Biology subjects

Marchand, A.

Publications and source records attributed to Marchand, A..

17 recordsLinked to original sources

bioRxiv2024

Mapping targetable sites on the human surfaceome for the design of novel binders

The human cell surfaceome, integral to cell communication and disease mechanisms, presents a prime target for therapeutic intervention. De novo protein binder design against these cell surface proteins offers a promising yet underexplored strategy for drug development. However, the vast search space and limited data on natural or competitive binders have historically limited experimental success. In this study, we systematically analyzed the entire human surfaceome, identifying approximately 4,500 targetable sites and introducing high-quality binder seeds tailored for protein design applications. To validate these seeds, we implemented two experimental approaches (protein scaffolding and peptide cyclization) on three representative targets (FGFR2, IFNAR2, and HER3). Our results revealed a high success rate, emphasizing the precision and therapeutic potential of these seeds, as well as the need for constant improvements of computational protein design pipelines utilizing machine learning and physics-based methods. Additionally, we present SURFACE-Bind, an interactive database offering open access to all generated data. The high-throughput computational design methods and target-specific binder seeds established here pave the way for a new generation of targeted therapeutics for the human surfaceome.

synthetic biology↗

bioRxiv2024

Drug-controlled CAR-T cells through the regulation of cell-cell interactions

CAR T-cell therapy is constrained by on-target, off-tumor toxicities as well as cellular exhaustion due to chronic antigen exposure. CARs comprising small-molecule controlled switches can enhance both safety and therapeutic efficacy but are limited by the scarcity of non-immunogenic protein elements responsive to non-immunosuppressive, clinically approved drugs with favorable pharmacodynamics. Here, we combine rational design and library-based optimization of a protein-protein interaction (PPI) of human origin to develop venetoclax-controlled Drug-Regulated Off-switch PPI (DROP)-CARs. DROP-CARs enable dose-dependent release of the tumor-targeting scFv and consequent T-cell dissociation from the target tumor cell. Additionally, we present proof-of-concept for a dual DROP-CAR controlled by different small molecules, as well as for logic-gated synthetic receptors enabling STAT3 signaling. We demonstrate in vitro and in vivo function of DROP-CAR T cells and conclude that the approach holds promise for clinical application.

synthetic biology↗

bioRxiv2024

Targeting protein-ligand neosurfaces using a generalizable deep learning approach

Molecular recognition events between proteins drive biological processes in living systems. However, higher levels of mechanistic regulation have emerged, where protein-protein interactions are conditioned to small molecules. Here, we present a computational strategy for the design of proteins that target neosurfaces, i.e. surfaces arising from protein-ligand complexes. To do so, we leveraged a deep learning approach based on learned molecular surface representations and experimentally validated binders against three drug-bound protein complexes. Remarkably, surface fingerprints trained only on proteins can be applied to neosurfaces emerging from small molecules, serving as a powerful demonstration of generalizability that is uncommon in deep learning approaches. The designed chemically-induced protein interactions hold the potential to expand the sensing repertoire and the assembly of new synthetic pathways in engineered cells.

biochemistry↗

bioRxiv2022

Rational design of chemically controlled antibodies and protein therapeutics

Protein-based therapeutics such as monoclonal antibodies and cytokines are important therapies in various pathophysiological conditions such as oncology, auto-immune disorders, and viral infections. However, the wide application of such protein therapeutics is often hindered by dose-limiting toxicities and adverse effects, namely cytokine storm syndrome, organ failure and others. Therefore, spatiotemporal control of the activities of these proteins is crucial to further expand their application. Here, we report the design and application of small molecule-controlled switchable protein therapeutics by taking advantage of a previously engineered OFF-switch system. We used Rosetta modeling suite to computationally optimize the affinity between B-cell lymphoma 2 (Bcl-2) protein and a previously developed computationally designed protein partner (LD3) to obtain a fast and efficient heterodimer disruption upon addition of a competing drug (Venetoclax). The incorporation of the engineered OFF-switch system into CTLA4, anti-HER2 antibodies or an Fc-fused IL-15 cytokine demonstrated an efficient disruption in vitro, as well as fast clearance in vivo upon addition of the competing drug Venetoclax. These results provide a proof-of-concept for the rational design of controllable biologics by introducing a drug-induced OFF-switch into existing protein-based therapeutics.

