Biology subjects

Djukic, S.

Publications and source records attributed to Djukic, S..

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↗

bioRxiv2024

TDP-43 pathology is sufficient to drive axon initial segment plasticity and hyperexcitability of spinal motoneurones in vivo in the TDP43-NLS model of Amyotrophic Lateral Sclerosis

A hyperexcitability of the motor system is consistently observed in Amyotrophic Lateral Sclerosis (ALS) and has been implicated in the disease pathogenesis. What drives this hyperexcitability in the vast majority of patients is unknown. This is important to know as existing treatments simply reduce all neuronal excitability and fail to distinguish between pathological changes and important homeostatic changes. Understanding what drives the initial pathological changes could therefore provide better treatments. One challenge is that patients represent a heterogeneous population and the vast majority of cases are sporadic. One pathological feature that almost all ([~]97%) cases (familial and sporadic) have in common is cytoplasmic aggregates of the protein TDP-43 which is normally located in the nucleus. In our experiments we investigated whether this pathology was sufficient to increase neuronal excitability and the mechanisms by which this occurs. We used the TDP-43({Delta}NLS) mouse model which successfully recapitulates this pathology in a controllable way. We used in vivo intracellular recordings in this model to demonstrate that TDP-43 pathology is sufficient to drive a severe hyper-excitability of spinal motoneurones. Reductions in soma size and a lengthening and constriction of axon initial segments were observed, which would contribute to enhanced excitability. Resuppression of the transgene resulted in a return to normal excitability parameters by 6-8 weeks. We therefore conclude that TDP-43 pathology itself is sufficient to drive a severe but reversible hyperexcitability of spinal motoneurones.

neuroscience↗

bioRxiv2023

Characterization of new highly selective pyrazolopyrimidine inhibitor of CDK7

Targeting cyclin-dependent kinase 7 (CDK7) provides an interesting therapeutic option in cancer therapy because this kinase participates in regulating the cell cycle and transcription. Here, we describe a new trisubstituted pyrazolo[4,3-d]pyrimidine derivative, LGR6768, that inhibits CDK7 in the nanomolar range and displays favourable selectivity across the CDK family. We determined the structure of fully active CDK2/cyclin A2 in complex with LGR6768 at 2.6 [A] resolution using X-ray crystallography, revealing conserved interactions within the active site. Structural analysis and comparison with LGR6768 docked to CDK7 provides an explanation of the observed biochemical selectivity, which is linked to a conformational difference in the biphenyl moiety. In cellular experiments, LGR6768 affected regulation of the cell cycle and transcription by inhibiting the phosphorylation of cell cycle CDKs and the carboxy-terminal domain of RNA polymerase II, respectively. LGR6768 limited the proliferation of several leukaemia cell lines, triggered significant changes in protein and mRNA levels related to CDK7 inhibition and induced apoptosis in dose- and time-dependent experiments. Our work supports previous findings and provides further information for the development of selective CDK7 inhibitors. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/529844v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1d13e99org.highwire.dtl.DTLVardef@64ea36org.highwire.dtl.DTLVardef@14c9705org.highwire.dtl.DTLVardef@14efc31_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Cytoplasmic TDP-43 accumulation drives changes in C-bouton number and size in a mouse model of sporadic Amyotrophic Lateral Sclerosis

An altered neuronal excitability of spinal motoneurones has consistently been implicated in Amyotrophic Lateral Sclerosis (ALS) leading to several investigations of synaptic input to these motoneurones. One such input that has repeatedly been shown to be affected is a population of large cholinergic synapses terminating mainly on the soma of the motoneurones referred to as C-boutons. Most research on these synapses during disease progression has used transgenic Superoxide Dismutase 1 (SOD1) mouse models of the disease which have not only produced conflicting findings, but also fail to recapitulate the key pathological feature seen in ALS; cytoplasmic accumulations of TAR DNA-binding protein 43 (TDP-43). Additionally, they fail to distinguish between slow and fast motoneurones, the latter of which have more C-boutons but are lost earlier in the disease. To circumvent these issues, we quantified the frequency and volume of C-boutons on traced soleus and gastrocnemius motoneurones, representing predominantly slow and fast motor pools respectively. Experiments were performed using the TDP-43{Delta}NLS mouse model that carries a transgenic construct of TDP-43 devoid of its nuclear localization signal, preventing its nuclear import. This results in the emergence of pathological TDP-43 inclusions in the cytoplasm, modelling the main pathology seen in this disorder, accompanied by a severe and lethal ALS phenotype. Our results confirmed changes in both the number and volume of C-boutons with a decrease in number on the more vulnerable, predominantly fast gastrocnemius motoneurones and an increase in number on the less vulnerable, predominantly slow soleus motoneurones. Importantly, these changes were only found in male mice. However, both sexes and motor pools showed a decrease in C-bouton volume. Our experiments confirm that cytoplasmic TDP-43 accumulation is sufficient to drive C-bouton changes.

neuroscience↗