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The nuclear receptor-related 1 protein, Nurr1, is a transcription factor critical for the development and maintenance of dopamine-producing neurons in the substantia nigra pars compacta, a cell population that progressively loses the ability to make dopamine and degenerates in Parkinson's disease. Recently, we demonstrated that Nurr1 binds directly to and is regulated by the endogenous dopamine metabolite 5,6-dihydroxyindole (DHI). Unfortunately, DHI is an unstable compound, and thus a poor tool for studying Nurr1 function. Here, we report that 5-chloroindole, an unreactive analog of DHI, binds directly to the Nurr1 ligand binding domain with micromolar affinity and stimulates the activity of Nurr1, including the transcription of genes governing the synthesis and packaging of dopamine.

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 β−1,4-galactosyltransferase-7 (β-GALT-7)—the first and second enzymes of GAG biosynthesis—when microinjected into a cell. In another study, 4-deoxy-4-fluoro-β-xylosides inhibited β-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 β-) xylosides and their corresponding hydrophilic inhibitors of XYLT-1 and β-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 IC(50) = 380 nM (prodrug), 122 μM (drug), and U87 cells at IC(50) = 10.57 μM (prodrug). Molecular docking studies were consistent with preferred binding of the α- versus β-nitro xyloside conformer to XYLT-1 and β-GALT-7 enzymes.

Non-invasive methods to study glucocorticoid receptor (GR) signaling are urgently needed to reveal the complexity of GR signaling in normal physiology and human disorders, as well as to identify selective GR modulators to treat diseases. Here, we report evidence supporting translational studies with (±)-[11C]-5-(4-fluorobenzyl)-10-methoxy-2,2,4-trimethyl-2,5-dihydro-1H-chromeno[3,4-f ]-quinoline (named as (±)-[11C]YJH08), a radioligand for positron emission tomography (PET) that engages the ligand binding domain on GR. Methods: (±)-[11C]YJH08 was synthesized by reacting the phenol precursor with [11C]methyl iodide. The biodistribution was studied in vivo with PET/CT and autoradiography. A library of analogues were synthesized and studied in vitro and in vivo to understand the (±)-[11C]YJH08 structure activity relationship. Rodent dosimetry studies were performed to estimate the human equivalent doses of (±)-[11C]YJH08. Results: (±)-[11C]YJH08 was synthesized by reaction of the phenolic precursor with [11C]methyl iodide giving a radiochemical yield of 51.7 ± 4.7% (decay corrected to starting [11C]methyl iodide). An analysis of the (±)-YJH08 structure-activity relationship with molecular dynamics simulations showed that (R)- and (S)-enantiomers occupy the ligand binding domain on GR with different binding modes and have indistinguishable patterns of biodistribution in vivo. Moreover, a focused chemical screen of analogues revealed that the aryl fluoride motif on YJH08 is essential for high affinity GR binding in vitro, high tissue uptake in vivo, and passage across the blood brain barrier. Lastly, we performed dosimetry studies in rodents, from which we showed estimated human equivalent doses of (±)-[11C]YJH08 to be commensurate with widely used carbon-11 and fluorine-18 tracers. Conclusion: In summary, these studies reveal the molecular determinants of a high affinity and selectivity ligand-receptor interaction and support the use of (±)-[11C]YJH08 PET to make the first measurements of GR expression in human subjects.

Progranulin (PGRN) is an evolutionarily conserved glycoprotein associated with several disease states, including neurodegeneration, cancer, and autoimmune disorders. This protein has recently been implicated in the regulation of lysosome function, whereby PGRN may bind to and promote the maturation and activity of the aspartyl protease cathepsin D (proCTSD, inactive precursor; matCTSD, mature, enzymatically active form). As the full-length PGRN protein can be cleaved into smaller peptides, called granulins, we assessed the function of these granulin peptides in binding to proCTSD and stimulating matCTSD enzyme activity in vitro. Here, we report that full-length PGRN and multi-granulin domain peptides bound to proCTSD with low to submicromolar binding affinities. This binding promoted proCTSD destabilization, the magnitude of which was greater for multi-granulin domain peptides than for full-length PGRN. Such destabilization correlated with enhanced matCTSD activity at acidic pH. The presence and function of multi-granulin domain peptides have typically been overlooked in previous studies. This work provides the first in vitro quantification of their binding and activity on proCTSD. Our study highlights the significance of multi-granulin domain peptides in the regulation of proCTSD maturation and enzymatic activity and suggests that attention to PGRN processing will be essential for the future understanding of the molecular mechanisms leading to neurodegenerative disease states with loss-of-function mutations in PGRN.

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 k(cat)/K(M) 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 k(cat)/K(M) 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 k(cat)/K(M) values (k(cat)/K(M) × 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 k(cat)/K(M) values, h12-LOX was markedly less effective than h15-LOX-1 in reacting with 17S-HpDHA, with a 55-fold lower effective k(cat)/K(M) in producing 16S,17S-epoxyDHA and a 27-fold lower effective k(cat)/K(M) 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 (K(d) = 5.9 μM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (K(d) = 2.5 μM), and 17S-hydroxy-13Z,15E,19Z-docosatrienoic acid (17S-HDTA) (K(d) = 1.4 μM), suggesting a possible regulatory pathway in reducing epoxide formation. Finally, 17S-HpDHA and PDX inhibited platelet aggregation, with EC(50) 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.

