Biology subjects

De Vita, E.

Publications and source records attributed to De Vita, E..

6 recordsLinked to original sources

bioRxiv2026

A potent macrocyclic peptide binder enables effector-free crystallization of the GTPase RAB27A

RAB27A is a member of a highly conserved family of small GTPases that regulate intracellular vesicle trafficking. Multiple studies have linked RAB27A cellular activity to exosome-mediated modulation of the tumour microenvironment and inflammatory signalling, highlighting it as an attractive target for drug discovery. However, identifying structurally validated and selective inhibitors of the RAB27A-effector interaction has proven challenging, partly because wild-type RAB27A crystallizes only in complex with effector proteins such as Slp2a. Using mRNA display, we identified macrocyclic peptide ligands that target the RAB27A effector-binding interface. Optimisation of initial hits led to IMP2660, a nanomolar-affinity RAB27A binder that effectively disrupts RAB27A-effector interactions. IMP2660 enabled determination of a 2.1[A] structure of active-state RAB27A bound to a noncovalent macrocyclic peptide in the absence of an effector, revealing structural features of the effector-binding surface and providing a platform for future structure-guided ligand development.

biochemistry↗

bioRxiv2026

Quantitative Susceptibility Mapping for Differentiating Hydroxyapatite and Calcium Oxalate Breast Calcifications at 3T: A Phantom Study

PurposeBreast microcalcifications trigger 70-80% of unnecessary biopsies because current imaging cannot distinguish malignancy-associated hydroxyapatite (HA) from benign-associated calcium oxalate (CaOx). Quantitative susceptibility mapping (QSM) could exploit the susceptibility contrast between these minerals (HA: {Delta}{chi} {approx} -7 ppm; CaOx: {Delta}{chi} {approx} -1 ppm relative to water), but no study has demonstrated compositional differentiation at clinical field strength. This work assessed susceptibility and R2* relaxation rate maps for microcalcification differentiation at 3 T using tissue-mimicking phantoms. MethodsA phantom comprising 12 tubes, each containing co-embedded HA and CaOx particles in BaCl2-crosslinked alginate gels (pure alginate, adipose-mimicking, and fibroglandular tissue-mimicking relaxation properties; n = 4 per type), were scanned at 0.70 mm and 0.86 mm isotropic resolution using a multi-echo gradient echo sequence. A consensus-aligned QSM pipeline and mono-exponential R2* fitting was developed. A digital twin phantom simulation quantified the contributions of partial volume effects and Total Variation (TV) regularisation to susceptibility underestimation. ResultsQSM detected HA in 18/24 measurements ({Delta}{chi}peak = -0.37 {+/-} 0.07 ppm in alginate at 0.70 mm) and CaOx in 0/24. R2* mapping detected HA in 23/24 and CaOx in 22/24. The digital twin identified TV regularisation as the dominant signal loss mechanism (57.5% loss), exceeding partial volume effects (24.3% loss). Combined parameters yielded three classification categories: QSM-positive with elevated R2* (HA), QSM-negative with moderate R2* (CaOx), and neither elevated (no calcification). ConclusionQSM at 3 T enables categorical HA detection while R2* provides complementary CaOx sensitivity, together enabling two-parameter microcalcification classification from a single multi-echo acquisition.

biophysics↗

bioRxiv2026

Discovery of stereoselective targeted covalent inhibitors of the RAB27-effector protein-protein interaction

RAB27A and RAB27B are homologous small GTPases that regulate intracellular vesicle trafficking, orchestrating endocytic and exocytic processes that affect cellular communication, immune responses, and dynamics of the cellular microenvironment. Through their interactions with effector proteins, RAB27A/B play roles in tumor metastasis and chronic inflammation. However, pharmacological modulation of their activity faces challenges typical of small GTPases, including a lack of well-defined pockets outside the conserved GTP binding site, and large RAB27-effector protein-protein interaction (PPI) surfaces. Here, we present the discovery and development of the first cell-active, rationally designed covalent inhibitors of the RAB27-effector interaction, targeting a non-conserved cysteine residue flanking the PPI interface. An electrophile-first biochemical screen led to a novel class of acrylamide covalent inhibitors, and X-ray crystallography structure-guided design led to optimized inhibitors and probes that enantioselectively target RAB27A/B-Cys123 in cells. Potency and selectivity were confirmed through biochemical and cellular assays, including chemical proteomics and phenotype recapitulation in melanocytes alongside a matched inactive enantioprobe control. In contrast, a previously reported compound, Nexinhib-20, was found to be toxic and to exert its activity through non-selective reactivity. This work provides the first toolbox of cell-active chemical probes for RAB27 which can be used in future studies to shed light on the function of this protein and its potential as a therapeutic target.

biochemistry↗

bioRxiv2024

Thalamic blood flow and EEG frequency band power during hyperventilation in idiopathic generalized epilepsy

