Distinct Ligand Binding Research Articles

Genomic analysis of Salmonella bacteriophages revealed multiple endolysin ORFs and importance of ligand binding site of receptor binding protein.

Salmonella is a prevalent foodborne pathogen causing millions of global cases annually. Antimicrobial resistance is a growing public health concern, leading to search for alternatives like bacteriophages. 97 bacteriophages, isolated from cattle farms (n= 48), poultry farms (n=37), and wastewater (n=5) samples in Türkiye, were subjected to host range analysis using 36 Salmonella isolates with 18 different serotypes. The broadest host range belonged to an Infantis phage (MET P1-091), lysing 28 hosts. Ten phages with the widest host range underwent further analysis, revealing 7 unique genomes (32 to 243kb), including a jumbophage (>200kb). Except for one with lysogenic properties, none of them harbored virulence or antibiotic resistance genes, making them potential Salmonella reducers in different environments. Examining ORFs of endolysin enzymes revealed surprising findings: 5 of 7 unique genomes contained multiple endolysin ORFs. Despite sharing same endolysin sequences, phages exhibited significant differences in host range. Detailed analysis unveiled diverse receptor binding protein sequences, with similar structures but distinct ligand binding sites. These findings emphasize the importance of ligand binding sites of receptor binding proteins. Additionally, bacterial reduction curve and virulence index revealed that Enteritidis phages inhibit bacterial growth even at low concentrations, unlike Infantis and Kentucky phages.

Structural basis of ligand recognition and design of antihistamines targeting histamine H4 receptor

The histamine H4 receptor (H4R) plays key role in immune cell function and is a highly valued target for treating allergic and inflammatory diseases. However, structural information of H4R remains elusive. Here, we report four cryo-EM structures of H4R/Gi complexes, with either histamine or synthetic agonists clobenpropit, VUF6884 and clozapine bound. Combined with mutagenesis, ligand binding and functional assays, the structural data reveal a distinct ligand binding mode where D943.32 and a π-π network determine the orientation of the positively charged group of ligands, while E1825.46, located at the opposite end of the ligand binding pocket, plays a key role in regulating receptor activity. The structural insight into H4R ligand binding allows us to identify mutants at E1825.46 for which the agonist clobenpropit acts as an inverse agonist and to correctly predict inverse agonism of a closely related analog with nanomolar potency. Together with the findings regarding receptor activation and Gi engagement, we establish a framework for understanding H4R signaling and provide a rational basis for designing novel antihistamines targeting H4R.

Differentiating the roles of Mycobacterium tuberculosis substrate binding proteins, FecB and FecB2, in iron uptake.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, poses a great threat to human health. With the emergence of drug resistant Mtb strains, new therapeutics are desperately needed. As iron is critical to the growth and survival of Mtb, mechanisms through which Mtb acquires host iron represent attractive therapeutic targets. Mtb scavenges host iron via Mtb siderophore-dependent and heme iron uptake pathways. While multiple studies describe the import of heme and ferric-siderophores and the export of apo-siderophores across the inner membrane, little is known about their transport across the periplasm and cell-wall environments. Mtb FecB and FecB2 are predicted periplasmic binding proteins implicated in host iron acquisition; however, their precise roles are not well understood. This study sought to differentiate the roles FecB and FecB2 play in Mtb iron acquisition. The crystallographic structures of Mtb FecB and FecB2 were determined to 2.0 Å and 2.2 Å resolution, respectively, and show distinct ligand binding pockets. In vitro ligand binding experiments for FecB and FecB2 were performed with heme and bacterial siderophores from Mtb and other species, revealing that both FecB and FecB2 bind heme, while only FecB binds the Mtb sideophore ferric-carboxymycobactin (Fe-cMB). Subsequent structure-guided mutagenesis of FecB identified a single glutamate residue-Glu339-that significantly contributes to Fe-cMB binding. A role for FecB in the Mtb siderophore-mediated iron acquisition pathway was corroborated by Mycobacterium smegmatis and Mtb pull-down assays, which revealed interactions between FecB and members of the mycobacterial siderophore export and import machinery. Similarly, pull-down assays with FecB2 confirms its role in heme uptake revealing interactions with a potential inner membrane heme importer. Due to ligand preference and protein partners, our data suggest that Mtb FecB plays a role in siderophore-dependent iron and heme acquisition pathways; in addition, we confirm that Mtb FecB2 is involved in heme uptake.