bioengineering↗

bioRxiv2022

De novo design of site-specific protein interactions with learned surface fingerprints

Physical interactions between proteins are essential for most biological processes governing life. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic, and structural data grows. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein-protein interaction (PPI) networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications. We exploit a geometric deep learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features critical to drive PPIs. We hypothesized these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof-of-principle, we computationally designed several de novo protein binders to engage four protein targets: SARS-CoV-2 spike, PD-1, PD-L1, and CTLA-4. Several designs were experimentally optimized while others were purely generated in silico, reaching nanomolar affinity with structural and mutational characterization showing highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling a novel approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.

bioengineering↗

bioRxiv2022

The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitor in vitro and confer resistance to nirmatrelvir

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with > 20x increase in EC50 values for ALG-097161, nirmatrelvir (PF-07321332) and PF-00835231. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6x to 72x). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. Abstract ImportancePaxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug resistant viruses. In order to guide the use of novel antivirals it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro. The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next generation SARS-CoV-2 3CLpro inhibitors.

microbiology↗

bioRxiv2021

Identification of novel Ebola virus inhibitors using biologically contained virus

Despite recent advancements in the development of vaccines and monoclonal antibody therapies for Ebola virus disease, treatment options remain limited. Moreover, management and containment of Ebola virus outbreaks is often hindered by the remote nature of the locations in which the outbreaks originate. Small-molecule compounds offer the advantage of being relatively cheap and easy to produce, transport and store, making them an interesting modality for the development of novel therapeutics against Ebola virus disease. Furthermore, the repurposing of small-molecule compounds, previously developed for alternative applications, can aid in reducing the time needed to bring potential therapeutics from bench to bedside. For this purpose, the Medicines for Malaria Venture provides collections of previously developed small-molecule compounds for screening against other infectious diseases. In this study, we used biologically contained Ebola virus to screen over 4,200 small-molecule drugs and drug-like compounds provided by the Medicines for Malaria Venture (i.e., the Pandemic Response Box and the COVID Box) and the Centre for Drug Design and Discovery (CD3, KU Leuven, Belgium). In addition to confirming known Ebola virus inhibitors, illustrating the validity of our screening assays, we identified eight novel selective Ebola virus inhibitors. Although the inhibitory potential of these compounds remains to be validated in vivo, they represent interesting compounds for the study of potential interventions against Ebola virus disease and might serve as a basis for the development of new therapeutics.

microbiology↗

bioRxiv2021

Functional analyses of two novel LRRK2 pathogenic variants in familial Parkinson's disease

BackgroundPathogenic variants in the LRRK2 gene are a common monogenic cause of Parkinsons disease. However, only seven variants have been confirmed to be pathogenic. ObjectivesWe identified two novel LRRK2 variants (H230R and A1440P) and performed functional testing. MethodsWe transiently expressed wildtype, the two new variants, or two known pathogenic mutants (G2019S and R1441G), in HEK-293T cells, with or without LRRK2 kinase inhibitor treatment. We characterized the phosphorylation and kinase activity of the mutants by western blotting. Thermal shift assays were performed to determine the folding and stability of the LRRK2 proteins. ResultsThe two variants were found in two large families and segregate with the disease. They display altered LRRK2 phosphorylation and kinase activity. ConclusionsWe identified two novel LRRK2 variants which segregate with the disease. The results of functional testing lead us to propose these two variants as novel causative mutations for familial Parkinsons disease.

genetics↗

bioRxiv2021

Cell cycle-independent integration of stress signals promotes Non-G1/G0 cellular quiescence