Absence seizures in idiopathic generalised epilepsy (IGE) involve thalamo-cortical circuits. Hyperventilation (HV) is a standard technique to trigger epileptiform discharges and absence seizures in IGE. HV also increases electroencephalography (EEG) delta band power and decreases global cerebral blood flow (CBF). The relationships between HV, EEG band power, and regional CBF have not been investigated in the same patients at the same time. We compared the effects of hyperventilation between 13 individuals with IGE and 18 healthy controls on thalamic CBF assessed with pseudo-continuous Arterial Spin Labelling (pCASL) simultaneously to EEG frequency band power during three periods of hyperventilation interleaved with three periods of rest, normalised to each participants pre-HV values. Pre-HV, there were no differences between patients and controls. During the three rest periods combined, patients had higher normalised respiratory rates but no difference in CBF or EEG band power compared with controls. During HV, CBF decreases were similar in controls and IGE patients in cortical gray matter (37.1{+/-}1.3% in controls, 37.9{+/-}2.0% in patients) and basal ganglia (35.6{+/-}2.0% in controls, 33.3{+/-}2.3% in patients). In the thalamus, CBF decreased by 35.5{+/-}1.9% in controls, but only by 27.1{+/-}2.5% in patients (p=0.011). Delta band power increased by 42% in patients, but only by 27% in patients (p<0.045). Both controls and patients thalamic CBF significantly decreased as a function of respiratory rate, but the relationship was weaker in patients (rho -0.38 [0.95 confidence interval lower limit -0.629; upper limit -0.059] vs -0.51 [-0.698; -0.255] in controls). Delta power correlated with thalamic CBF only in controls (rho -0.21 [-0.388; -0.017]) but not in patients (rho -0.1 [-0.345; 0.157]). EEG power and respiratory rate were not significantly correlated in either group. This first study of the interrelationships between HV, blood flow, and EEG band power suggests that regional thalamic abnormalities of blood flow regulation, or a neurophysiological abnormality leading to smaller reactive blood flow changes, may underlie HV-associated EEG activation. Graphical abstract textIn this first investigation of combined hyperventilation, blood flow and EEG band power measurements in idiopathic generalised epileptic patients and healthy subjects, we revealed that localized thalamic blood flow regulatory problems or neurophysiological disorders caused minor reactive blood flow variations and may cause hyperventilation-associated EEG activation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/586742v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@12a5ebeorg.highwire.dtl.DTLVardef@b22944org.highwire.dtl.DTLVardef@b335dforg.highwire.dtl.DTLVardef@195c5e2_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Structure-guided design and optimization of covalent VHL-targeted sulfonyl fluoride PROTACs

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/545773v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@da22e5org.highwire.dtl.DTLVardef@1290278org.highwire.dtl.DTLVardef@1611c0eorg.highwire.dtl.DTLVardef@7902d2_HPS_FORMAT_FIGEXP M_FIG C_FIG Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that have emerged as a therapeutic modality to induce targeted protein degradation (TPD) by harnessing cellular proteolytic degradation machinery. PROTACs which ligand the E3 ligase in a covalent manner have attracted intense interest, however, covalent PROTACs with a broad protein of interest (POI) scope have proven challenging to discover by design. Here, we report structure-guided design and optimization of Von Hippel-Lindau (VHL) protein-targeted sulfonyl fluorides which covalently bind Ser110 in the HIF1 binding site. We demonstrate that their incorporation in bifunctional degraders induces targeted protein degradation of BRD4 or androgen receptor (AR) without further linker optimization. Our study discloses the first covalent VHL ligands which can be implemented directly in bifunctional degrader design expanding the substrate scope of covalent E3 ligase PROTACs.

molecular biology↗

bioRxiv2021

A suite of activity-based probes to dissect the KLK activome in drug-resistant prostate cancer

Kallikrein-related peptidases (KLKs) are a family of secreted serine proteases, which form a network - the KLK activome - with an important role in proteolysis and signaling. In prostate cancer (PCa), increased KLK activity promotes tumor growth and metastasis through multiple biochemical pathways, and specific quantification and tracking of changes in the KLK activome could contribute to validation of KLKs as potential drug targets. Herein we report a technology platform based on novel activity-based probes (ABPs) and inhibitors with unprecedented potency and selectivity enabling simultaneous orthogonal analysis of KLK2, KLK3 and KLK14 activity in hormone-responsive PCa cell lines and tumor homogenates. Using selective inhibitors and multiplexed fluorescent activity-based protein profiling (ABPP) we dissect the KLK activome in PCa cells and show that increased KLK14 activity leads to a migratory phenotype. Furthermore, using biotinylated ABPs we show that active KLK molecules are secreted into the bone microenvironment by PCa cells following stimulation by osteoblasts suggesting KLK-mediated signaling mechanisms could contribute to PCa metastasis to bone. Together our findings show that ABPP is a powerful approach to dissect dysregulation of the KLK activome as a promising and previously underappreciated therapeutic target in advanced PCa.

biochemistry↗