Gonadotropin Receptor Cross-Talk and Altered Functions in Gonadal and Non-Gonadal Tissues

Reproduction depends on the responses of gonadotropins through their specific receptors. The gonadotropin family has three members; Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), and Human Chorionic Gonadotropin (hCG). These glycoprotein hormones comprise two subunits, an identical α-subunit and a hormone-specific-β subunit. Their cognate receptors (FSHR and LHCGR) are two adrenergic receptor-like family A/rhodopsin-like G-Protein Coupled Receptors (GPCRs) with structurally distinct ligand binding domains. The hCG binds to LHCGR but has a longer half-life and higher affinity to LHCGR. The expression of FSHR and LHCGR is observed in both gonadal and nongonadal cells. In this review, we will be emphasizing the differential expression of gonadotropin receptors in different cells of the human body, their specific responses through cross-talk, and how a defect in the expression and activity of FSHR and LHCGR may alter the responses of FSH and LH/hCG leading to diseases like PCOS, cancer and metabolic disorders.

Structural basis of α1A-adrenergic receptor activation and recognition by an extracellular nanobody

The α1A-adrenergic receptor (α1AAR) belongs to the family of G protein-coupled receptors that respond to adrenaline and noradrenaline. α1AAR is involved in smooth muscle contraction and cognitive function. Here, we present three cryo-electron microscopy structures of human α1AAR bound to the endogenous agonist noradrenaline, its selective agonist oxymetazoline, and the antagonist tamsulosin, with resolutions range from 2.9 Å to 3.5 Å. Our active and inactive α1AAR structures reveal the activation mechanism and distinct ligand binding modes for noradrenaline compared with other adrenergic receptor subtypes. In addition, we identified a nanobody that preferentially binds to the extracellular vestibule of α1AAR when bound to the selective agonist oxymetazoline. These results should facilitate the design of more selective therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

A deep encoder-decoder framework for identifying distinct ligand binding pathways.

The pathway(s) that a ligand would adopt en route to its trajectory to the native pocket of the receptor protein act as a key determinant of its biological activity. While Molecular Dynamics (MD) simulations have emerged as the method of choice for modeling protein-ligand binding events, the high dimensional nature of the MD-derived trajectories often remains a barrier in the statistical elucidation of distinct ligand binding pathways due to the stochasticity inherent in the ligand's fluctuation in the solution and around the receptor. Here, we demonstrate that an autoencoder based deep neural network, trained using an objective input feature of a large matrix of residue-ligand distances, can efficiently produce an optimal low-dimensional latent space that stores necessary information on the ligand-binding event. In particular, for a system of L99A mutant of T4 lysozyme interacting with its native ligand, benzene, this deep encoder-decoder framework automatically identifies multiple distinct recognition pathways, without requiring user intervention. The intermediates involve the spatially discrete location of the ligand in different helices of the protein before its eventual recognition of native pose. The compressed subspace derived from the autoencoder provides a quantitatively accurate measure of the free energy and kinetics of ligand binding to the native pocket. The investigation also recommends that while a linear dimensional reduction technique, such as time-structured independent component analysis, can do a decent job of state-space decomposition in cases where the intermediates are long-lived, autoencoder is the method of choice in systems where transient, low-populated intermediates can lead to multiple ligand-binding pathways.

Structural analysis of human G-protein-coupled receptor 17 ligand binding sites.

