Discovery of natural-product-derived sequanamycins as potent oral anti-tuberculosis agents.

The emergence of drug-resistant tuberculosis has created an urgent need for new anti-tubercular agents. Here, we report the discovery of a series of macrolides called sequanamycins with outstanding in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb). Sequanamycins are bacterial ribosome inhibitors that interact with the ribosome in a similar manner to classic macrolides like erythromycin and clarithromycin, but with binding characteristics that allow them to overcome the inherent macrolide resistance of Mtb. Structures of the ribosome with bound inhibitors were used to optimize sequanamycin to produce the advanced lead compound SEQ-9. SEQ-9 was efficacious in mouse models of acute and chronic TB as a single agent, and it demonstrated bactericidal activity in a murine TB infection model in combination with other TB drugs. These results support further investigation of this series as TB clinical candidates, with the potential for use in new regimens against drug-susceptible and drug-resistant TB.

Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction.

The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity.

Selective inhibition of mutant isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an allosteric small molecule.

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.

Structural and biochemical rationale for Beta variant protein booster vaccine broad cross-neutralization of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, uses a surface expressed trimeric spike glycoprotein for cell entry. This trimer is the primary target for neutralizing antibodies making it a key candidate for vaccine development. During the global pandemic circulating variants of concern (VOC) caused several waves of infection, severe disease, and death. The reduced efficacy of the ancestral trimer-based vaccines against emerging VOC led to the need for booster vaccines. Here we present a detailed characterization of the Sanofi Beta trimer, utilizing cryo-EM for structural elucidation. We investigate the conformational dynamics and stabilizing features using orthogonal SPR, SEC, nanoDSF, and HDX-MS techniques to better understand how this antigen elicits superior broad neutralizing antibodies as a variant booster vaccine. This structural analysis confirms the Beta trimer preference for canonical quaternary structure with two RBD in the up position and the reversible equilibrium between the canonical spike and open trimer conformations. Moreover, this report provides a better understanding of structural differences between spike antigens contributing to differential vaccine efficacy.

The use of virtual screening and differential scanning fluorimetry for the rapid identification of fragments active against MEK1.

We report the analysis of an in-house fragment screening campaign for the oncology target MEK1. The application of virtual screening (VS) as a primary fragment screening approach, followed by biophysical validation using differential screening fluorimetry (DSF), with resultant binding mode determination by X-ray crystallography (X-ray), is presented as the most time and cost-effective combination of in silico and in vitro methods to identify fragments. We demonstrate the effectiveness of the VS-DSF workflow for the early identification of fragments to both 'jump-start' the drug discovery project and to complement biochemical screening data.

The PX-BAR membrane-remodeling unit of sorting nexin 9.

Sorting nexins (SNXs) form a family of proteins known to interact with components in the endosomal system and to regulate various steps of vesicle transport. Sorting nexin 9 (SNX9) is involved in the late stages of clathrin-mediated endocytosis in non-neuronal cells, where together with the GTPase dynamin, it participates in the formation and scission of the vesicle neck. We report here crystal structures of the functional membrane-remodeling unit of SNX9 and show that it efficiently tubulates lipid membranes in vivo and in vitro. Elucidation of the protein superdomain structure, together with mutational analysis and biochemical and cell biological experiments, demonstrated how the SNX9 PX and BAR domains work in concert in targeting and tubulation of phosphoinositide-containing membranes. The study provides insights into the SNX9-induced membrane modulation mechanism.

Fast Mek1 Hit Identification with TRIC Technology Correlates Well with Other Biophysical Methods.

The variety and complexity of drug targets are expanding rapidly. At the same time, there is significant interest in exploring a larger chemical space to identify new candidates. Fragment-based screening (FBS) has emerged as a popular alternative to traditional high-throughput screening campaigns to identify such drug candidates. FBS identifies hit fragments that exhibit weak interactions with the target of interest, thereby enabling the rational design of small-molecule compounds from the identified hit fragments, which serve as building blocks. This strategy reduces the number of molecules to screen while also allowing the exploration of a greater chemical space.Here we use temperature-related intensity change (TRIC) technology to perform FBS against the target MAPK/ERK kinase-1 (Mek1). TRIC describes the change in fluorescence intensity of a fluorescently labeled molecule upon a change in temperature. This intensity variation is dependent on the physicochemical environment in the vicinity of the dye and strongly affected by binding events. Thus, the detection of binding events is independent of mass, making TRIC an ideal tool for FBS.Using only 150 pmol of labeled Mek1, the authors screened 193 fragments from a prescreened library in less than 1 h of measurement time, leading to 66 hits. Among those hits, they identified more than 80% of the published top hits found using orthogonal techniques. Furthermore, TRIC allowed the identification of fragments that were of poor solubility but could be mistaken as false-positive hits in other methods.