The nuclear receptor-related 1 protein, Nurr1, is a transcription factor critical for the development and maintenance of dopamine-producing neurons in the substantia nigra pars compacta, a cell population that progressively loses the ability to make dopamine and degenerates in Parkinson’s disease. Recently, we demonstrated that Nurr1 binds directly to and is regulated by the endogenous dopamine metabolite 5,6-dihydroxyindole (DHI). Unfortunately, DHI is an unstable compound, and thus a poor tool for studying Nurr1 function. Here we report that 5-chloroindole, an unreactive analog of DHI, binds directly to the Nurr1 ligand binding domain with micromolar affinity and stimulates the activity of Nurr1, including the transcription of genes governing the synthesis and packaging of dopamine.

The unfolded protein response (UPR) maintains protein folding homeostasis in the endoplasmic reticulum (ER). In metazoan cells, the Ire1 branch of the UPR initiates two functional outputs— non-conventional mRNA splicing and selective mRNA decay (RIDD). By contrast, Ire1 orthologs from Saccharomyces cerevisiae and Schizosaccharomyces pombe are specialized for only splicing or RIDD, respectively. The functional specialization lies in Ire1’s RNase activity, which is either stringently splice-site specific (as in S. cerevisiae) or promiscuous (as in S. pombe). Here, we developed an assay that reports on Ire1’s RNase promiscuity. We found that conversion of two amino acids within the RNase domain of S. cerevisiae Ire1 to their S. pombe counterparts rendered it promiscuous. Using biochemical assays and computational modeling, we show that the mutations rewired a pair of salt bridges at Ire1 RNase domain’s dimer interface, changing its protomer alignment. Thus, Ire1 protomer alignment affects its substrates specificity.

Small molecule colloidal aggregates adsorb and partially denature proteins, inhibiting them artifactually. Oddly, this inhibition is typically time-dependent. Two mechanisms might explain this: low concentrations of the colloid and enzyme might mean low encounter rates, or colloid-based protein denaturation might impose a kinetic barrier. These two mechanisms should have different concentration dependencies. Perplexingly, when enzyme concentration was increased, incubation times actually lengthened, inconsistent with both models and with classical chemical kinetics of solution species. We therefore considered molecular crowding, where colloids with lower protein surface density demand a shorter incubation time than more crowded colloids. To test this, we grew and shrank colloid surface area. As the surface area shrank, the incubation time lengthened, while as it increased, the converse was true. These observations support a crowding effect on protein binding to colloidal aggregates. Implications for drug delivery and for detecting aggregation-based inhibition will be discussed.

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.

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 k(cat)/K(M) 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.

International audience; Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics1. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

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.

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 k(cat)/K(M) 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 k(cat)/K(M) 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 (K(d) = 6.0 μM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (K(d) = 3.5 μM), and 14S-hydroxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic acid (14S-HDPA(ω-3)) (K(d) = 4.0 μM) were shown to decrease 13S,14S-epoxy-DHA production. h12-LOX was also shown to be allosterically regulated by 14S-HpDHA (K(d) = 3.5 μM) and 14S-HDPA(ω-3) (K(d) = 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.

The oxylipins, 7S,14S-diHDHA and 7S,17S-diHDHA (RvD5), have been found in macrophages exudates and are believed to function as specialized pro-resolving mediators (SPM’s). Their biosynthesis is thought to proceed through sequential oxidations of docosahexaenoic acid (DHA) by lipoxygenase enzymes, specifically by h5-LOX first to 7S-HDHA, followed by h12-LOX to form 7S,14S-diHDHA or h15-LOX-1 to form 7S,17S-diHDHA (RvD5). In this work, we determined that oxidation of 7S-HpDHA to 7S,14S-diHDHA can be performed by either h12-LOX or h15-LOX-1, with similar kinetics. The oxidation at C14 of DHA by h12-LOX was expected, but the non-canonical reaction of h15-LOX-1 to make primarily 7S,14S-diHDHA was unexpected. Computer modeling suggests the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of I399, forcing the hydrogen abstraction from C12 to oxygenate on C14, and not C17. This result raised questions regarding synthesis of 7S,17S-diHDHA (RvD5). Strikingly, we find h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages, suggests it may have a greater role in biosynthesizing SPM’s than previously thought. We also determined that the reactions of h5-LOX with 14S-HpDHA and 17S-HpDHA are kinetically slow compared to DHA, suggesting these may be minor biosynthetic routesin-vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micro-molar potencies, which could directly regulate clot resolution.

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 ± 0.05 μM for MLS000327069, 0.53 ± 0.04 μM for MLS000327186 and 0.87 ± 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.

