Chemical screening by time-resolved X-ray scattering to discover allosteric probes.

Drug discovery relies on efficient identification of small-molecule leads and their interactions with macromolecular targets. However, understanding how chemotypes impact mechanistically important conformational states often remains secondary among high-throughput discovery methods. Here, we present a conformational discovery pipeline integrating time-resolved, high-throughput small-angle X-ray scattering (TR-HT-SAXS) and classic fragment screening applied to allosteric states of the mitochondrial import oxidoreductase apoptosis-inducing factor (AIF).

Applying HT-SAXS to chemical ligand screening.

Chemical probes are invaluable tools for investigating essential biological processes. Understanding how small-molecule probes engage biomolecular conformations is critical to developing their functional selectivity. High-throughput solution X-ray scattering is well-positioned to profile target-ligand complexes during probe development, bringing conformational insight and selection to traditional ligand binding assays.

Vitamin E Enhances Cancer Immunotherapy by Reinvigorating Dendritic Cells via Targeting Checkpoint SHP1.

Unlabelled: Despite the popular use of dietary supplements during conventional cancer treatments, their impacts on the efficacies of prevalent immunotherapies, including immune-checkpoint therapy (ICT), are unknown. Surprisingly, our analyses of electronic health records revealed that ICT-treated patients with cancer who took vitamin E (VitE) had significantly improved survival. In mouse models, VitE increased ICT antitumor efficacy, which depended on dendritic cells (DC).

An efficient chemical screening method for structure-based inhibitors to nucleic acid enzymes targeting the DNA repair-replication interface and SARS CoV-2.

We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis as exemplified here by specific targeting of Poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose glycohydrolase ARH3 macrodomains plus SARS CoV-2 nonstructural protein 3 (Nsp3) Macrodomain 1 (Mac1) and Nsp15 nuclease. As opposed to the classical massive effort employing libraries with large numbers of compounds against single proteins, we make inhibitor design for multiple targets efficient.

GRB2 enforces homology-directed repair initiation by MRE11.

DNA double-strand break (DSB) repair is initiated by MRE11 nuclease for both homology-directed repair (HDR) and alternative end joining (Alt-EJ). Here, we found that GRB2, crucial to timely proliferative RAS/MAPK pathway activation, unexpectedly forms a biophysically validated GRB2-MRE11 (GM) complex for efficient HDR initiation. GRB2-SH2 domain targets the GM complex to phosphorylated H2AX at DSBs.

An effective human uracil-DNA glycosylase inhibitor targets the open pre-catalytic active site conformation.

Human uracil DNA-glycosylase (UDG) is the prototypic and first identified DNA glycosylase with a vital role in removing deaminated cytosine and incorporated uracil and 5-fluorouracil (5-FU) from DNA. UDG depletion sensitizes cells to high APOBEC3B deaminase and to pemetrexed (PEM) and floxuridine (5-FdU), which are toxic to tumor cells through incorporation of uracil and 5-FU into DNA. To identify small-molecule UDG inhibitors for pre-clinical evaluation, we optimized biochemical screening of a selected diversity collection of >3,000 small-molecules.

Targeting SARS-CoV-2 Nsp3 macrodomain structure with insights from human poly(ADP-ribose) glycohydrolase (PARG) structures with inhibitors.

Arrival of the novel SARS-CoV-2 has launched a worldwide effort to identify both pre-approved and novel therapeutics targeting the viral proteome, highlighting the urgent need for efficient drug discovery strategies. Even with effective vaccines, infection is possible, and at-risk populations would benefit from effective drug compounds that reduce the lethality and lasting damage of COVID-19 infection. The CoV-2 MacroD-like macrodomain (Mac1) is implicated in viral pathogenicity by disrupting host innate immunity through its mono (ADP-ribosyl) hydrolase activity, making it a prime target for antiviral therapy.

Fragment- and structure-based drug discovery for developing therapeutic agents targeting the DNA Damage Response.

Cancer will directly affect the lives of over one-third of the population. The DNA Damage Response (DDR) is an intricate system involving damage recognition, cell cycle regulation, DNA repair, and ultimately cell fate determination, playing a central role in cancer etiology and therapy. Two primary therapeutic approaches involving DDR targeting include: combinatorial treatments employing anticancer genotoxic agents; and synthetic lethality, exploiting a sporadic DDR defect as a mechanism for cancer-specific therapy.

Selective small molecule PARG inhibitor causes replication fork stalling and cancer cell death.

Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Here, we found that PARG expression is upregulated in many cancers. We employed chemical library screening to identify and optimize methylxanthine derivatives as selective bioavailable PARG inhibitors.

Targeting Allostery with Avatars to Design Inhibitors Assessed by Cell Activity: Dissecting MRE11 Endo- and Exonuclease Activities.

For inhibitor design, as in most research, the best system is question dependent. We suggest structurally defined allostery to design specific inhibitors that target regions beyond active sites. We choose systems allowing efficient quality structures with conformational changes as optimal for structure-based design to optimize inhibitors.

