHA), followed by human platelet 12-LOX (h12-LOX) to form 7(S),14(S)-dihydroxy-4Z,8E,10Z,12E,16Z,19Z-DHA (7S,14S-diHDHA) or human reticulocyte 15-LOX-1 (h15-LOX-1) to form RvD5. In this work, we determined that oxidation of 7(S)-hydroperoxy-4Z,8E,10Z,13Z,16Z,19Z-DHA to 7S,14S-diHDHA is performed with similar kinetics by either h12-LOX or h15-LOX-1. The oxidation at C14 of DHA by h12-LOX was expected, but the noncanonical reaction of h15-LOX-1 to make over 80% 7S,14S-diHDHA was larger than expected. Results of computer modeling suggested that the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of Ile399, forcing the hydrogen abstraction from C12 to oxygenate on C14 but not C17. This result raised questions regarding the synthesis of RvD5. Strikingly, we found that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than does h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages suggests that it may have a greater role in biosynthesizing SPMs than previously thought. We also determined that the reactions of h5-LOX with 14(S)-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-DHA and 17(S)-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-DHA are kinetically slow compared with DHA, suggesting that these reactions may be minor biosynthetic routes in vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micromolar potencies, which could directly regulate clot resolution.

Biosynthesis of the Maresin Intermediate, 13S,14S-Epoxy-DHA, by Human 15-Lipoxygenase and 12-Lipoxygenase and Its Regulation through Negative Allosteric Modulators.

Human reticulocyte 15-lipoxygenase-1 (h15-LOX-1 or ALOX15) and platelet 12-lipoxygenase (h12-LOX or ALOX12) catalysis of docosahexaenoic acid (DHA) and the maresin precursor, 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-HpDHA), were investigated to determine their product profiles and relative rates in the biosynthesis of the key maresin intermediate, 13S,14S-epoxy-4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid (13S,14S-epoxy-DHA). Both enzymes converted DHA to 14S-HpDHA, with h12-LOX having a 39-fold greater kcat/KM value (14.0 ± 0.8 s-1 µM-1) than that of h15-LOX-1 (0.36 ± 0.08 s-1 µM-1) and a 1.8-fold greater 14S-HpDHA product selectivity, 81 and 46%, respectively. However, h12-LOX was markedly less effective at producing 13S,14S-epoxy-DHA from 14S-HpDHA than h15-LOX-1, with a 4.6-fold smaller kcat/KM value, 0.0024 ± 0.0002 and 0.11 ± 0.006 s-1 µM-1, respectively. This is the first evidence of h15-LOX-1 to catalyze this reaction and reveals a novel in vitro pathway for maresin biosynthesis. In addition, epoxidation of 14S-HpDHA is negatively regulated through allosteric oxylipin binding to h15-LOX-1 and h12-LOX. For h15-LOX-1, 14S-HpDHA (Kd = 6.0 µM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (Kd = 3.5 µM), and 14S-hydroxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic acid (14S-HDPAω-3) (Kd = 4.0 µM) were shown to decrease 13S,14S-epoxy-DHA production. h12-LOX was also shown to be allosterically regulated by 14S-HpDHA (Kd = 3.5 µM) and 14S-HDPAω-3 (Kd = 4.0 µM); however, 12S-HETE showed no effect, indicating for the first time an allosteric response by h12-LOX. Finally, 14S-HpDHA inhibited platelet aggregation at a submicrololar concentration, which may have implications in the benefits of diets rich in DHA. These in vitro biosynthetic pathways may help guide in vivo maresin biosynthetic investigations and possibly direct therapeutic interventions.

Probing the Electrostatic and Steric Requirements for Substrate Binding in Human Platelet-Type 12-Lipoxygenase.