Cellular quiescence is a non-proliferative state required for cell survival under stress and during development. In most quiescent cells, proliferation is stopped in a reversible state of low Cdk1 kinase activity; in many organisms, however, quiescent states with high Cdk1 activity can also be established through still uncharacterized stress or developmental mechanisms. Here, we used a microfluidics approach coupled to phenotypic classification by machine learning to identify stress pathways associated with starvation-triggered high-Cdk1 quiescent states in Saccharomyces cerevisiae. We found that low- and high-Cdk1 quiescent states shared a core of stress-associated processes, such as autophagy, protein aggregation, and mitochondrial upregulation, but differed in the nuclear accumulation of the stress transcription factors Xbp1, Gln3, and Sfp1. The decision between low- or high-Cdk1 quiescence was controlled by cell cycle-independent accumulation of Xbp1, which acted as a time-delayed integrator of the duration of stress stimuli. Our results show how cell cycle-independent stress-activated factors promote cellular quiescence outside of G1/G0.

cell biology↗

bioRxiv2021

Landscape connectivity alters the evolution of density-dependent dispersal during pushed range expansions

AO_SCPLOWBSTRACTC_SCPLOWAs human influence reshapes communities worldwide, many species expand or shift their ranges as a result, with extensive consequences across levels of biological organization. Range expansions can be ranked on a continuum going from pulled dynamics, in which low-density edge populations provide the "fuel" for the advance, to pushed dynamics in which high-density rear populations "push" the expansion forward. While theory suggests that evolution during range expansions could lead pushed expansions to become pulled with time, empirical comparisons of phenotypic divergence in pushed vs. pulled contexts are lacking. In a previous experiment using Trichogramma brassicae wasps as a model, we showed that expansions were more pushed when connectivity was lower. Here we used descendants from these experimental landscapes to look at how the range expansion process and connectivity interact to shape phenotypic evolution. Interestingly, we found no clear and consistent phenotypic shifts, whether along expansion gradients or between reference and low connectivity replicates, when we focused on low-density trait expression. However, we found evidence of changes in density-dependence, in particular regarding dispersal: populations went from positive to negative density-dependent dispersal at the expansion edge, but only when connectivity was high. As positive density-dependent dispersal leads to pushed expansions, our results confirm predictions that evolution during range expansions may lead pushed expansions to become pulled, but add nuance by showing landscape conditions may slow down or cancel this process. This shows we need to jointly consider evolution and landscape context to accurately predict range expansion dynamics and their consequences.

ecology↗

bioRxiv2021

ALG-097111, a potent and selective SARS-CoV-2 3-chymotrypsin-like cysteine protease inhibitor exhibits in vivo efficacy in a Syrian Hamster model

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY There is an urgent need for antivirals targeting the SARS-CoV-2 virus to fight the current COVID-19 pandemic. The SARS-CoV-2 main protease (3CLpro) represents a promising target for antiviral therapy. The lack of selectivity for some of the reported 3CLpro inhibitors, specifically versus cathepsin L, raises potential safety and efficacy concerns. ALG-097111 potently inhibited SARS-CoV-2 3CLpro (IC50 = 7 nM) without affecting the activity of human cathepsin L (IC50 > 10 M). When ALG-097111 was dosed in hamsters challenged with SARS-CoV-2, a robust and significant 3.5 log10 (RNA copies/mg) reduction of the viral RNA copies and 3.7 log10 (TCID50/mg) reduction in the infectious virus titers in the lungs was observed. These results provide the first in vivo validation for the SARS-CoV-2 3CLpro as a promising therapeutic target for selective small molecule inhibitors.