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.

Resistance to clinical antiandrogens has plagued the evolution of effective therapeutics for advanced prostate cancer. As with the first-line therapeutic bicalutamide (Casodex), resistance to newer antiandrogens (enzalutamide, ARN-509) develops quickly in patients, despite the fact that these drugs have ∼10-fold better affinity for the androgen receptor than bicalutamide. Improving affinity alone is often not sufficient to prevent resistance, and alternative strategies are needed to improve antiandrogen efficacy. Covalent and reversible covalent drugs are being used to thwart drug resistance in other contexts, and activated aryl nitriles are among the moieties being exploited for this purpose. We capitalized on the presence of an aryl nitrile in bicalutamide, and the existence of a native cysteine residue (Cys784) in the androgen receptor ligand binding pocket, to develop 5N-bicalutamide, a cysteine-reactive antiandrogen. 5N-bicalutamide exhibits a 150-fold improvement in Ki and 20-fold improvement in IC5...

Macrocycles are appealing drug candidates due to their high affinity, specificity, and favorable pharmacological properties. In this study, we explored the effects of chemical modifications to a natural product macrocycle upon its activity, 3D geometry, and conformational entropy. We chose thiocillin as a model system, a thiopeptide in the ribosomally encoded family of natural products that exhibits potent antimicrobial effects against Gram-positive bacteria. Since thiocillin is derived from a genetically encoded peptide scaffold, site-directed mutagenesis allows for rapid generation of analogues. To understand thiocillin's structure-activity relationship, we generated a site-saturation mutagenesis library covering each position along thiocillin's macrocyclic ring. We report the identification of eight unique compounds more potent than wild-type thiocillin, the best having an 8-fold improvement in potency. Computational modeling of thiocillin's macrocyclic structure revealed a striking requirement for a low-entropy macrocycle for activity. The populated ensembles of the active mutants showed a rigid structure with few adoptable conformations while inactive mutants showed a more flexible macrocycle which is unfavorable for binding. This finding highlights the importance of macrocyclization in combination with rigidifying post-translational modifications to achieve high-potency binding.

Natural products serve as chemical blueprints for the majority of classes of antibiotics in our clinical arsenal. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class. Virginiamycin acetyltransferases (Vats) are resistance proteins that provide protection against streptogramins, potent Gram-positive antibiotics that inhibit the bacterial ribosome. Due to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogs that overcome Vat resistance have not been previously accessible. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with unprecedented structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogs to the bacterial ribosome at high resolution (2.4-2.8 Å), revealing new binding interactions that extend into the peptidyl tRNA binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. Two of these analogs have excellent activity against a streptogramin-resistant strain of S. aureus expressing VatA and exhibit decreased acetylation rates in vitro. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

Human immunodeficiency virus-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 (A3) antiviral proteins. Vif folds when it binds Cullin-RING E3 ligase 5 and the transcription cofactor CBF-β. A five-protein complex containing the substrate receptor (Vif, CBF-β, Elongin-B, Elongin-C (VCBC)) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use molecular dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and an A3 protein bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, whereas VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation-optimized spectroscopy NMR data, which indicates that the VCBC complex without CUL5 is dynamic on the μs-ms timescale. Our NMR data also show that the VCBC complex is more conformationally restricted when bound to the antiviral APOBEC3F (one of the A3 proteins), consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjunction with the global dynamics of the complex. Like other substrate receptors, VCBC can exist alone or in complex with CUL5 and other proteins in cells. Accordingly, the VCBC complex could be a good target for therapeutics that would inhibit full assembly of the ubiquitination complex by stabilizing an alternate VCBC conformation.

Protein kinase CK2 has emerged as an attractive cancer therapeutic target. Previous studies have highlighted the challenge of optimizing CK2 ATP-competitive inhibitors that have low druggability due to their polycyclic ring scaffolds. Therefore the development of novel inhibitors with non-polycyclic scaffolds emerges as a promising strategy for drug discovery targeting CK2. In this current study, based on the similar predicted binding poses of the linear 2-propenone scaffold of isoliquiritigenin with that of the polycyclic inhibitor CX-4945, a series of 2-propenone derivatives containing an amine-substituted five-membered heterocycle and a benzoic acid were designed, synthesized and evaluated for their in vitro CK2 inhibition and anti-cancer activity. Compound 8b was found to be the most potent CK2 inhibitor (IC50 = 0.6 μM) with the anti-proliferative activity on HepG2 cancer cells (IC50 = 14 μM), compared to the activity of isoliquiritigenin (IC50 = 17 μM and 51 μM, respectively). Molecular docking was performed to understand the binding modes of the newly designed 2-propenone derivatives with CK2. Compound 8b formed the most favorable network of hydrogen bonds with both the hinge region and positive area. Our results indicate that CK2 derivatives with a linear 2-propenone scaffold are promising candidates for anti-cancer drug discovery.