The human G protein coupled membrane receptor (GPR17), the sensor of brain damage, is identified as a biomarker for many neurological diseases. In human brain tissue, GPR17 exist in two isoforms, long and short. While cryo-electron microscopy technology has provided the structure of the long isoform of GPR17 with Gi complex, the structure of the short isoform and its activation mechanism remains unclear. Recently, we theoretically modeled the structure of the short isoform of GPR17 with Gi signaling protein and identified novel ligands. In the present work, we demonstrated the presence of two distinct ligand binding sites in the short isoform of GPR17. The molecular docking of GPR17 with endogenous (UDP) and synthetic ligands (T0510.3657, MDL29950) found the presence of two distinct binding pockets. Our observations revealed that endogenous ligand UDP can bind stronger in two different binding pockets as evidenced by glide and autodock vina scores, whereas the other two ligand's binding with GPR17 has less docking score. The analysis of receptor-UDP interactions shows complexes' stability in the lipid environment by 100 ns atomic molecular dynamics simulations. The amino acid residues VAL83, ARG87, and PHE111 constitute ligand binding site 1, whereas site 2 constitutes ASN67, ARG129, and LYS232. Root mean square fluctuation analysis showed the residues 83, 87, and 232 with higher fluctuations during molecular dynamics simulation in both binding pockets. Our findings imply that the residues of GPR17's two binding sites are crucial, and their interaction with UDP reveals the protein's hidden signaling and communication properties. Furthermore, this finding may assist in the development of targeted therapies for the treatment of neurological diseases.

Glycosaminoglycan Analysis: Purification, Structural Profiling, and GAG-Protein Interactions.

Glycosaminoglycans (GAGs) are long, linear polysaccharides that are ubiquitously expressed on the cell surface and in the extracellular matrix of all animal cells. These complex carbohydrates are composed of alternating glucosamine and uronic acids that can be heterogeneously N- and O-sulfated. The arrangement and orientation of the sulfated sugar residues specify the location of distinct ligand binding sites on the cell surface, and their capacity to bind ligands impacts cell growth and development, the ability to form tissues and organs, and normal physiology. The heterogeneous nature of GAGs and their inherent structural diversity across different tissues, cell types, and disease states creates challenges to characterizing their structure and function. Here, we describe detailed methods to investigate GAG-protein interactions in vitro and evaluate the structural composition of two classes of sulfated GAGs, heparan sulfate and chondroitin/dermatan sulfate, using liquid chromatography, mass spectrometry, and radiolabeling techniques. Overall, these methods facilitate the evaluation of GAG structure and function to uncover the unique roles these molecules play in cell biology and human disease.

Use of NanoBiT and NanoBRET to monitor fluorescent VEGF-A binding kinetics to VEGFR2/NRP1 heteromeric complexes in living cells.

VEGF-A is a key mediator of angiogenesis, primarily signalling via VEGF receptor 2 (VEGFR2). Endothelial cells also express the co-receptor neuropilin-1 (NRP1) that potentiates VEGF-A/VEGFR2 signalling. VEGFR2 and NRP1 had distinct real-time ligand binding kinetics when monitored using BRET. We previously characterised fluorescent VEGF-A isoforms tagged at a single site with tetramethylrhodamine (TMR). Here, we explored differences between VEGF-A isoforms in living cells that co-expressed both receptors. Receptor localisation was monitored in HEK293T cells expressing both VEGFR2 and NRP1 using membrane-impermeant HaloTag and SnapTag technologies. To isolate ligand binding pharmacology at a defined VEGFR2/NRP1 complex, we developed an assay using NanoBiT complementation technology whereby heteromerisation is required for luminescence emissions. Binding affinities and kinetics of VEGFR2-selective VEGF165 b-TMR and non-selective VEGF165 a-TMR were monitored using BRET from this defined complex. Cell surface VEGFR2 and NRP1 were co-localised and formed a constitutive heteromeric complex. Despite being selective for VEGFR2, VEGF165 b-TMR had a distinct kinetic ligand binding profile at the complex that largely remained elevated in cells over 90 min. VEGF165 a-TMR bound to the VEGFR2/NRP1 complex with kinetics comparable to those of VEGFR2 alone. Using a binding-dead mutant of NRP1 did not affect the binding kinetics or affinity of VEGF165 a-TMR. This NanoBiT approach enabled real-time ligand binding to be quantified in living cells at 37°C from a specified complex between a receptor TK and its co-receptor for the first time.