Human platelet ALOX12 (hALOX12 or h12-LOX) has been implicated in a variety of human diseases. The present study investigates the active site of hALOX12 to more thoroughly understand how it positions the substrate and achieves nearly perfect regio- and stereospecificities (i.e., 100 ± 5% of the 12(S)-hydroperoxide product), utilizing site-directed mutagenesis. Specifically, we have determined that Arg402 is not as important in substrate binding as previously seen for hALOX15 but that His596 may play a role in anchoring the carboxy terminal of the arachidonic acid during catalysis. In addition, Phe414 creates a π-stacking interaction with a double bond of arachidonic acid (Δ11), and Ala417/Val418 define the bottom of the cavity. However, the influence of Ala417/Val418 on the profile is markedly less for hALOX12 than that seen in hALOX15. Mutating these two residues to larger amino acids (Ala417Ile/Val418Met) only increased the generation of 15-HpETE by 24 ± 2%, but conversely, smaller residues at these positions converted hALOX15 to almost 100% hALOX12 reactivity [Gan et al. (1996) J. Biol. Chem. 271, 25412-25418]. However, we were able to increase 15-HpETE to 46 ± 3% by restricting the width of the active site with the Ala417Ile/Val418Met/Ser594Thr mutation, indicating both depth and width of the active site are important. Finally, residue Leu407 is shown to play a critical role in positioning the substrate correctly, as seen by the increase of 15-HpETE to 21 ± 1% for the single Leu407Gly mutant. These results outline critical differences between the active site requirements of hALOX12 relative to hALOX15 and explain both their product specificity and inhibitory differences.

Inhibitor binding mode and allosteric regulation of Na+-glucose symporters.

Sodium-dependent glucose transporters (SGLTs) exploit sodium gradients to transport sugars across the plasma membrane. Due to their role in renal sugar reabsorption, SGLTs are targets for the treatment of type 2 diabetes. Current therapeutics are phlorizin derivatives that contain a sugar moiety bound to an aromatic aglycon tail. Here, we develop structural models of human SGLT1/2 in complex with inhibitors by combining computational and functional studies. Inhibitors bind with the sugar moiety in the sugar pocket and the aglycon tail in the extracellular vestibule. The binding poses corroborate mutagenesis studies and suggest a partial closure of the outer gate upon binding. The models also reveal a putative Na+ binding site in hSGLT1 whose disruption reduces the transport stoichiometry to the value observed in hSGLT2 and increases inhibition by aglycon tails. Our work demonstrates that subtype selectivity arises from Na+-regulated outer gate closure and a variable region in extracellular loop EL5.

Exploration of Benzothiazole Rhodacyanines as Allosteric Inhibitors of Protein-Protein Interactions with Heat Shock Protein 70 (Hsp70).

Cancer cells rely on the chaperone heat shock protein 70 (Hsp70) for survival and proliferation. Recently, benzothiazole rhodacyanines have been shown to bind an allosteric site on Hsp70, interrupting its binding to nucleotide-exchange factors (NEFs) and promoting cell death in breast cancer cell lines. However, proof-of-concept molecules, such as JG-98, have relatively modest potency (EC50 ≈ 0.7-0.4 µM) and are rapidly metabolized in animals. Here, we explored this chemical series through structure- and property-based design of ∼300 analogs, showing that the most potent had >10-fold improved EC50 values (∼0.05 to 0.03 µM) against two breast cancer cells. Biomarkers and whole genome CRISPRi screens confirmed members of the Hsp70 family as cellular targets. On the basis of these results, JG-231 was found to reduce tumor burden in an MDA-MB-231 xenograft model (4 mg/kg, ip). Together, these studies support the hypothesis that Hsp70 may be a promising target for anticancer therapeutics.

High-throughput screen for inhibitors of protein-protein interactions in a reconstituted heat shock protein 70 (Hsp70) complex.