Macrocycles pose challenges for computer-aided drug design due to their conformational complexity. One fundamental challenge is identifying all low-energy conformations of the macrocyclic ring, which is important for modeling target binding, passive membrane permeation, and other conformation-dependent properties. Macrocyclic polyketides are medically and biologically important natural products characterized by structural and functional diversity. Advances in synthetic biology and semisynthetic methods may enable creation of an even more diverse set of non-natural product polyketides for drug discovery and other applications. However, the conformational sampling of these flexible compounds remains demanding. We developed and optimized a dihedral angle-based macrocycle conformational sampling method for macrocycles of arbitrary structure, and here we apply it to diverse polyketide natural products. First, we evaluated its performance using a data set of 37 polyketides with available crystal structures, with 9-22 rotatable bonds in the macrocyclic ring. Our optimized protocol was able to reproduce the crystal structure of polyketides' aglycone backbone within 0.50 Å RMSD for 31 out of 37 polyketides. Consistent with prior structural studies, our analysis suggests that polyketides tend to have multiple distinct low-energy structures, including the bioactive (target-bound) conformation as well as others of unknown significance. For this reason, we also introduce a strategy to improve both efficiency and accuracy of the conformational search by utilizing torsional restraints derived from NMR vicinal proton couplings to restrict the conformational search. Finally, as a first application of the method, we made blinded predictions of the passive membrane permeability of a diverse set of polyketides, based on their predicted structures in low- and high-dielectric media.

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.

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.

Tau, a member of the MAP2/tau family of microtubule-associated proteins, stabilizes and organizes axonal microtubules in healthy neurons. In neurodegenerative tauopathies, tau dissociates from microtubules and forms neurotoxic extracellular aggregates. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. Here, we used molecular dynamics, microtubule-binding experiments, and live-cell microscopy, revealing that highly-conserved histidine residues near the C terminus of each microtubule-binding repeat are pH sensors that can modulate tau-microtubule interaction strength within the physiological intracellular pH range. We observed that at low pH (7.5), tau deprotonation decreased binding to microtubules both in vitro and in cells. Electrostatic and hydrophobic characteristics of histidine were both required for tau-microtubule binding, as substitutions with constitutively and positively charged nonaromatic lysine or uncharged alanine greatly reduced or abolished tau-microtubule binding. Consistent with these findings, tau-microtubule binding was reduced in a cancer cell model with increased intracellular pH but was rapidly restored by decreasing the pH to normal levels. These results add detailed insights into the intracellular regulation of tau activity that may be relevant in both normal and pathological conditions.

Tau, a member of the MAP2/tau family of microtubule-associated proteins, functions to stabilize and organize axonal microtubules in healthy neurons. In contrast, tau dissociates from microtubules and forms neurotoxic extracellular aggregates in neurodegenerative tauopathies. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. We use molecular dynamics, microtubule-binding experiments and live cell microscopy to show that highly conserved histidine residues near the C terminus of each MT-binding repeat are pH sensors that can modulate tau-MT interaction strength within the physiological intracellular pH range. At lower pH, these histidines are positively charged and form cation-π interactions with phenylalanine residues in a hydrophobic cleft between adjacent tubulin dimers. At higher pH, tau deprotonation decreases microtubule-binding both in vitro and in cells. However, electrostatic and hydrophobic characteristics of histidine are required for tau-MT-binding as substitution with constitutively positively charged, non-aromatic lysine or uncharged alanine greatly reduces or abolishes tau-MT binding. Consistent with these findings, tau-MT binding is reduced in a cancer cell model with increased intracellular pH but is rapidly rescued by decreasing pH to normal levels. Thus, these data add a new dimension to the intracellular regulation of tau activity and could be relevant in normal and pathological conditions.

HIV-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 antiviral proteins. Vif folds when it binds the Cullin-RING E3 ligase CRL5 and the transcription cofactor CBF-β. A five-protein complex containing the substrate receptor (Vif, CBF-β, Elongin-B, Elongin-C) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use Molecular Dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and APOBEC3 bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, while VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation optimized spectroscopy (methyl-TROSY) NMR data, which indicates that the entire VCBC complex without CUL5 is dynamic on the μs-ms timescale. Vif binds APOBEC3 to recruit it to the complex, and methyl-TROSY NMR shows that the VCBC complex is more conformationally restricted when bound to APOBEC3F, consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjuction with the global dynamics of the complex. Like other ubiquitin substrate receptors, VCBC can exist alone or in complex with CUL5 in cells. Accordingly, the VCBC complex could be a good target for therapeutics that would inhibit full assembly of the ubiquitination complex by stabilizing an alternate VCBC conformation.

The progressive failure of protein homeostasis is a hallmark of aging and a common feature in neurodegenerative disease. As the enzymes executing the final stages of autophagy, lysosomal proteases (or cathepsins) are key contributors to maintenance of protein homeostasis with age. Here, we identify the cysteine-rich granulin peptides as a new class of regulators of lysosomal aspartyl protease activity. Granulins are produced in an age and stress-dependent manner through cleavage of the neurodegenerative disease protein, progranulin. Once liberated, granulins selectively interact with the aspartyl protease ASP-3/cathepsin D to impair enzymatic activity. Consequently, protein homeostasis and lysosome function is disrupted, prompting cells to activate a compensatory transcriptional program. Our results support a model in which granulin production modulates a critical transition between the normal, physiological regulation of protease activity and the impairment of lysosomal function that can occur with age and disease.