Allosteric Nanobodies Modulate EGFR Functioning by Tuning Receptor Dynamics

Membrane-spanning receptors are crucial for sensing and transducing signals across the plasma membrane. While increasing evidence suggests that these processes are characterized by distinct conformations and ligand binding properties, the role of receptor dynamics in signal transduction remains elusive. An example refers to the epidermal growth factor receptor, that activates signaling pathways crucial for the regulation of cell proliferation, migration and differentiation. We employed nanobodies to directly examine the relationship between EGFR dynamics and function. Using a combination of cell biology and solid-state NMR methods, we find that allosteric nanobody binding reduces extracellular domain (ECD) motion and EGF binding affinity, leading to inhibition of kinase activation and receptor internalization. By introducing point mutations, we can increase the dynamic state of the ECD, thereby enhancing kinase activity and receptor internalization. Our findings suggest that EGFR functioning can not only be controlled by activating ligands, but also by modulation of receptor dynamics.

Genome‐wide Analysis of Heparan Sulfate Biosynthesis

Heparan sulfate proteoglycans (HSPGs) are expressed on virtually all animal cells and are involved in many important biological processes. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N‐ and O‐sulfated. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites on the cell surface, and these modifications can vary temporally during development and spatially across tissues. While most of the enzymes involved in HS biosynthesis have been studied extensively, much less information exists regarding the specific mechanisms that give rise to the variable composition and binding properties of HS. A genome‐wide CRISPR/Cas9 screen was developed to uncover novel genes other than those encoding known HS biosynthetic enzymes. A lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human malignant melanoma cell line. Subsequently, high‐throughput screens were adapted to identify sgRNAs that induce resistance to cytotoxins whose action depends on HSPGs and decrease binding of HS‐dependent ligands to the cell surface. Among the resulting targets, we identified well‐known HS biosynthesis enzymes as well as candidate genes with a previously unrecognized link to HS biosynthesis. The top overlapping hits from the screens were then characterized based on gene function and validated to regulate HS assembly. Overall, these studies demonstrate novel approaches to uncovering regulatory factors and provide a better understanding of the genetic regulation of HS biosynthesis. Furthermore, these studies lead us to new methods to manipulate heparan sulfate and its activities in other cellular processes that go awry in human diseases.Support or Funding InformationR01GM33063 (to J.E.), R21CA199292 (to J.E. and N.L.), R35GM119850 (to N.L.), and K12HL141956 (to R.W.)

Cryo-EM structures of PAC1 receptor reveal ligand binding mechanism

The pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R) belongs to the secretin receptor family and is widely distributed in the central neural system and peripheral organs. Abnormal activation of the receptor mediates trigeminovascular activation and sensitization, which is highly related to migraine, making PAC1R a potential therapeutic target. Elucidation of PAC1R activation mechanism would benefit discovery of therapeutic drugs for neuronal disorders. PAC1R activity is governed by pituitary adenylate cyclase-activating polypeptide (PACAP), known as a major vasodilator neuropeptide, and maxadilan, a native peptide from the sand fly, which is also capable of activating the receptor with similar potency. These peptide ligands have divergent sequences yet initiate convergent PAC1R activity. It is of interest to understand the mechanism of PAC1R ligand recognition and receptor activity regulation through structural biology. Here we report two near-atomic resolution cryo-EM structures of PAC1R activated by PACAP38 or maxadilan, providing structural insights into two distinct ligand binding modes. The structures illustrate flexibility of the extracellular domain (ECD) for ligands with distinct conformations, where ECD accommodates ligands in different orientations while extracellular loop 1 (ECL1) protrudes to further anchor the ligand bound in the orthosteric site. By structure-guided molecular modeling and mutagenesis, we tested residues in the ligand-binding pockets and identified clusters of residues that are critical for receptor activity. The structures reported here for the first time elucidate the mechanism of specificity and flexibility of ligand recognition and binding for PAC1R, and provide insights toward the design of therapeutic molecules targeting PAC1R.