The human formin FHOD1 contains a bipartite structure of FH3 and GTPase-binding domains required for activation.

Formins induce the nucleation and polymerization of unbranched actin filaments. They share three homology domains required for profilin binding, actin polymerization, and regulation. Diaphanous-related formins (DRFs) are activated by GTPases of the Rho/Rac family, whose interaction with the N-terminal formin domain is thought to displace a C-terminal Diaphanous-autoregulatory domain (DAD). We have determined the structure of the N-terminal domains of FHOD1 consisting of a GTPase-binding domain (GBD) and the DAD-recognition domain FH3. In contrast to the formin mDia1, the FHOD1-GBD reveals a ubiquitin superfold as found similarly in c-Raf1 or PI3 kinase. This GBD is recruited by Rac and Ras GTPases in cells and plays an essential role for FHOD1-mediated actin remodeling. The FHOD1-FH3 domain is composed of five armadillo repeats, similarly to other formins. Mutation of one residue in the predicted DAD-interaction surface efficiently activates FHOD1 in cells. These results demonstrate that DRFs have evolved different molecular solutions to govern their autoregulation and GTPase specificity.

Genetically detoxified pertussis toxin displays near identical structure to its wild-type and exhibits robust immunogenicity.

The mutant gdPT R9K/E129G is a genetically detoxified variant of the pertussis toxin (PTx) and represents an attractive candidate for the development of improved pertussis vaccines. The impact of the mutations on the overall protein structure and its immunogenicity has remained elusive. Here we present the crystal structure of gdPT and show that it is nearly identical to that of PTx. Hydrogen-deuterium exchange mass spectrometry revealed dynamic changes in the catalytic domain that directly impacted NAD+ binding which was confirmed by biolayer interferometry. Distal changes in dynamics were also detected in S2-S5 subunit interactions resulting in tighter packing of B-oligomer corresponding to increased thermal stability. Finally, antigen stimulation of human whole blood, analyzed by a previously unreported mass cytometry assay, indicated broader immunogenicity of gdPT compared to pertussis toxoid. These findings establish a direct link between the conserved structure of gdPT and its ability to generate a robust immune response.

Discovery of 6-(2,4-Dichlorophenyl)-5-[4-[(3S)-1-(3-fluoropropyl)pyrrolidin-3-yl]oxyphenyl]-8,9-dihydro-7H-benzo[7]annulene-2-carboxylic acid (SAR439859), a Potent and Selective Estrogen Receptor Degrader (SERD) for the Treatment of Estrogen-Receptor-Positive Breast Cancer.

More than 75% of breast cancers are estrogen receptor alpha (ERα) positive (ER+), and resistance to current hormone therapies occurs in one-third of ER+ patients. Tumor resistance is still ERα-dependent, but mutations usually confer constitutive activation to the hormone receptor, rendering ERα modulator drugs such as tamoxifen and aromatase inhibitors ineffective. Fulvestrant is a potent selective estrogen receptor degrader (SERD), which degrades the ERα receptor in drug-resistant tumors and has been approved for the treatment of hormone-receptor-positive metastatic breast cancer following antiestrogen therapy. However, fulvestrant shows poor pharmacokinetic properties in human, low solubility, weak permeation, and high metabolism, limiting its administration to inconvenient intramuscular injections. This Drug Annotation describes the identification and optimization of a new series of potent orally available SERDs, which led to the discovery of 6-(2,4-dichlorophenyl)-5-[4-[(3S)-1-(3-fluoropropyl)pyrrolidin-3-yl]oxyphenyl]-8,9-dihydro-7H-benzo[7]annulene-2-carboxylic acid (43d), showing promising antitumor activity in breast cancer mice xenograft models and whose properties warranted clinical evaluation.

Fast NMR Methods for Measuring in the Direct and/or Competition Mode the Dissociation Constants of Chemical Fragments Interacting with a Receptor.

Ligand-based NMR screening represents a powerful method in fragment-based drug discovery for the identification of chemical matter interacting with the receptor of interest. The large dynamic range of these methods allows the detection of weakly binding ligands. However, the methodology has not been extensively used for quantifying the strength of these interactions. This knowledge is important for ranking fragments according to their binding strength and for prioritizing structure-based and medicinal chemistry activities. Rapid NMR methods for measuring the dissociation constant in direct and competition modes are presented here. The theory underpinning these methods are presented, along with their application to the measurement of the binding affinities of several ligands of the heat shock protein 90.