SUMMARY Multiple posttranslational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate passage through the transcription cycle. The crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac), a modification only found in higher eukaryotes, regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. Using mass spectrometry, we identified Regulator of Pre-mRNA Domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced in vitro binding of CTD peptides to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins and inversely regulated S5p levels genome-wide. Treatment with deacetylase inhibitors globally enhanced levels of K7ac‐ and decreased levels of S5-phosphorylated polymerases ≥500 base pairs downstream of transcription start sites of expressed genes, consistent with acetylation-dependent S5 dephosphorylation via a previously identified RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p, but also enhanced K7ac levels, indicating that RPRD proteins recruit a K7 deacetylase. Collectively, our data identify RPRD CIDs as K7ac reader domains and reveal auto-regulatory crosstalk between K7ac and S5p via RPRD proteins at the transition from transcription initiation to elongation in higher eukaryotes.

KIT is a type-3 receptor tyrosine kinase that is frequently mutated at exon 11 or 17 in a variety of cancers. First-generation KIT tyrosine kinase inhibitors (TKI) are ineffective against KIT exon 17 mutations, which favor an active conformation that prevents these TKIs from binding. The ATP-competitive inhibitors, midostaurin and avapritinib, which target the active kinase conformation, were developed to inhibit exon 17–mutant KIT. Because secondary kinase domain mutations are a common mechanism of TKI resistance and guide ensuing TKI design, we sought to define problematic KIT kinase domain mutations for these emerging therapeutics. Midostaurin and avapritinib displayed different vulnerabilities to secondary kinase domain substitutions, with the T670I gatekeeper mutation being selectively problematic for avapritinib. Although gatekeeper mutations often directly disrupt inhibitor binding, we provide evidence that T670I confers avapritinib resistance indirectly by inducing distant conformational changes in the phosphate-binding loop. These findings suggest combining midostaurin and avapritinib may forestall acquired resistance mediated by secondary kinase domain mutations. Significance: This study identifies potential problematic kinase domain mutations for next-generation KIT inhibitors midostaurin and avapritinib.

Single-copy loss-of-function mutations in the progranulin gene (PGRN) underlie the neurodegenerative disease frontotemporal lobar degeneration, while homozygous loss-of-function of PGRN results in the lysosomal storage disorder neuronal ceroid lipofuscinosis. Despite evidence that normal PGRN levels are critical for neuronal health, the function of this protein is not yet understood. Here, we show that PGRN stimulates the in vitro maturation of the lysosomal aspartyl protease cathepsin D (CTSD). CTSD is delivered to the endolysosomal system as an inactive precursor (proCTSD) and requires sequential cleavage steps via intermediate forms to achieve the mature state (matCTSD). In co-immunoprecipitation experiments, PGRN interacts predominantly with immature pro- and intermediate forms of CTSD. PGRN enhances in vitro conversion of proCTSD to matCTSD in a concentration-dependent manner. Differential scanning fluorimetry shows a destabilizing effect induced by PGRN on proCTSD folding (∆ Tm = − 1.7 °C at a 3:1 molar ratio). We propose a mechanism whereby PGRN binds to proCTSD, destabilizing the propeptide from the enzyme catalytic core and favoring conversion to mature forms of the enzyme. Further understanding of the role of PGRN in CTSD maturation will assist in the development of targeted therapies for neurodegenerative disease.

Protein kinase CK2 is considered as an emerging target in cancer therapy, and recent efforts have been made to develop its ATP-competitive inhibitors, but achieving selectivity with respect to related kinases remains challenging because of the highly conserved ATP-binding pocket of kinases. Non-ATP competitive inhibitors might solve this challenge; one such strategy is to identify compounds that target the CK2α/CK2β interface as CK2 holoenzyme antagonists. Here we improved the binding affinity to CK2α and cell-based anti-cancer activity of a CK2β-derived cyclic peptide (Pc) by combining structure-based computational design with experimental evaluation. By analyzing molecular dynamics simulations of Pc bound to CK2α, a series of Pc-derived peptides was rationally designed and synthesized to evaluate their binding affinity to CK2α, as well as anti-proliferative and pro-apoptotic effects against HepG2 cancer cell line. One amino acid substitutions on Pc, I192F, exhibited over 10-fold improvement in the predicted binding affinity to CK2α when compared to Pc, and a cell-permeable version, I192F-Tat, also demonstrated more potent anti-proliferative and pro-apoptotic effects against HepG2 compared to Pc. A second modification of Pc, H193W, also led to more potent cell-based activity, despite having weaker binding affinity (∼5×) to CK2α. The discovery of the I192F and H193W peptides provides new insights for further optimization of CK2 antagonist candidates as anti-cancer leads.