Protein-protein interactions (PPIs) are an important category of putative drug targets. Improvements in high-throughput screening (HTS) have significantly accelerated the discovery of inhibitors for some categories of PPIs. However, methods suitable for screening multiprotein complexes (e.g. those composed of three or more different components) have been slower to emerge. Here, we explored an approach that uses reconstituted multiprotein complexes (RMPCs). As a model system, we chose heat shock protein 70 (Hsp70), which is an ATP-dependent molecular chaperone that interacts with co-chaperones, including DnaJA2 and BAG2. The PPIs between Hsp70 and its co-chaperones stimulate nucleotide cycling. Thus, to re-create this ternary protein system, we combined purified human Hsp70 with DnaJA2 and BAG2 and then screened 100,000 diverse compounds for those that inhibited co-chaperone-stimulated ATPase activity. This HTS campaign yielded two compounds with promising inhibitory activity. Interestingly, one inhibited the PPI between Hsp70 and DnaJA2, whereas the other seemed to inhibit the Hsp70-BAG2 complex. Using secondary assays, we found that both compounds inhibited the PPIs through binding to allosteric sites on Hsp70, but neither affected Hsp70's intrinsic ATPase activity. Our RMPC approach expands the toolbox of biochemical HTS methods available for studying difficult-to-target PPIs in multiprotein complexes. The results may also provide a starting point for new chemical probes of the Hsp70 system.

GluA1 signal peptide determines the spatial assembly of heteromeric AMPA receptors.

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and predominantly assemble as heterotetramers in the brain. Recently, the crystal structures of homotetrameric GluA2 demonstrated that AMPARs are assembled with two pairs of conformationally distinct subunits, in a dimer of dimers formation. However, the structure of heteromeric AMPARs remains unclear. Guided by the GluA2 structure, we performed cysteine mutant cross-linking experiments in full-length GluA1/A2, aiming to draw the heteromeric AMPAR architecture. We found that the amino-terminal domains determine the first level of heterodimer formation. When the dimers further assemble into tetramers, GluA1 and GluA2 subunits have preferred positions, possessing a 1-2-1-2 spatial assembly. By swapping the critical sequences, we surprisingly found that the spatial assembly pattern is controlled by the excisable signal peptides. Replacements with an unrelated GluK2 signal peptide demonstrated that GluA1 signal peptide plays a critical role in determining the spatial priority. Our study thus uncovers the spatial assembly of an important type of glutamate receptors in the brain and reveals a novel function of signal peptides.

Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors.

A potent and selective inhibitor targeting human and murine 12/15-LOX.

Human reticulocyte 12/15-lipoxygenase (h12/15-LOX) is a lipid-oxidizing enzyme that can directly oxidize lipid membranes in the absence of a phospholipase, leading to a direct attack on organelles, such as the mitochondria. This cytotoxic activity of h12/15-LOX is up-regulated in neurons and endothelial cells after a stroke and thought to contribute to both neuronal cell death and blood-brain barrier leakage. The discovery of inhibitors that selectively target recombinant h12/15-LOX in vitro, as well as possessing activity against the murine ortholog ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke. Herein, we report a new family of inhibitors discovered in a High Throughput Screen (HTS) that are selective and potent against recombinant h12/15-LOX and cellular mouse 12/15-LOX (m12/15-LOX). MLS000099089 (compound 99089), the parent molecule, exhibits an IC50 potency of 3.4±0.5 µM against h12/15-LOX in vitro and an ex vivo IC50 potency of approximately 10 µM in a mouse neuronal cell line, HT-22. Compound 99089 displays greater than 30-fold selectivity versus h5-LOX and COX-2, 15-fold versus h15-LOX-2 and 10-fold versus h12-LOX, when tested at 20 µM inhibitor concentration. Steady-state inhibition kinetics reveals that the mode of inhibition of 99089 against h12/15-LOX is that of a mixed inhibitor with a Kic of 1.0±0.08 µM and a Kiu of 6.0±3.3 µM. These data indicate that 99089 and related derivatives may serve as a starting point for the development of anti-stroke therapeutics due to their ability to selectively target h12/15-LOX in vitro and m12/15-LOX ex vivo.

Structure-Bioactivity Relationship for Benzimidazole Thiophene Inhibitors of Polo-Like Kinase 1 (PLK1), a Potential Drug Target in Schistosoma mansoni.

