Structure-based Rational Design Research Articles

High-Affinity Alkynyl Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT).

Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that methylates nicotinamide (NAM) using cofactor S-adenosylmethionine (SAM). NNMT overexpression has been linked to diabetes, obesity, and various cancers. In this work, structure-based rational design led to the development of potent and selective alkynyl bisubstrate inhibitors of NNMT. The reported nicotinamide-SAM conjugate (named NS1) features an alkyne as a key design element that closely mimics the linear, 180° transition state geometry found in the NNMT-catalyzed SAM → NAM methyl transfer reaction. NS1 was synthesized in 14 steps and found to be a high-affinity, subnanomolar NNMT inhibitor. An X-ray cocrystal structure and SAR study revealed the ability of an alkynyl linker to span the methyl transfer tunnel of NNMT with ideal shape complementarity. The compounds reported in this work represent the most potent and selective NNMT inhibitors reported to date. The rational design principle described herein could potentially be extended to other methyltransferase enzymes.

Structure-based rational design of staurosporine-based fluorescent probe with broad-ranging kinase affinity for kinase panel application.

Selectivity profiling of compounds is important for kinase drug discovery. To this end, we aimed to develop a broad-range protein kinase assay by synthesizing a novel staurosporine-derived fluorescent probe based on staurosporine and kinase-binding related structural information. Upon structural analysis of staurosporine with kinases, a 4'-methylamine moiety of staurosporine was found to be located on the solvent side of the kinases, to which several linker units can be conjugated by either alkylation or acylation. However, such conjugation was suggested to reduce the binding affinities of the modified compound for several kinases, owing to the elimination of hydrogen bond donor moiety of NH-group from 4'-methylamine and/or steric hindrance by acyl moiety. Based on this structural information, we designed and synthesized a novel staurosporine-based probe without methyl group in order to retain the hydrogen bond donor, similar to unmodified staurosporine. The broad range of the kinase binding assay demonstrated that our novel fluorescent probe is an excellent tool for developing broad-ranging kinase binding assay.

Virtual Pharmacophore Screening Identifies Small-Molecule Inhibitors of the Rev1-CT/RIR Protein-Protein Interaction.

Translesion synthesis (TLS) has emerged as a mechanism through which several forms of cancer develop acquired resistance to first-line genotoxic chemotherapies by allowing replication to continue in the presence of damaged DNA. Small molecules that inhibit TLS hold promise as a novel class of anticancer agents that can serve to enhance the efficacy of these front-line therapies. We previously used a structure-based rational design approach to identify the phenazopyridine scaffold as an inhibitor of TLS that functions by disrupting the protein-protein interaction (PPI) between the C-terminal domain of the TLS DNA polymerase Rev1 (Rev1-CT) and the Rev1 interacting regions (RIR) of other TLS DNA polymerases. To continue the identification of small molecules that disrupt the Rev1-CT/RIR PPI, we generated a pharmacophore model based on the phenazopyridine scaffold and used it in a structure-based virtual screen. In vitro analysis of promising hits identified several new chemotypes with the ability to disrupt this key TLS PPI. In addition, several of these compounds were found to enhance the efficacy of cisplatin in cultured cells, highlighting their anti-TLS potential.

Lead Optimization Yields High Affinity Frizzled 7-Targeting Peptides That Modulate Clostridium difficile Toxin B Pathogenicity in Epithelial Cells.

Frizzled 7 (FZD7) receptors have been shown to play a central role in intestinal stem cell regeneration and, more recently, in Clostridium difficile pathogenesis. Yet, targeting FZD7 receptors with small ligands has not been explored as an approach to block C. difficile pathogenesis. Here, we report the discovery of high affinity peptides that selectively bind to FZD7 receptors. We describe an integrated approach for lead optimization, utilizing structure-based rational design and directed evolution, to enhance the peptide binding affinity while still maintaining FZD7 receptor selectivity. This work yielded new peptide leads with picomolar binding constants to FZD7 as measured by biophysical methods. The new peptides block the interaction between C. difficile toxin B (TcdB) and FZD receptors and perturb C. difficile pathogenesis in epithelial cells. As such, our findings provide a proof of concept that targeting FZD receptors could be a viable pharmacological approach to protect epithelial cells from TcdB pathogenicity.