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 (EC(50) ~0.7 to 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 EC(50) 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. Based on these results, JG-231 was found to reduce tumor burden in an MDA-MB-231 xenograft model (4 mg/kg; i.p.). Together, these studies support the hypothesis that Hsp70 may be a promising target for anti-cancer therapeutics.

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.

The effect of peptide-to-peptoid substitutions on the passive membrane permeability of an N-methylated cyclic hexapeptide is examined. In general, substitutions maintained permeability but increased conformational heterogeneity. Diversification with nonproteinogenic side chains increased permeability up to 3-fold. Additionally, the conformational impact of peptoid substitutions within a β-turn are explored. Based on these results, the strategic incorporation of peptoid residues into cyclic peptides can maintain or improve cell permeability, while increasing access to diverse side-chain functionality.

Cyclic peptide natural products contain a variety of conserved, nonproteinogenic structural elements such as d-amino acids and amide N-methylation. In addition, many cyclic peptides incorporate γ-amino acids and other elements derived from polyketide synthases. We hypothesized that the position and orientation of these extended backbone elements impact the ADME properties of these hybrid molecules, especially their ability to cross cell membranes and avoid metabolic degradation. Here we report the synthesis of cyclic hexapeptide diastereomers containing γ-amino acids (e.g., statines) and systematically investigate their structure-permeability relationships. These compounds were much more water-soluble and, in many cases, were both more membrane permeable and more stable to liver microsomes than a similar non-statine-containing derivative. Permeability correlated well with the extent of intramolecular hydrogen bonding observed in the solution structures determined in the low-dielectric solvent CDCl3, and one compound showed an oral bioavailability of 21% in rat. Thus, the incorporation of γ-amino acids offers a route to increase backbone diversity and improve ADME properties in cyclic peptide scaffolds.

Farnesyl diphosphate synthase catalyzes the sequential chain elongation reactions between isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to form geranyl diphosphate (GPP) and between IPP and GPP to give farnesyl diphosphate (FPP). Bisubstrate analogues containing the allylic and homoallylic substrates were synthesized by joining fragments for IPP and the allylic diphosphates with a C-C bond between the methyl group at C3 in IPP and the Z-methyl group at C3 in DMAPP (3-OPP) and GPP (4-OPP), respectively. These constructs placed substantial limits on the conformational space available to the analogues relative to the two substrates. The key features of the synthesis of bisubstrate analogues 3-OPP and 4-OPP are a regioselective C-alkylation of the dianion of 3-methyl-3-buten-1-ol (5), a Z-selective cuprate addition of alkyl groups to an α,β-alkynyl ester intermediate, and differential activation of allylic and homoallylic alcohols in the analogues, followed by a simultaneous displacement of the leaving groups with tris(tetra-n-butylammonium) hydrogen diphosphate to give the corresponding bisdiphosphate analogues. The bisubstrate analogues were substrates for FPP synthase, giving novel seven-membered ring analogues of GPP and FPP. The catalytic efficiencies for cyclization of 3-OPP and 4-OPP were similar to those for chain elongation with IPP and DMAPP.

The genome of Labrenzia aggregata IAM 12614 encodes an uncharacterized member of the muconate lactonizing enzyme (MLE) subgroup of the enolase superfamily (UniProt ID A0NXQ8 ). The gene encoding A0NXQ8 is located between genes that encode members of the proline racemase superfamily, 4R-hydroxyproline 2-epimerase (UniProt ID A0NXQ7 ; 4HypE) and trans-3-hydroxy-l-proline dehydratase (UniProt ID A0NXQ9 ; t3LHypD). A0NXQ8 was screened with a library of proline analogues; two reactions were observed with cis-3-hydroxy-l-proline (c3LHyp), competing 2-epimerization to trans-3-hydroxy-d-proline (1,1-proton transfer) and dehydration to Δ(1)-pyrroline-2-carboxylate (β-elimination; c3LHyp dehydratase), with eventual total dehydration. The genome context encoding A0NXQ8 both (1) confirms its novel c3LHyp dehydratase function and (2) provides evidence for metabolic pathways that allow L. aggregata to utilize several isomeric 3- and 4-hydroxyprolines as sole carbon sources.

The chemokine receptor CXCR7 is an attractive target for a variety of diseases. While several small-molecule modulators of CXCR7 have been reported, peptidic macrocycles may provide advantages in terms of potency, selectivity, and reduced off-target activity. We produced a series of peptidic macrocycles that incorporate an N-linked peptoid functionality where the peptoid group enabled us to explore side-chain diversity well beyond that of natural amino acids. At the same time, theoretical calculations and experimental assays were used to track and reduce the polarity while closely monitoring the physicochemical properties. This strategy led to the discovery of macrocyclic peptide–peptoid hybrids with high CXCR7 binding affinities (Ki 5 × 10–6 cm/s). Moreover, bioactive peptide 25 (Ki = 9 nM) achieved oral bioavailability of 18% in rats, which was commensurate with the observed plasma clearance values upon intravenous administration.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation–contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro. However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38−/− mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38−/− myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of β-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38−/− myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38−/− mice. Whole hearts isolated from CD38−/− mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of β-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.