Two Distinct Ligand Binding Sites in Monoamine Transporters Monitored by Nanopharmacological Force Sensing

Two Distinct Ligand Binding Sites in Monoamine Transporters Monitored by Nanopharmacological Force Sensing

Activity and Affinity of Pin1 Variants

Pin1 is a peptidyl-prolyl isomerase responsible for isomerizing phosphorylated S/T-P motifs. Pin1 has two domains that each have a distinct ligand binding site, but only its PPIase domain has catalytic activity. Vast evidence supports interdomain allostery of Pin1, with binding of a ligand to its regulatory WW domain impacting activity in the PPIase domain. Many diverse studies have made mutations in Pin1 in order to elucidate interactions that are responsible for ligand binding, isomerase activity, and interdomain allostery. Here, we summarize these mutations and their impact on Pin1′s structure and function.

TLR8 Is a Sensor of RNase T2 Degradation Products

TLR8 is among the highest-expressed pattern-recognition receptors in the human myeloid compartment, yet its mode of action is poorly understood. TLR8 engages two distinct ligand binding sites to sense RNA degradation products, although it remains unclear how these ligands are formed in cellulo in the context of complex RNA molecule sensing. Here, we identified the lysosomal endoribonuclease RNase T2 as anon-redundant upstream component of TLR8-dependent RNA recognition. RNase T2 activity is required for rendering complex single-stranded, exogenous RNA molecules detectable for TLR8. This is due to RNase T2's preferential cleavage of single-stranded RNA molecules between purine and uridine residues, which critically contributes to the supply of catabolic uridine and the generation of purine-2',3'-cyclophosphate-terminated oligoribonucleotides. Thus-generated molecules constitute agonistic ligands for the first and second binding pocket of TLR8. Together, these results establish the identity and origin of the RNA-derived molecular pattern sensed by TLR8.

Structural determinants of ligand binding in truncated hemoglobins: Resonance Raman spectroscopy of the native states and their carbon monoxide and hydroxide complexes

The ligand binding characteristics of heme-containing proteins are determined by a number of factors, including the nature and conformation of the distal residues and their capability to stabilize the heme-bound ligand via hydrogen-bonding and electrostatic interactions. In this regard, the heme pockets of truncated hemoglobins (TrHbs) constitute an interesting case study as they share many common features, including a number of polar cavity residues. In this review, we will focus on three proteins of group II TrHbs, from Thermobifida fusca (Tf-HbO) and Pseudoalteromonas haloplanktis TAC125 (Ph-HbO). Although the residues in positions G8 (Trp) and B10 (Tyr) are conserved in all three proteins, the CD1 residue is a Tyr in T. fusca and a His in P. haloplanktis. Comparison of the ligand binding characteristics of these proteins, in particular the hydroxo and CO ligands by means of resonance Raman spectroscopy, reveals that this single difference in the key heme cavity residues markedly affects their ligand binding capability and conformation. Furthermore, although the two Ph-HbOs (Ph-HbO-2217 and Ph-HbO-0030) have identical key cavity residues, they display distinct ligand binding properties.