Structure-based drug target prioritisation and rational drug design for targeting Chlamydia trachomatis eye infections

Chlamydia trachomatis (C.t) is a major causative of infectious blindness in world. It is a real challenge to combat Chlamydial infection as it is an intracellular pathogen. Hence, it is essential to determine the most potential targets of C.t in order to inhibit or suppress its virulence during its infectious phase. Thus, in this study, the highly expressed-cum-most essential genes reported through our earlier study were reprioritized by structure-based comparative binding site analysis with host proteome. Therefore, computational approaches involving molecular modelling, large-scale binding site prediction and comparison, molecular dynamics simulation studies were performed to narrow down the most potential targets. Furthermore, high-throughput virtual screening and ADMETox were also performed to identify potential hits that shall efficiently inhibit the prioritised targets. Hence, by this study we report Pyruvoyl-dependent arginine decarboxylase (PvlArgDC), DNA-repair protein (RecO) and porin (outer membrane protein) as the most viable targets of C.t which can be potentially targeted by compounds, NSC_13086, MFCD00276409, MFCD05662003, respectively.AbbreviationsC.tChlamydia trachomatisSTDSexually transmitted diseaseHTVSHigh-throughput virtual screeningADMEToxAbsorption, Distribution, Metabolism, Excretion and ToxicityPMPocketMatchMDMolecular Dynamics simulationSPStandard precisionXPExtra precisionMMGBSAMolecular mechanics energies combined with generalised Born and surface area continuum solvationOMPOuter membrane proteinPvlArgDCPyruvoyl-dependent arginine decarboxylaseRecORecombination protein O.Communicated by Ramaswamy H. Sarma

Structure-based rational design of a novel chimeric PD1-NKG2D receptor for natural killer cells

Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have the potential to provide the potential for the implementation of allogeneic “off-the-shelf” cellular therapy against cancers. Currently, most CARs are not optimized for NK cells, so new NK-tailored CARs are needed. Here, a major activating receptor of NK cells, NKG2D was harnessed to design different chimeric receptors that mediate strong NK cell signaling. In these NKG2D signaling-based chimeric receptors, the extracellular domain of inhibitory receptor PD-1 was employed to reverse the immune escape mediated by PD-1 ligands in the solid tumors. To achieve the rational design of chimeric PD1-NKG2D receptors, we developed a transmembrane protein tertiary structure prediction program (PredMP & I-TASSER) and optimized the conformation of the PD-1 ectodomain by genetically altering the sequences encoding the hinge and intracellular domain. Finally, we identified a chimeric PD1-NKG2D receptor containing NKG2D hinge region and 4-1BB co-stimulatory domain to exhibit stable surface expression and mediate in vitro cytotoxicity of NK92 cells against various tumor cells. This strategy now provides a promising approach for the computer-aided design (CAD) of potent NK cell-tailored chimeric receptors with NKG2D signaling.

Robust Lithium Metal Anodes Realized by Lithiophilic 3D Porous Current Collectors for Constructing High-Energy Lithium-Sulfur Batteries.

Lithium-sulfur (Li-S) batteries are attractive candidates for next-generation rechargeable batteries. With the steady development of sulfur cathodes, the recent revival of research on dendrite-free Li metal anodes offers opportunities to improve the stabilities and safety of Li-S batteries. However, the low capacities and low Li utilizations of current Li anodes hinder the improvement of the energy densities of Li-S batteries. Here, we present a facile approach to fabricate lithiophilic three-dimensional porous current collectors by modifying commercial metal foams with yolk-shell structured N-doped porous carbon nanosheets. Benefiting from the structure-based rational design, this current collector is able to generate dendrite-free Li anodes with improved Coulombic efficiencies and life spans, enabling carbon/sulfur cathodes to exhibit significantly enhanced stabilities (e.g., 78.1% of capacity retention after 1400 cycles). More importantly, we successfully constructed a high-areal-capacity Li-S full cell (9.84 mAh cm-2) with 82% Li utilization. This work provides a promising route toward high-energy-density Li-S batteries.