[Developability assessment with case studies highlighting the decision taking]. / Exemples d'études de développabilité apportant un éclairage à la prise de décision.

Therapeutic antibodies generation has to be faster with less development costs. This requires combination of in silico predictions associated with cutting edge screening and characterization technologies. Here, non-exhaustive examples illustrate this simultaneity need.

TITLE: Exemples d'études de développabilité apportant un éclairage à la prise de décision. ABSTRACT: De nos jours, la génération d'anticorps thérapeutiques doit être plus rapide avec des coûts de développement moins importants. Pour cela, des prédictions in silico sont associées à des technologies de criblage et de caractérisation de pointe. Les exemples choisis ici sont non-exhaustifs mais illustrent ce besoin de travailler en parallèle.

Therapeutic Opportunities with Pharmacological Inhibition of CD38 with Isatuximab.

CD38 is a transmembrane glycoprotein with ectoenzymatic activity involved in regulation of migration, signal transduction, and receptor-mediated adhesion. CD38 is highly expressed on various malignant cells, including multiple myeloma (MM), and at relatively low levels in other tissues, making it a suitable target for therapeutic antibodies. Several anti-CD38 therapies have been, or are being, developed for the treatment of MM, including daratumumab and isatuximab (SAR650984), respectively. Studies have shown that anti-CD38 therapies are effective in the treatment of relapsed/refractory MM and are well tolerated, with infusion reactions being the most common side effects. They can be used as monotherapy or in combination with immunomodulatory agents, such as pomalidomide, or proteasome inhibitors to potentiate their activity. Here we examine isatuximab and several anti-CD38 agents in development that were generated using new antibody engineering techniques and that may lead to more effective CD38 targeting. We also summarize trials assessing these antibodies in MM, other malignancies, and solid organ transplantation. Finally, we propose that further research on the mechanisms of resistance to anti-CD38 therapy and the development of biomarkers and new backbone regimens with CD38 antibodies will be important steps in building more personalized treatment for patients with MM.

A combinatorial approach to crystallization of PX-BAR unit of the human Sorting Nexin 9.

Sorting nexins (SNXs) form a family of proteins known to interact with endosomal vesicles and to regulate various steps of vesicle transport. Sorting Nexin 9 (SNX9) is involved in the interface of endocytic, actin polymerizing, and signal transduction events in the cell. Here we report crystallization of the SNX9 PX-BAR domain protein. Initially we used an ordinary protein construct design, and protein crystallization approaches resulted in obtaining granular crystal-like precipitation. SDS-PAGE and MS analysis of the crystal-like precipitation followed by protein construct optimization and using of macro seeding technique resulted in X-ray quality diffracting crystals. The crystals belonged to P2(1)2(1)2(1) space group (a=65.6 A, b=117.5 A, c=145.8 A) with two protein molecules per asymmetric unit. A complete SAD data set from Se-Methionine derived crystal (3.2 A) has been collected to solve the structure.

Sec2 is a highly efficient exchange factor for the Rab protein Sec4.

Sec2 is a reversibly membrane associated multi-domain protein with guanine nucleotide exchange activity towards the yeast Rab-protein Sec4. Both proteins are localized to secretory vesicles destined for exocytosis. We have used transient kinetic methods to show that Sec2 is a highly active exchange factor, in contrast to other proteins previously characterized as Rab exchange factors. With a K(d) value for the Sec2:Sec4.GDP interaction of ca 70 microM and a maximal rate of GDP displacement of ca 15 s(-1), it is 100-1000-fold more effective than other proteins showing exchange activity towards Rabs (MSS4, DSS4, Vps9) and ca tenfold faster than Cdc25 as a Ras specific exchanger, although still 100-fold slower than the fastest systems studied so far, EF-Tu/Ef-Ts and Ran/RCC1. A comparison with other proteins showing Rab exchange activity shows that maximal rates of GDP dissociation catalyzed by Sec2 are orders of magnitude faster. When comparing Sec2 with DSS4, which also acts on Sec4, the difference was particularly dramatic. Another difference is seen in the kinetics of association of GTP with the Sec4:Sec2 complex, a process which is extremely slow for DSS4/MSS4 complexes with cognate Rabs but in the range observed for other GTPase:exchanger complexes for Sec4:Sec2., It is suggested that systems such as Ef-Tu/Ef-Ts and Ran/RCC1 have evolved for maximal possible activity for the interaction between two soluble proteins, whereas other evolutionary constraints which are connected to the spatial and temporal coordination of events in vesicular transport and other regulatory networks have determined the detailed kinetic properties of the other systems.