Cancer can be viewed as a set of different diseases with distinctions based on tissue origin, driver mutations, and genetic signatures. Accordingly, each of these distinctions have been used to classify cancer subtypes and to reveal common features. Here, we present a different analysis of cancer based on amino acid mutation signatures. Non-negative Matrix Factorization and principal component analysis of 29 cancers revealed six amino acid mutation signatures, including four signatures that were dominated by either arginine to histidine (Arg>His) or glutamate to lysine (Glu>Lys) mutations. Sample-level analyses reveal that while some cancers are heterogeneous, others are largely dominated by one type of mutation. Using a non-overlapping set of samples from the COSMIC somatic mutation database, we validate five of six mutation signatures, including signatures with prominent arginine to histidine (Arg>His) or glutamate to lysine (Glu>Lys) mutations. This suggests that our classification of cancers based on amino acid mutation patterns may provide avenues of inquiry pertaining to specific protein mutations that may generate novel insights into cancer biology.

Electrophilic probes that covalently modify a cysteine thiol often show enhanced pharmacological potency and selectivity. Although reversible Michael acceptors have been reported, the structural requirements for reversibility are poorly understood. Here, we report a novel class of acrylonitrile-based Michael acceptors, activated by aryl or heteroaryl electron-withdrawing groups. We demonstrate that thiol adducts of these acrylonitriles undergo β-elimination at rates that span more than 3 orders of magnitude. These rates correlate inversely with the computed proton affinity of the corresponding carbanions, enabling the intrinsic reversibility of the thiol-Michael reaction to be tuned in a predictable manner. We apply these principles to the design of new reversible covalent kinase inhibitors with improved properties. A cocrystal structure of one such inhibitor reveals specific noncovalent interactions between the 1,2,4-triazole activating group and the kinase. Our experimental and computational study enables the design of new Michael acceptors, expanding the palette of reversible, cysteine-targeted electrophiles.

The genome of Agrobacterium tumefaciens C58 encodes 12 members of the enolase superfamily (ENS), eight of which are members of the mandelate racemase (MR) subgroup and, therefore, likely to be acid sugar dehydratases. Using a library of 77 acid sugars for high-throughput screening, one protein (UniProt entry A9CG74; locus tag Atu4196) showed activity with both m-galactarate and d-galacturonate. Two families of galactarate dehydratases had been discovered previously in the ENS, GalrD/TalrD [Yew, W. S., et al. (2007) Biochemistry 46, 9564-9577] and GalrD-II [Rakus, J. F., et al. (2009) Biochemistry 48, 11546-11558]; these have different active site acid/base catalysis and have no activity with d-galacturonate. A9CG74 dehydrates m-galactarate to form 2-keto-3-deoxy-galactarate but does not dehydrate d-galacturonate as expected. Instead, when A9CG74 is incubated with d-galacturonate, 3-deoxy-d-xylo-hexarate or 3-deoxy-d-lyxo-hexarate is formed. In this reaction, instead of abstracting the C5 proton α to the carboxylate group, the expected reaction for a member of the ENS, the enzyme apparently abstracts the proton α to the aldehyde group to form 3-deoxy-d-threo-hexulosuronate that undergoes a 1,2-hydride shift similar to the benzylic acid rearrangement to form the observed product. A. tumefaciens C58 does not utilize m-galactarate as a carbon source under the conditions tested in this study, although it does utilize d-galacturonate, which is a likely precursor to m-galactarate. The gene encoding A9CG74 and several genome proximal genes were upregulated with d-galacturonate as the carbon source. One of these, a member of the dihydrodipicolinate synthase superfamily, catalyzes the dehydration and subsequent decarboxylation of 2-keto-3-deoxy-d-galactarate to α-ketoglutarate semialdehyde, thereby providing a pathway for the conversion of m-galactarate to α-ketoglutarate semialdehyde.

Enzymes in the glutathione transferase (GST) superfamily catalyze the conjugation of glutathione (GSH) to electrophilic substrates. As a consequence they are involved in a number of key biological processes, including protection of cells against chemical damage, steroid and prostaglandin biosynthesis, tyrosine catabolism, and cell apoptosis. Although virtual screening has been used widely to discover substrates by docking potential noncovalent ligands into active site clefts of enzymes, docking has been rarely constrained by a covalent bond between the enzyme and ligand. In this study, we investigate the accuracy of docking poses and substrate discovery in the GST superfamily, by docking 6738 potential ligands from the KEGG and MetaCyc compound libraries into 14 representative GST enzymes with known structures and substrates using the PLOP program [JacobsonProteins2004, 55, 35115048827]. For X-ray structures as receptors, one of the top 3 ranked models is within 3 Å all-atom root mean square deviation (RMSD) of the native complex in 11 of the 14 cases; the enrichment LogAUC value is better than random in all cases, and better than 25 in 7 of 11 cases. For comparative models as receptors, near-native ligand–enzyme configurations are often sampled but difficult to rank highly. For models based on templates with the highest sequence identity, the enrichment LogAUC is better than 25 in 5 of 11 cases, not significantly different from the crystal structures. In conclusion, we show that covalent docking can be a useful tool for substrate discovery and point out specific challenges for future method improvement.