Abstract 1235: Targeting RNA-binding protein HuR to inhibit human breast cancer invasion and metastasis

Abstract The majority of breast cancer-related mortality is due to metastasis. Patients diagnosed with metastatic breast cancer have a dismal 5-year survival rate of only 24%. The RNA-binding protein Hu antigen R (HuR) is overexpressed in breast cancer. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by regulating numerous proto-oncogenes, growth factors and cytokines that implicate major tumor hallmarks including invasion and metastasis. Knocking out HuR by CRISPR/CAS9 technology inhibits cell invasion in breast cancer cells. Therefore, HuR is an emerging target for breast cancer therapy, especially the lethal metastatic breast cancer. RNA-binding proteins had previously been considered “undruggable” due to lack of a well-defined binding pocket for target RNAs. Using high throughput screening followed by structure-based rational design and lead optimization, we have identified small molecules that inhibit HuR-mRNA interaction at nM to sub-µM potency. Our lead compound, KH-3, potently inhibits breast cancer cell growth and decreases cell invasion in vitro similar to HuR knockout, as well as increasing the expression of epithelial marker E-cadherin. In the study of mechanism of action, a transcription factor, FOXQ1, which is recently revealed to implicate in breast cancer invasion and metastasis processes, is found for the first time to be a direct mRNA target of HuR and one of the top genes that are reduced by KH-3 treatment. Exogenous introduction of FOXQ1 can rescue cell invasive capability impaired by HuR knockout and abolish the effect of KH-3 on inhibiting cell invasion in breast cancer cells. Moreover, KH-3 disrupts HuR-FOXQ1 interaction in RNP-IP, RNA pull down and FOXQ1 3′-UTR luciferase reporter assays. In vivo efficacy studies show that KH-3 not only exhibits potent antitumor efficacy in an orthotopic xenograft model of breast cancer, but also efficiently inhibits lung metastasis and improves mouse survival in an experimental metastasis model. Our data provide a proof-of-principle that HuR inhibition by KH-3 may be developed as a promising molecular therapy for inhibiting progression and metastasis of breast cancer with high HuR. Citation Format: Xiaoqing Wu, Gulhumay Gardashova, Lan Lan, Shuang Han, Cuncong Zhong, Ragul Gowthaman, John Karanicolas, Dan A. Dixon, Danny R. Welch, Ling Li, Min Ji, Jeffrey Aubé, Liang Xu. Targeting RNA-binding protein HuR to inhibit human breast cancer invasion and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1235.

α-Helical Motif as Inhibitors of Toxic Amyloid-β Oligomer Generation via Highly Specific Recognition of Amyloid Surface.

SummaryAmyloid fibril surfaces can convert soluble proteins into toxic oligomers and are attractive targets for intervention of protein aggregation diseases. Thus far, molecules identified with inhibitory activity are either large proteins or flat cyclic compounds lacking in specificity. The main design difficulty is flatness of amyloid surfaces and the lack of knowledge on binding interfaces. Here, we demonstrate, for the first time, a rational design of alpha-helical peptide inhibitors targeting the amyloid-beta 40 (Aβ40) fibril surfaces, based on our in silico finding that a helical fragment of Aβ40 interacts in a unique way with side-chain arrays on the fibril surface. We strengthen the fragment's binding capability through mutations and helicity enhancement with our Terminal Aspartic acid strategy. The resulting inhibitor shows micromolar affinity for the fibril surface, effectively impedes the surface-mediated oligomerization of Aβ40, and mitigates its cytotoxicity. This work opens up an avenue to designing aggregation modulators for amyloid diseases.