Structural and biochemical characterization of ligand recognition by CysB, the master regulator of sulfate metabolism

CysB, a member of LysR-type transcriptional regulators, up-regulates the expression of genes associated with sulfate metabolism and cysteine biosynthesis. CysB is activated under sulfur limiting conditions by O-acetylserine (OAS) and N-acetylserine (NAS), but the activation mechanism of CysB remain unknown. Here, we report four crystal structures of ligand binding domains of CysB (CysB-LBD) in apo form and in complex with sulfate, OAS, and NAS. Our results show that CysB has two distinct allosteric ligand binding sites; a sulfate and NAS specific site-1 and a second, NAS and OAS specific site-2. All three ligands bind through the induced-fit mechanism. Surprisingly, OAS remodels the site-1 by binding to site-2, suggesting that site-1 and site-2 are coupled allosterically. Using DNA binding and site-directed mutagenesis approach, we show that OAS enhances NAS mediated activation and mutation at site-1 has no effect on site-2 mediated OAS activation. Results indicate that inducer binding triggered signals from OAS-Specific site-2 are relayed to DBD through site-1. Together, results presented here suggest that induced-fit binding and allosteric coupling between two ligand binding sites and DBD underlie the key feature of CysB activation. Further, this study provides first structural glimpse into recognition of inducer ligands by CysB and provides a general framework to understand how LTTR family regulators respond to dual activators.

Abstract 5945: CRISPR-Cas9 dissection of heparan sulfate

Abstract Heparan sulfate proteoglycans (HSPGs) are expressed on virtually all animal cells and play important roles in tumor growth and metastasis. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N- and O-sulfated. These complex cell surface carbohydrates regulate important biological processes including cell proliferation and development through their interaction with a large number of matrix proteins and growth factors. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites on the cell surface, and these modifications can vary temporally during development and spatially across tissues. Previous studies have shown that HSPGs can modulate tumor growth kinetics and are abundantly expressed on the cell surface of many types of cancer. Additionally, genetically reducing the sulfation of HS has been shown to selectively inhibit tumor angiogenesis and lymph node metastasis. While most of the enzymes involved in HS biosynthesis have been studied extensively, much less information exists regarding the specific mechanisms that give rise to the variable composition and binding properties of HS. The overall goal of this project is to uncover and characterize novel genes whose expression influences HS-mediated regulatory networks in tumor growth and metastasis. A genome-wide CRISPR/Cas9-mediated screen was developed to uncover and characterize novel genes other than those encoding known HS biosynthetic enzymes. A lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human malignant melanoma cell line. Subsequently, a high-throughput screening assay was adapted to identify lentiviral-encoded sgRNAs that induce resistance to cytotoxins whose action depends on HSPGs. Parallel screens using alternative HS-dependent toxins or plant lectins that cause cytotoxicity dependent on other types of glycosylation were performed in order to sort genes that selectively affect HS biosynthesis. From the toxin screens, we identified previously studied genes essential for HS formation and factors involved in the intoxication of cells by diphtheria toxin, an HS-dependent exotoxin. Furthermore, we uncovered potential candidate genes whose function is unknown relative to HS biosynthesis. Top hits from the screens were characterized and categorized based on their predicted gene functions and are currently being individually validated and examined for their potential involvement in the regulation of HS biosynthesis. Overall, these studies will provide a better understanding of the genetic regulatory factors involved in HS biogenesis. Additionally, the factors we identify could reveal novel targets for anti-cancer therapies, as well as lead us to methods to manipulate HS and its activities in other cellular processes that go awry in human diseases. Citation Format: Ryan J. Weiss, Philipp N. Spahn, Nathan E. Lewis, Jeffrey D. Esko. CRISPR-Cas9 dissection of heparan sulfate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5945. doi:10.1158/1538-7445.AM2017-5945

Abstract LB-228: Identifying small molecule inhibitors of Fascin 1 using fragment-based drug discovery