microbiology↗

bioRxiv2020

Small-molecule antibiotic inhibitors of post-translational protein secretion

The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. The Sec preprotein export pathway is an attractive potential alternative target. It is essential for bacterial viability and includes components that are absent from eukaryotes. Here we used a new high throughput in vivo screen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, fourteen HTS secretion inhibitors (HSI), belonging to 8 structural classes, were identified (IC50 <60 M). The inhibitors were also evaluated as antibacterials against 19 Gram- and Gram+ bacterial species (including those from the WHO top pathogens list). Seven of them, HSI#6, 9; HSI#1, 5, 10 and HSI#12, 14 representing three structural families were microbicidals. HSI#6 was the most potent (IC50 of 0.4-8.7 M), against 13 species of both Gram- and Gram+ bacteria. HSI#1, 5, 9 and 10 inhibited viability of Gram+ bacteria with IC50 ~6.9-77.8 M. HSI#9, 12 and 14 inhibited viability of E. coli strains with IC50 <65 M. Moreover, HSI#1, 5 and 10 inhibited viability of an E. coli strain missing TolC to improve permeability with IC50 4-14 M, indicating their inability to penetrate the outer membrane. In vitro assays revealed that antimicrobial activity was not related to inhibition of the SecA component of the translocase and hence HSI molecules may target new unknown components that affect secretion. The results provide proof of principle for our approach, and new starting compounds for optimization.

microbiology↗

bioRxiv2019

CTCF controls imprinted gene activity at the mouse Dlk1-Dio3 and Igf2- H19 domains by modulating allele-specific sub-TAD structure

Mammalian genomic imprinting is essential for development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. Here, we compared chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints--the Igf2-H19 and the Dlk1-Dio3 domains--and assessed the involvement of the insulator protein CTCF. At both domains, CTCF binds the maternal allele of a differentially-methylated region (DMR), in addition to multiple instances of bi-allelic CTCF binding in their surrounding TAD (Topologically Associating Domain). On the paternal chromosome, bi-allelic CTCF binding alone is sufficient to structure a first level of sub-TAD organization. Maternal-specific CTCF binding at the DMRs adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal sub-TAD organisation. Genome-editing experiments at the Dlk1-Dio3 locus confirm that the maternal sub-TADs are essential during development to maintain the imprinted Dlk1 gene in an inactive state on the maternal chromosome.

molecular biology↗

bioRxiv2019

Probing Ligand and Cation Binding Sites in G-Quadruplex Nucleic Acids by Mass Spectrometry and Electron Photodetachment Dissociation Sequencing

Mass spectrometry provides exquisite detail on ligand and cation binding stoichiometries with a DNA target. The next important step is to develop reliable methods to determine the cation and ligand binding sites in each complex separated by the mass spectrometer. To circumvent the caveat of ligand derivatization for cross-linking, which may alter the ligand binding mode, we explored a tandem mass spectrometry (MS/MS) method that does not require ligand derivatization, and is therefore also applicable to localize metal cations. By obtaining more negative charge states for the complexes using supercharging agents, and by creating radical ions by electron photodetachment, oligonucleotide bonds become weaker than the DNA-cation or DNA-ligand noncovalent bonds upon collision-induced dissociation of the radicals. This electron photodetachment (EPD) method allows to locate the binding regions of cations and ligands by top-down sequencing of the oligonucleotide target. The very potent G-quadruplex ligands 360A and PhenDC3 were found to replace a potassium cation and bind close to the central loop of 4-repeat human telomeric sequences.

biophysics↗

bioRxiv2018

Dopamine regulates the exploration-exploitation trade-off in rats

In a volatile environment where rewards are uncertain, successful performance requires a delicate balance between exploitation of the best option and exploration of alternative choices. It has theoretically been proposed that dopamine controls this exploration-exploitation trade-off, specifically that the higher the level of tonic dopamine, the more exploitation is favored. We demonstrate here that there is a formal relationship between the rescaling of dopamine positive reward prediction errors and the exploration-exploitation trade-off in simple non-stationary multi-armed bandit tasks. We further show in rats performing such a task that systemically antagonizing dopamine receptors greatly increases the number of random choices without affecting learning capacities. Simulations and comparison of a set of different computational models (an extended Q-learning model, a directed exploration model, and a meta-learning model) fitted on each individual confirm that, independently of the model, decreasing dopaminergic activity does not affect learning rate but is equivalent to an increase in exploration rate. This study shows that dopamine could adapt the exploration-exploitation trade-off in decision making when facing changing environmental contingencies.