Molecular Recognition of the Hybrid-Type G-Quadruplexes in Human Telomeres.

G-quadruplex (G4) DNA secondary structures formed in human telomeres have been shown to inhibit cancer-specific telomerase and alternative lengthening of telomere (ALT) pathways. Thus, human telomeric G-quadruplexes are considered attractive targets for anticancer drugs. Human telomeric G-quadruplexes are structurally polymorphic and predominantly form two hybrid-type G-quadruplexes, namely hybrid-1 and hybrid-2, under physiologically relevant solution conditions. To date, only a handful solution structures are available for drug complexes of human telomeric G-quadruplexes. In this review, we will describe two recent solution structural studies from our labs. We use NMR spectroscopy to elucidate the solution structure of a 1:1 complex between a small molecule epiberberine and the hybrid-2 telomeric G-quadruplex, and the structures of 1:1 and 4:2 complexes between a small molecule Pt-tripod and the hybrid-1 telomeric G-quadruplex. Structural information of small molecule complexes can provide important information for understanding small molecule recognition of human telomeric G-quadruplexes and for structure-based rational drug design targeting human telomeric G-quadruplexes.

Small-Molecule Targeting of Oncogenic FTO Demethylase in Acute Myeloid Leukemia

FTO, an mRNA N6-methyladenosine (m6A) demethylase, was reported to promote leukemogenesis. Using structure-based rational design, we have developed two promising FTO inhibitors, namely FB23 and FB23-2, which directly bind to FTO and selectively inhibit FTO's m6A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells invitro. Moreover, FB23-2 significantly inhibits the progression of human AML cell lines and primary cells in xeno-transplanted mice. Collectively, our data suggest that FTO is a druggable target and that targeting FTO by small-molecule inhibitors holds potential to treat AML.

Improving catalytic efficiency and maximum activity at low pH of Aspergillus neoniger phytase using rational design.

A preferable phytase for use in animal feeds for industrial applications should have high, optimal activity at low pH in the monogastric gut environment and high thermostability. To obtain enzymes with enhanced catalytic efficiency (pH 5.5) and excellent activity in acidic pH range, we performed structure-based rational design of a thermostable phytase (PhyAn). For this, six mutants based on different rational design strategies were constructed and heterologously expressed in Pichia pastoris. Particularly, the extracellular enzymatic activity was assessed to ensure that the produced enzymes met requirements of further analyses. Several positive mutants with enhanced catalytic efficiency or pH-profile shifts were carefully examined. Biochemical and kinetic investigations of purified mutants revealed that E79K, E80K, E79K + E80K and D68K had higher catalytic efficiency than the parent enzyme by approximately 49%, 67%, 86% and 15%, respectively. Moreover, the optimum pH of mutant Y65H was shifted from 5.0 to 3.0, and the peak of D68K shifted to pH 5.5. Analysis of the structural-functional relationships revealed that changes in amino acid charges, structural flexibility and space hindrance could significantly influence certain enzyme characteristics. Our results illustrate the feasibility and present a structural foundation for enhancing the phytase-catalytic efficiency and acid resistance by assembling mutations derived using rational design.

Structure-Based Peptide Inhibitor Design of Amyloid-β Aggregation.

Many human neurodegenerative diseases are associated with amyloid fibril formation. Inhibition of amyloid formation is of importance for therapeutics of the related diseases. However, the development of selective potent amyloid inhibitors remains challenging. Here based on the structures of amyloid β (Aβ) fibrils and their amyloid-forming segments, we designed a series of peptide inhibitors using RosettaDesign. We further utilized a chemical scaffold to constrain the designed peptides into β-strand conformation, which significantly improves the potency of the inhibitors against Aβ aggregation and toxicity. Furthermore, we show that by targeting different Aβ segments, the designed peptide inhibitors can selectively recognize different species of Aβ. Our study developed an approach that combines the structure-based rational design with chemical modification for the development of amyloid inhibitors, which could be applied to the development of therapeutics for different amyloid-related diseases.