RNF4 interacts with both SUMO and nucleosomes to promote the DNA damage response.

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO.

DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities.

MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by nonhomologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors, we examined repair pathway choice at DSBs generated in G2 following radiation exposure.

MRE11 facilitates the removal of human topoisomerase II complexes from genomic DNA.

Topoisomerase II creates a double-strand break intermediate with topoisomerase covalently coupled to the DNA via a 5'-phosphotyrosyl bond. These intermediate complexes can become cytotoxic protein-DNA adducts and DSB repair at these lesions requires removal of topoisomerase II. To analyse removal of topoisomerase II from genomic DNA we adapted the trapped in agarose DNA immunostaining assay.

Mre11 ATLD17/18 mutation retains Tel1/ATM activity but blocks DNA double-strand break repair.

The Mre11 complex (Mre11-Rad50-Nbs1 or MRN) binds double-strand breaks where it interacts with CtIP/Ctp1/Sae2 and ATM/Tel1 to preserve genome stability through its functions in homology-directed repair, checkpoint signaling and telomere maintenance. Here, we combine biochemical, structural and in vivo functional studies to uncover key properties of Mre11-W243R, a mutation identified in two pediatric cancer patients with enhanced ataxia telangiectasia-like disorder. Purified human Mre11-W243R retains nuclease and DNA binding activities in vitro.

Crystal structure of the first eubacterial Mre11 nuclease reveals novel features that may discriminate substrates during DNA repair.

Mre11 nuclease plays a central role in the repair of cytotoxic and mutagenic DNA double-strand breaks. As X-ray structural information has been available only for the Pyrococcus furiosus enzyme (PfMre11), the conserved and variable features of this nuclease across the domains of life have not been experimentally defined. Our crystal structure and biochemical studies demonstrate that TM1635 from Thermotoga maritima, originally annotated as a putative nuclease, is an Mre11 endo/exonuclease (TmMre11) and the first such structure from eubacteria.

p38alpha MAP kinase C-terminal domain binding pocket characterized by crystallographic and computational analyses.

The mitogen-activated protein (MAP) kinase protein family has a critical role in cellular signaling events, with MAP kinase p38alpha acting in inflammatory processes and being an important drug discovery target. MAP kinase drug design efforts have focused on small-molecule inhibitors of the ATP catalytic site, which exhibit dose-limiting adverse effects. Therefore, characterizing other potential sites that bind substrates, inhibitors, or allosteric effectors is of great interest.

Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair.

Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends.

Supramolecular sensing with phosphonate cavitands.

Phosphonate cavitands are an emerging class of synthetic receptors for supramolecular sensing. The molecular recognition properties of the third-generation tetraphosphonate cavitands toward alcohols and water at the gas-solid interface have been evaluated by means of three complementary techniques and compared to those of the parent mono- and diphosphonate cavitands. The combined use of ESI-MS and X-ray crystallography defined precisely the host-guest association at the interface in terms of type, number, strength, and geometry of interactions.

Molecular dynamic investigation of the interaction of supported affinity ligands with monoclonal antibodies.

Diagnostics and therapeutic treatments based on monoclonal antibodies have been attaining an increasing importance in the past decades, but their large scale employment requires the optimization of purification processes. To obtain this goal, research is focusing on affinity chromatography techniques and the development of new synthetic ligands. In this work we present a computational investigation aimed at obtaining some guidelines for the rational design of affinity ligands, through the study of their interactions with both monoclonal antibodies (modeled as the FC domain of human IgG) and a model support material (agarose).

Oxoanion binding by guanidiniocarbonylpyrrole cations in water: a combined DFT and MD investigation.

Structures and properties of nonbonding interactions involving guanidinium-functionalized hosts and carboxylate substrates were investigated by a combination of ab initio and molecular dynamics approaches. The systems under study are on one hand intended to be a model of the arginine-anion bond, so often observed in proteins and nucleic acids, and on the other to provide an opportunity to investigate the influence of molecular structure on the formation of supramolecular complexes in detail. Use of DFT calculations, including extended basis sets and implicit water treatment, allowed us to determine minimum-energy structures and binding enthalpies that compared well with experimental data.

Measuring H-bonding in supramolecular complexes by gas phase ion-molecule reactions.

H/D and guest-exchange ion-molecule reactions have been used as a new tool to elucidate the operation of multiple hydrogen bonding in gas-phase complexes formed between phosphonate cavitands and ethyl-substituted ammonium ions.

Investigation of the influence of spacer arm on the structural evolution of affinity ligands supported on agarose.

The influence of the spacer arm on the interaction between agarose and a supported ligand was investigated through molecular dynamics for a combination of several spacers. The spacers differ for degree of hydrophobicity, length, and chemical composition, which was varied through insertion of thio, ether, and CH(2) groups. Agarose was modeled through a modified Glycam force field, whose parameters were determined through ab initio calculations.