BACKGROUND: Schistosoma flatworm parasites cause schistosomiasis, a chronic and debilitating disease of poverty in developing countries. Praziquantel is employed for treatment and disease control. However, its efficacy spectrum is incomplete (less active or inactive against immature stages of the parasite) and there is a concern of drug resistance. Thus, there is a need to identify new drugs and drug targets. METHODOLOGY/PRINCIPAL FINDINGS: We show that RNA interference (RNAi) of the Schistosoma mansoni ortholog of human polo-like kinase (huPLK)1 elicits a deleterious phenotypic alteration in post-infective larvae (schistosomula or somules). Phenotypic screening and analysis of schistosomula and adult S. mansoni with small molecule inhibitors of huPLK1 identified a number of potent anti-schistosomals. Among these was a GlaxoSmithKline (GSK) benzimidazole thiophene inhibitor that has completed Phase I clinical trials for treatment of solid tumor malignancies. We then obtained GSKs Published Kinase Inhibitor Sets (PKIS) 1 and 2, and phenotypically screened an expanded series of 38 benzimidazole thiophene PLK1 inhibitors. Computational analysis of controls and PLK1 inhibitor-treated populations of somules demonstrated a distinctive phenotype distribution. Using principal component analysis (PCA), the phenotypes exhibited by these populations were mapped, visualized and analyzed through projection to a low-dimensional space. The phenotype distribution was found to have a distinct shape and topology, which could be elicited using cluster analysis. A structure-activity relationship (SAR) was identified for the benzimidazole thiophenes that held for both somules and adult parasites. The most potent inhibitors produced marked phenotypic alterations at 1-2 µM within 1 h. Among these were compounds previously characterized as potent inhibitors of huPLK1 in cell assays. CONCLUSIONS/SIGNIFICANCE: The reverse genetic and chemical SAR data support a continued investigation of SmPLK1 as a possible drug target and/or the prosecution of the benzimidazole thiophene chemotype as a source of novel anti-schistosomals.

A high throughput screen identifies potent and selective inhibitors to human epithelial 15-lipoxygenase-2.

Lipoxygenase (LOX) enzymes catalyze the hydroperoxidation of arachidonic acid and other polyunsaturated fatty acids to hydroxyeicosatetraenoic acids with varying positional specificity to yield important biological signaling molecules. Human epithelial 15-lipoxygenase-2 (15-LOX-2) is a highly specific LOX isozyme that is expressed in epithelial tissue and whose activity has been correlated with suppression of tumor growth in prostate and other epithelial derived cancers. Despite the potential utility of an inhibitor to probe the specific role of 15-LOX-2 in tumor progression, no such potent/specific 15-LOX-2 inhibitors have been reported to date. This study employs high throughput screening to identify two novel, specific 15-LOX-2 inhibitors. MLS000545091 is a mixed-type inhibitor of 15-LOX-2 with a Ki of 0.9+/-0.4 µM and has a 20-fold selectivity over 5-LOX, 12-LOX, 15-LOX-1, COX-1, and COX-2. MLS000536924 is a competitive inhibitor with a Ki of 2.5+/-0.5 µM and also possesses 20-fold selectivity toward 15-LOX-2 over the other oxygenases, listed above. Finally, neither compound possesses reductive activity towards the active-site ferrous ion.

Leveraging structure for enzyme function prediction: methods, opportunities, and challenges.

The rapid growth of the number of protein sequences that can be inferred from sequenced genomes presents challenges for function assignment, because only a small fraction (currently <1%) has been experimentally characterized. Bioinformatics tools are commonly used to predict functions of uncharacterized proteins. Recently, there has been significant progress in using protein structures as an additional source of information to infer aspects of enzyme function, which is the focus of this review. Successful application of these approaches has led to the identification of novel metabolites, enzyme activities, and biochemical pathways. We discuss opportunities to elucidate systematically protein domains of unknown function, orphan enzyme activities, dead-end metabolites, and pathways in secondary metabolism.