Abstract B037: Tunable thermal bioswitches for control of cell-based therapeutics

Abstract Temperature is a unique input signal that could be used by therapeutic agents, including engineered proteins and cells, to sense and respond to patient conditions or spatially targeted external triggers such as focused ultrasound. A critical capability for many envisioned applications is the ability to control the function of such therapies in situ to enable spatially and temporally specific activation at tumors and surrounding local tissue. However, among existing control methods, systemic chemical administration typically lacks the spatial precision needed to modulate activity at specific anatomic locations, while optical approaches suffer from poor light penetration into tissues. On the other hand, temperature can be controlled both globally and locally—at depth—using technologies such as focused ultrasound, infrared light and magnetic particle hyperthermia. In addition, body temperature can serve as an indicator of the patient’s condition. We have previously described the development of temperature-dependent protein actuators for control of gene expression in prokaryotic hosT-cells (1). Here, we generalize this technology to control protein-protein interactions with the aim of modulating arbitrary cell signaling pathways, including those of engineered therapeutic mammalian cells.To develop temperature-responsive protein-protein interaction domains, we turned to the thermo-sensitive bacterial transcriptional repressor TlpA. We demonstrated that this protein exhibits a tunable activation threshold which can be modulated within the biomedically relevant range of 32–46 °C, and that genes controlled by this repressor activate upon exposure to focused ultrasound hyperthermia applied noninvasively to a murine host (1). We then utilized a structure-based rational design strategy to convert the homodimeric TlpA protein into a pair of obligate heterodimers. We subsequently demonstrated that these heterodimers can be fused to sub-cellular anchoring moieties and cargo domains to conditionally sequester their payload into various compartments of human cells. Release of the cargo can be triggered via a mild thermal elevation, and the release temperature can be tuned via mutagenesis. In this study, we demonstrate that the temperature-sensing components of a bacterial transcriptional repressor can be isolated and repurposed into a generalized strategy to exert direct and rapid control over protein function in the context of both bacterial and mammalian cells. We aim to harness this technology to regulate therapeutically relevant processes in mammalian cells such as transcriptional activation and enzymatic signaling cascades. This class of bioswitches can be utilized to engineer a broad range of research tools and biological therapies with actuation driven by spatiotemporally precise noninvasively applied stimuli or by real-time sensing of host conditions.Reference: 1. Piraner DI, Abedi MH, Moser BA, Lee-Gosselin A, Shapiro MG. Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nat Chem Biol 2016;13(1):75–80. Citation Format: Dan Ilya Piraner, Mikhail G. Shapiro. Tunable thermal bioswitches for control of cell-based therapeutics [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B037.

Dissecting a novel allosteric mechanism of cruzain: A computer-aided approach.

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected infection affecting millions of people in tropical regions. There are several chemotherapeutic agents for the treatment of this disease, but most of them are highly toxic and generate resistance. Currently, the development of allosteric inhibitors constitutes a promising research field, since it can improve the accessibility to more selective and less toxic medicines. To date, the allosteric drugs prediction is a state-of-the-art topic in rational structure-based computational design. In this work, a simulation strategy was developed for computational discovery of allosteric inhibitors, and it was applied to cruzain, a promising target and the major cysteine protease of T. cruzi. Molecular dynamics simulations, binding free energy calculations and network-based modelling of residue interactions were combined to characterize and compare molecular distinctive features of the apo form and the cruzain-allosteric inhibitor complexes. By using geometry-based criteria on trajectory snapshots, we predicted two main allosteric sites suitable for drug targeting. The results suggest dissimilar mechanisms exerted by the same allosteric site when binding different potential allosteric inhibitors. Finally, we identified the residues involved in suboptimal paths linking the identified site and the orthosteric site. The present study constitutes the first approximation to the design of cruzain allosteric inhibitors and may serve for future pharmacological intervention. Here, no major effects on active site structure were observed due to compound binding (modification of distance and angles between catalytic residues), which indicates that allosteric regulation in cruzain might be mediated via alterations of its dynamical properties similarly to allosteric inhibition of human cathepsin K (HCatK). The current findings are particularly relevant for the design of allosteric modulators of papain-like cysteine proteases.