Abstract The actin-bundling protein fascin 1 is markedly overexpressed in a range of invasive tumors and is believed to play a critical role in cancer cell metastasis. Targeting fascin is however very challenging owing to its mechanism of protein-protein interaction and a lack of knowledge regarding the crucial actin-binding sites. By combining a fragment-based approach, biophysical assay screening and X-ray crystallography, we have been able to identify and optimize novel fascin 1 inhibitors that show nanomolar affinity in biochemical binding and bundling assays. We now show the development of functional cell assays in which the lead fascin inhibitor compounds have been tested. For the first time we disclose the structures of the most advanced compounds and show their effects in 2D and 3D cell culture. Fascin 1 binds and cross-links filamentous actin (F-actin) into parallel bundles that are used in the formation of dynamic cellular protrusions (such as lamellipodia and filopodia) used during cell migration, and in the formation of invadopodia used by tumor cell lines to degrade the tumor extracellular matrix (ECM). Whereas fascin 1 expression is low or absent in normal epithelia, its expression is dramatically increased in a variety of invasive tumor types including colon, lung, ovarian and pancreas, with increased expression being an independent prognostic indicator of poor clinical outcome in the most aggressive and metastatic tumor types. Knockdown of fascin expression has been shown to reduce tumor cell invasion both in vitro and in vivo, thereby highlighting fascin as an important drug discovery target. Small molecule inhibitors of fascin 1 were identified by screening our fragment library (~1000 compounds) using surface plasmon resonance (SPR). X-ray co-crystallography with these hit compounds showed four distinct ligand binding sites within the fascin protein. Optimized site 2 binders induce a substantial conformational change, with the deeply enclosed pocket between fascin domains 1 and 2 opening a channel that accesses the surface of the protein. A virtual screen identified the compound BDP-00010834, which binds in this pocket with an SPR Kd of 29.7µM and also inhibits (IC50=50µM) the functional activity of fascin measured in an F-actin bundling assay. Structure-based optimization in combination with X-ray co-crystallography has enabled the generation of compounds with more than 300-fold increase in both binding affinity and functional activity over this screening hit. Our current best-in-series compounds include BDP-00013544 (Kd=29nM, IC50=185nM). These lead compounds have now been tested in a number of cell based invasion assays including both 2D and 3D cultures. Citation Format: Daniel Croft, Stuart Francis, Justin Bower, Andrea Gohlke, Gillian Goodwin, Christopher Gray, Jennifer Konczal, Sophie Macconnachie, Patricia McConnell, Laura McDonald, Heather McKinnon, Mokdad Mezna, Charles Parry, Nikki Paul, Angelo Pugliese, Alexander Schuettelkopf, Laura Machesky, Martin Drysdale. Identifying small molecule inhibitors of Fascin 1 using fragment-based drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-228. doi:10.1158/1538-7445.AM2017-LB-228

High-Level Production and Properties of the Cysteine-Depleted Cytochrome P450 3A4.

Human drug-metabolizing cytochrome P450 3A4 (CYP3A4) is a dynamic enzyme with a large and highly malleable active site that can fit structurally diverse compounds. Despite extensive investigations, structure-function relationships and conformational dynamics in CYP3A4 are not fully understood. This study was undertaken to engineer a well-expressed and functionally active cysteine-depleted CYP3A4 that can be used in biochemical and biophysical studies. cDNA codon optimization and screening mutagenesis were utilized to boost the level of bacterial expression of CYP3A4 and identify the least harmful substitutions for all six non-heme-ligating cysteines. The C58A/C64M/C98A/C239T/C377A/C468S (Cys-less) mutant was found to be expressed as highly as the optimized wild-type (opt-WT) CYP3A4. The high-resolution X-ray structures of opt-WT and Cys-less CYP3A4 revealed that gene optimization leads to a different folding in the Phe108 and Phe189 regions and promotes binding of the active site glycerol that interlocks Ser119 and Arg212, critical for ligand association, and the hydrophobic cluster adjacent to Phe108. Crowding and decreased flexibility of the active site, as well as structural alterations observed at the C64M, C239T, and C468S mutational sites, might be responsible for the distinct ligand binding behavior of opt-WT and Cys-less CYP3A4. Nonetheless, the Cys-less mutant could be used for structure-function investigations because it orients bromoergocryptine and ritonavir (a high-affinity substrate and a high-potency inhibitor, respectively) like the WT and has a higher activity toward 7-benzyloxy(4-trifluoromethyl)coumarin.