SUMMARYNurr1, a nuclear receptor essential for the development, maintenance, and survival of midbrain dopaminergic neurons, is a potential therapeutic target for Parkinson’s disease, a neurological disorder characterized by the degeneration of these same neurons. Efforts to identify Nurr1 agonists have been hampered by the recognition that it lacks several classic regulatory elements of nuclear receptor function, including the canonical ligand-binding pocket. Here we report that the dopamine metabolite 5,6-dihydroxyindole (DHI) binds directly to and modulates the activity of Nurr1. Using biophysical assays and x-ray crystallography we show that DHI binds to the ligand binding domain within a non-canonical pocket, forming a covalent adduct with Cys566. In cultured cells and zebrafish, DHI stimulates Nurr1 activity, including the transcription of target genes underlying dopamine homeostasis. These findings suggest avenues for developing synthetic Nurr1 ligands to ameliorate the symptoms and progression of Parkinson’s disease.

The variation of the diffusion coefficient (D) of DNA molecules with their size and topology is an important problem. Experiments have elucidated key scaling factors for diffusion for DNA molecules in the size range of 5.9–287.1 kilo base pairs. In this Letter, we have investigated the dependence of D for both linear and supercoiled DNAs (from 300 to about 2300 base pairs) using Brownian dynamics simulation. We show that the dependence is similar for linear and supercoiled DNA, similar to larger DNA. Our calculated D values were, in general, in excellent agreement with experiments.

During prion diseases, a normally benign, host protein, denoted PrPC, undergoes alternative folding into the aberrant isoform, PrPSc. We used ELISA assays to identify and confirm hits in order to develop leads that reduce PrPSc in prion-infected dividing and stationary-phase mouse neuroblastoma (ScN2a-cl3) cells. We tested 52,830 diverse small molecules in dividing cells and 49,430 in stationary-phase cells. This led to 3,100 HTS and 970 single point confirmed (SPC) hits in dividing cells, 331 HTS and 55 confirmed SPC hits in stationary-phase cells as well as 36 confirmed SPC hits active in both. Fourteen chemical leads were identified from confirmed SPC hits in dividing cells and three in stationary-phase cells. From more than 682 compounds tested in concentration-effect relationships in dividing cells to determine potency (EC50), 102 had EC50 values between 1–10 µM and 50 had EC50 values of

SummaryRemoval of the 5′ cap structure by Dcp2 is a major step in several 5′–3′ mRNA decay pathways. The activity of Dcp2 is enhanced by Dcp1 and bound coactivators, yet the details of how these interactions are linked to chemistry are poorly understood. Here, we report three crystal structures of the catalytic Nudix hydrolase domain of Dcp2 that demonstrate binding of a catalytically essential metal ion, and enzyme kinetics are used to identify several key active site residues involved in acid/base chemistry of decapping. Using nuclear magnetic resonance and molecular dynamics, we find that a conserved metal binding loop on the catalytic domain undergoes conformational changes during the catalytic cycle. These findings describe key events during the chemical step of decapping, suggest local active site conformational changes are important for activity, and provide a framework to explain stimulation of catalysis by the regulatory domain of Dcp2 and associated coactivators.

The focus of CNS drug pharmacokinetics programs has recently shifted from determining the total concentrations in brain and blood to considering also unbound fractions and concentrations. Unfortunately, assessing unbound brain exposure experimentally requires demanding in vivo and in vitro studies. We propose a physical model, based on lipid binding and pH partitioning, to predict in silico the unbound volume of distribution in the brain. The model takes into account the partition of a drug into lipids, interstitial fluid and intracellular compartments of the brain. The results are in good agreement with the experimental data, suggesting that the contributions of lipid binding and pH partitioning are important in determining drug exposure in brain. The predicted values are used, together with predictions for plasma protein binding, as corrective terms in a second model to derive the unbound brain to plasma concentration ratio starting from experimental values of total concentration ratio. The calculated values of brain free fraction and passive permeability are also used to qualitatively determine the brain to plasma equilibration time in a model that shows promising results but is limited to a very small set of compounds. The models we propose are a step forward in understanding and predicting pharmacologically relevant exposure in brain starting from compounds' chemical structure and neuropharmacokinetics, by using experimental total brain to plasma ratios, in silico calculated properties and simple physics-based approaches. The models can be used in central nervous system drug discovery programs for a fast and cheap assessment of unbound brain exposure. For existing compounds, the unbound ratios can be derived from experimental values of total brain to plasma ratios. For both existing and hypothetical compounds, the unbound volume of distribution due to lipid binding and pH partitioning can be calculated starting only from the chemical structure.