Structure-based rational design, synthesis, crystal structure, DFT and molecular docking of 1,4-benzene dicarboxamide isomers with application as hardeners

Molecular structures of two isomers have been investigated by SXRD analysis and DFT calculations and the isomers are assessed for their antimicrobial properties and as curing agents for epoxy resins.

Quantifying drug-target engagement in live cells using sulfonyl fluoride chemical probes.

Phenotypic screening in disease-relevant models identifies small molecule hits with desirable efficacy but often with unknown modes of action. Target identification and validation are integral to successful biomedical research. Technologies are required to validate the biological target (or targets) through which a pharmacological agent is proposed to exert its effects. This work details the rational structure-based design, synthetic preparation and cell-based application of a clickable sulfonyl fluoride chemical probe to directly report on the mechanism of a series of compounds previously discovered in a reporter gene assay. Quantification of drug-target occupancy in living human primary cells enabled a deeper understanding of the molecular pharmacology of the chemotype. The technology described herein should be of broad interest to those involved in chemical biology research and the drug discovery endeavor.

Opportunities for Improvement: The Lot of the Inhibitor Patient Is Not a Happy One

INTRODUCTIONHemophilia A or B carries significant morbidity - progressive throughout life. The mainstay of treatment is regular factor replacement to treat or prevent bleeding episodes. Up to 30% of Hemophilia A and up to 5% of Hemophilia B develop neutralizing antibodies (inhibitors) rendering factor replacement therapy ineffective. Hemophilia patients with inhibitors are managed with bypassing agents for treatment or prevention of bleeds.Prophylaxis is not adequate in inhibitor patients using currently available bypassing products due to their very short half-life (2-3 hours for rFVIIa; 5-6 hours for aPCC) so they are treated on an episodic basis for bleeds. On-demand treatment results in poor quality of life, significantly higher mortality and inferior musculoskeletal outcomes in inhibitor patients when compared to patients without inhibitors. The quality of life of hemophilia has been evaluated using EQ-5D and Haem-A-QOL and impaired activity has been measured with Haemophilia Activity List (HAL). The EQ-5D is a widely used internationally validated general-purpose quality of life instrument however it lacks granularity and is not specific to Hemophilia. Haem A-QOL (Mackensen et al 2004) and Hemophilia Activities List (HAL) (Van Genderen FR et al 2006.) were developed and validated specifically to address the typical disabilities and life issues faced by patients with Hemophilia.Marzeptacog alfa (activated) (MarzAA) was created using a structure-based rational protein design and has 4 amino acid substitutions to enhance the biological properties of FVIIa. This variant molecule has substantially (6-7x) greater potency than wild-type FVIIa, a greater half-life and sufficient bioavailability when given subcutaneously (SQ).There is currently a paucity of data on quality of life (QoL) of inhibitor patients. We evaluated the baseline QoL and functional activity of inhibitor patients enrolled in the MAA-201 study using the EQ-5D , Haem-A-QoL and HAL compared the results to those of reference hemophilia patients without inhibitors.METHODS A phase 2 / 3 open-label study evaluating safety and efficacy of MarzAA in hemophilia patients with inhibitors is underway MAA-201, (NCT03407651)Subject eligibility required an annualized bleeding rate of >12 and documented inhibitor to replacement factor. The primary aim of the trial was the complete prevention of breakthrough bleeding for 50 days by the daily administration of a fixed SQ dose of MarzAA. If breakthrough bleeding occurred, up to three dose escalations were permitted. At baseline and study conclusion, each subject completed quality of life assessment tools the EQ-5D, Haem-A-QOL and the Hemophilia Activities ListHaem-A-QOL assesses subjects across ten domains and provides a summarized score. Subjects in the present trial (MARZAAPOP) were compared to baseline values for subjects with severe hemophilia but without inhibitors recruited into a long-term prophylaxis trial (The A-LONG trial - ALONGREFPOP).Mean baseline Haem-A-QOL summed score in the A-LONG trial was 29.3 ±15.7 contrasting sharply with a mean baseline summed score of 42 ±15.2 for subjects in the present trial (Table 1)HAL is more motor function oriented and assesses patients across ten domains of which seven assess basic functionality and three are composites intended to assess disability in performing complex tasks. HAL provides both raw and normalized scores. Normalized scores provide meaningful output in the case of missing data elements. For each domain and for the sum score, a raw score of 0 and a normalized score of 100 indicates no functional deficit. Figure 1 provides a visual comparison between recruited subjects (MARZAAPOP) in the present trial and the population used to validate the HAL tool. (HALREFPOP).CONCLUSION: In examining recruits into the present trial, it is clear that inhibitor patients have generally worse functional scores than either of two reference groups. Effective long-term prophylaxis is expected to produce measurable improvement in QOL scores in this hard-to-treat population. DisclosuresBooth:Catalyst Biosciences: Consultancy. Mahlangu:Sanofi: Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; LFB: Consultancy; NovoNordisk: Consultancy, Research Funding, Speakers Bureau; CSL Behring: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy; Catalyst Biosciences: Consultancy, Research Funding; Biomarin: Research Funding, Speakers Bureau; Biogen: Research Funding, Speakers Bureau; Bayer: Research Funding; Amgen: Consultancy; Alnylam: Consultancy, Research Funding, Speakers Bureau; Shire: Consultancy, Research Funding, Speakers Bureau; Sobi: Research Funding, Speakers Bureau; Spark: Consultancy, Research Funding. Levy:Catalyst Biosciences: Employment, Equity Ownership.