Flow cytometry for real-time measurement of guanine nucleotide binding and exchange by Ras-like GTPases.

Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here we report a bead-based flow cytometric assay that quantitatively measures the nucleotide binding properties of glutathione-S-transferase (GST) chimeras for prototypical Ras family members Rab7 and Rho. Measurements are possible in the presence or absence of Mg(2+), with magnesium cations principally increasing affinity and slowing nucleotide dissociation rates 8- to 10-fold. GST-Rab7 exhibited a 3-fold higher affinity for guanosine diphosphate (GDP) relative to guanosine triphosphate (GTP) that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used gamma-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real time and to quantitatively assess differences between GTPases.

Estimation of Drug-Target Residence Times by τ-Random Acceleration Molecular Dynamics Simulations.

Drug-target residence time (τ), one of the main determinants of drug efficacy, remains highly challenging to predict computationally and, therefore, is usually not considered in the early stages of drug design. Here, we present an efficient computational method, τ-random acceleration molecular dynamics (τRAMD), for the ranking of drug candidates by their residence time and obtaining insights into ligand-target dissociation mechanisms. We assessed τRAMD on a data set of 70 diverse drug-like ligands of the N-terminal domain of HSP90α, a pharmaceutically important target with a highly flexible binding site, obtaining computed relative residence times with an accuracy of about 2.3τ for 78% of the compounds and less than 2.0τ within congeneric series. Analysis of dissociation trajectories reveals features that affect ligand unbinding rates, including transient polar interactions and steric hindrance. These results suggest that τRAMD will be widely applicable as a computationally efficient aid to improving drug residence times during lead optimization.

Purification, crystallization and preliminary structural characterization of the N-terminal region of the human formin-homology protein FHOD1.

Formins are key regulators of actin cytoskeletal dynamics that constitute a diverse protein family that is present in all eukaryotes examined. They typically consist of more than 1000 amino acids and are defined by the presence of two conserved regions, namely the formin homology 1 and 2 domains. Additional conserved domains comprise a GTPase-binding domain for activation, a C-terminal autoregulation motif and an N-terminal recognition domain. In this study, the N-terminal region (residues 1-339) of the human formin homology domain-containing protein 1 (FHOD1) was purified and crystallized from 20%(w/v) PEG 4000, 10%(v/v) glycerol, 0.3 M magnesium chloride and 0.1 M Tris-HCl pH 8.0. Native crystals belong to space group P1, with unit-cell parameters a = 35.4, b = 73.9, c = 78.7 A, alpha = 78.2, beta = 86.2, gamma = 89.7 degrees. They contain two monomers of FHOD1 in the asymmetric unit and diffract to a resolution of 2.3 A using a synchrotron-radiation source.

Purification, crystallization and preliminary X-ray crystallographic analysis of mammalian MSS4-Rab8 GTPase protein complex.

Rab GTPases function as ubiquitous key regulators of membrane-vesicle transport in eukaryotic cells. MSS4 is an evolutionarily conserved protein that binds to exocytotic Rabs and facilitates nucleotide release. The MSS4 protein in complex with nucleotide-free Rab8 GTPase has been purified and crystallized in a form suitable for structure analysis. The crystals belonged to space group P1, with unit-cell parameters a = 40.92, b = 49.85, c = 83.48 A, alpha = 102.88, beta = 97.46, gamma = 90.12 degrees. A complete data set has been collected to 2 A resolution.

Rab-subfamily-specific regions of Ypt7p are structurally different from other RabGTPases.

The GTPase Ypt7p from S. cerevisiae is involved in late endosome-to-vacuole transport and homotypic vacuole fusion. We present crystal structures of the GDP- and GppNHp-bound conformation of Ypt7p solved at 1.35 and 1.6 A resolution, respectively. Despite the similarity of the overall structure to other Ypt/Rab proteins, Ypt7p displays small but significant differences. The Ypt7p-specific residues Tyr33 and Tyr37 cause a difference in the main chain trace of the RabSF2 region and form a characteristic surface epitope. Ypt7p*GppNHp does not display the helix alpha2, characteristic of the Ras-superfamily, but instead possess an extended loop L4/L5. Due to insertions in loops L3 and L7, the neighboring RabSF1 and RabSF4 regions are different in their conformations to those of other Ypt/Rab proteins.