Biology subjects

Geist, L.

Publications and source records attributed to Geist, L..

4 recordsLinked to original sources

bioRxiv2025

Tuning the open-close equilibrium of Cereblon with small molecules influences protein degradation

Most PROTACs and molecular glue degraders currently approved or in clinical trials recruit Cereblon (CRBN) as the ubiquitin E3 ligase. Upon binding ligands and molecular glues, CRBN undergoes a significant structural rearrangement from an open to closed state, defined by the positioning of the thalidomide-binding domain (TBD) with respect to the Lon domain. However, the exact molecular basis for this ligand-induced conformational change and its implication to neo-substrate degradation remain elusive. During our campaign to discover novel CRBN binders, we found hits exhibiting distinct biophysical behaviour from classical thalidomide-based ligands. By combining orthogonal biophysical methods of differential scanning fluorimetry, isothermal titration calorimetry, and small-angle X-ray scattering, supported by X-ray crystallography and cryo-EM structures of ligand-bound complexes, we classify CRBN binders between those that can induce CRBN closure, and those that cannot. Mutational studies identify key residues in the CRBN ligand binding pocket and N-terminal belt that are essential for the Lon and TBD domains to come together in the closed state. Finally, we show that the probability to yield active degrader molecules is greatly influenced by whether binders can or cannot induce CRBN closure. Together, our study reveals new molecular insights into the structural basis for how CRBN open-closed equilibrium is directly modulated by compound binding and impact target degradability by CRBN, with important implications to the design of PROTACs and molecular glue degraders.

biochemistry↗

bioRxiv2023

Sex differences in offspring risk and resilience following 11β-hydroxylase antagonism in a rodent model of maternal immune activation

Maternal immune activation (MIA) puts offspring at greater risk for neurodevelopmental disorders associated with impaired social behavior. While it is known that immune signaling through maternal, placental, and fetal compartments contributes to these phenotypical changes, it is unknown to what extent the stress response to illness is involved and how it can be harnessed for potential interventions. To this end, on gestational day 15, pregnant rat dams were administered the bacterial mimetic lipopolysaccharide (LPS; to induce MIA) alongside metyrapone, a clinically available 11{beta}-hydroxylase inhibitor used to treat hypercortisolism in pregnant and neonatal populations. Maternal, placental, and fetal CNS levels of corticosterone and placental 11{beta}HSD enzymes type 1 and 2 were measured 3-hrs post treatment. Offspring social behaviors were evaluated across critical phases of development. MIA was associated with increased maternal, placental, and fetal CNS corticosterone concentrations that were diminished with metyrapone exposure. Metyrapone protected against reductions in placental 11{beta}HSD2 in males only, suggesting that less corticosterone was inactivated in female placentas. Behaviorally, metyrapone-exposure attenuated MIA-induced social disruptions in juvenile, adolescent, and adult males, while females were unaffected or performed worse. Metyrapone-exposure reversed MIA-induced transcriptional changes in monoamine-, glutamate-, and GABA-related genes in the ventral hippocampus of adult males, but not females. Taken together, these findings illustrate that MIA-induced HPA responses act alongside the immune system to produce behavioral deficits. As a clinically available drug, the sex-specific benefits and constraints of metyrapone should be investigated further as a potential means of reducing neurodevelopmental risks due to gestational MIA.

neuroscience↗

bioRxiv2021

Inverse PI by NMR: Analysis of Ligand 1H-Chemical Shifts in the Protein-Bound State

The study of protein-ligand interactions via protein-based NMR generally relies on the detection of chemical-shift changes induced by ligand binding. However, the chemical shift of the ligand when bound to the protein is rarely discussed, since it is not readily detectable. In this work we use protein deuteration in combination with [1H-1H]-NOESY NMR to extract 1H chemical shift values of the ligand in the bound state. The chemical shift perturbations (CSPs) experienced by the proton ligand resonances (free vs bound) are an extremely rich source of information on protein-ligand complexes. Besides allowing for the detection of intermolecular CH-{pi} interactions and elucidation of the protein-bound ligand conformation, the CSP information can be used to analyse (de)solvation effects in a site-specific manner. In conjunction with crystal structure information, it is possible to distinguish protons whose desolvation penalty is compensated for upon protein-binding, from those that are not. Combined with the previously reported PI by NMR technique for the protein-based detection of intermolecular CH-{pi} interactions, this method represents another important step towards the ultimate goal of Interaction-Based Drug Discovery.

biophysics↗

bioRxiv2020

Crystal Structure of the Werner's Syndrome Helicase

Werner syndrome helicase (WRN) plays important roles in multiple pathways of DNA repair and the maintenance of genome integrity. Recently, loss of WRN was identified as a strong synthetic lethal interaction for microsatellite instable (MSI) cancers making WRN a promising drug target. Yet, structural information for the helicase domain is lacking, which prevents structure-based design of drug molecules. In this study, we show that ATP binding and hydrolysis in the helicase domain are required for genome integrity and viability of MSI cancer cells. We then determined the crystal structure of an ADP bound form of the WRN helicase core at 2.2 [A] resolution. The structure features an atypical mode of nucleotide binding with extensive contacts formed by motif VI, which in turn defines the relative positioning of the two RecA like domains. The structure features a novel additional {beta}-hairpin in the second RecA and an unusual helical hairpin in the Zn2+ binding domain, and modelling DNA substrates based on existing RecQ DNA complexes suggests roles for these features in the binding of alternative DNA structures. We have further analysed possible interfaces formed from the interactions between the HRDC domain and the helicase core by NMR. Together, this study will facilitate the structure-based design of inhibitors against WRN helicase.

biochemistry↗