Computer-Aided Discovery of Small Molecule Inhibitors of Transcriptional Activity of TLX (NR2E1) Nuclear Receptor.

Orphan nuclear receptor TLX (NR2E1) plays a critical role in the regulation of neural stem cells (NSC) as well as in the development of NSC-derived brain tumors. In the last years, new data have emerged implicating TLX in prostate and breast cancer. Therefore, inhibitors of TLX transcriptional activity may have a significant impact on the treatment of several critical malignancies. However, the TLX protein possesses a non-canonical ligand-binding domain (LBD), which lacks a ligand-binding pocket (conventionally targeted in case of nuclear receptors) that complicates the development of small molecule inhibitors of TLX. Herein, we utilized a rational structure-based design approach to identify small molecules targeting the Atro-box binding site of human TLX LBD. As a result of virtual screening of ~7 million molecular structures, 97 compounds were identified and evaluated in the TLX-responsive luciferase reporter assay. Among those, three chemicals demonstrated 40–50% inhibition of luciferase-detected transcriptional activity of the TLX orphan nuclear receptor at a dose of 35 µM. The identified compounds represent the first class of small molecule inhibitors of TLX transcriptional activity identified via methods of computer-aided drug discovery.

GPCR drug discovery: integrating solution NMR data with crystal and cryo-EM structures.

The 826 G protein-coupled receptors (GPCRs) in the human proteome regulate key physiological processes and thus have long been attractive drug targets. With the crystal structures of more than 50 different human GPCRs determined over the past decade, an initial platform for structure-based rational design has been established for drugs that target GPCRs, which is currently being augmented with cryo-electron microscopy (cryo-EM) structures of higher-order GPCR complexes. Nuclear magnetic resonance (NMR) spectroscopy in solution is one of the key approaches for expanding this platform with dynamic features, which can be accessed at physiological temperature and with minimal modification of the wild-type GPCR covalent structures. Here, we review strategies for the use of advanced biochemistry and NMR techniques with GPCRs, survey projects in which crystal or cryo-EM structures have been complemented with NMR investigations and discuss the impact of this integrative approach on GPCR biology and drug discovery.