neuroscience↗

bioRxiv2018

Thermal Denaturation of DNA G-Quadruplexes and their Complexes with Ligands: Thermodynamic Analysis of the Multiple States Revealed by Native Mass Spectrometry

As the idea that G-quadruplex nucleic acid structures are involved in cellular processes is gaining support, it becomes important to develop ligands that specifically target G-quadruplexes. However, ligand design is complicated because there are multiple G-quadruplex target sequences, some sequences are polymorphic, and very few ligand-quadruplex structures in solution were solved to date. Further, structure alone does not reveal the driving forces for ligand binding. To know why a ligand binds, the thermodynamics of binding must be characterized. Electrospray mass spectrometry makes it possible to detect and quantify each specific stoichiometry in terms of number of strands, number of specific cations, and number of ligands, and thus allows one to simultaneously determine the equilibrium constants for the formation of each complex. We designed and built a temperature-controlled nano-electrospray source to monitor thermal denaturation by mass spectrometry (\"MS-melting\"). We studied the thermal denaturation of G-quadruplexes, including the c-myc promoter and several telomeric sequence variants, and their complexes with popular ligands (Phen-DC3, TrisQ, TMPyP4, Cu-ttpy). From the temperature dependence of the equilibrium constants, we determined the enthalpic and entropic contributions to the formation of each stoichiometric state. In absence of ligand, we untangled the potassium-induced G-quadruplex folding thermodynamics, one potassium ion at a time. The formation of each quartet-K+-quartet units is strongly enthalpy driven, with entropy penalty. In contrast, the formation of quartet-K+-triplet units is entropically driven. For this reason, such misfolded structures can become more abundant as the temperature increases. In the presence of ligands, mass spectrometry also revealed new states at intermediate temperatures. For example, even in cases where only a 1:1 (ligand:quadruplex) is observed at room temperature, a 2:1 complex predominates at intermediate temperatures. Mass spectrometry also makes it easy to distinguish ligand bound to the 2-quartet structures (containing 1 K+), the 3-quartet structures (containing 2 K+) and to the unfolded strand (no specific K+). We confirm that TrisQ binds preferably, but not exclusively, to 3-quartet structures, Phen-DC3 binds to a 2-quartet structure, while the porphyrin ligand TMPyP4 is characterized as non-selective, because it binds to all forms including the unfolded one. The thermodynamics of ligand binding to each form, one ligand at a time, provides unprecedented detail on the interplay between ligand binding and changes in G-quadruplex topology.\n\nTOC Graphics\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC=\"FIGDIR/small/370254_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (20K):\norg.highwire.dtl.DTLVardef@e624adorg.highwire.dtl.DTLVardef@171fa1dorg.highwire.dtl.DTLVardef@1dbc32eorg.highwire.dtl.DTLVardef@eb301f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2018

Differential Antiviral Activities of RSV Inhibitors in Human Airway Epithelium

We report the use of reconstituted 3D-human airway epithelium cells of bronchial origin (HuAEC) in an air-liquid interface to study respiratory syncytial virus (RSV) infection and to assess the efficacy of RSV inhibitors in (pre-)clinical development. RSV-A replicates efficiently in HuAEC and viral RNA is shed for weeks after infection. RSV infection reduces the ciliary beat frequency of the ciliated cells as of 4 days post infection, with complete ciliary dyskinesia observed by day 10. Treatment with RSV fusion inhibitors resulted in an antiviral effect only when added at the time of infection. In contrast, the use of replication inhibitors (both nucleoside and non-nucleosides) elicited a marked antiviral effect even when start of treatment was delayed until one or even three days after infection. Levels of the inflammation marker RANTES (mRNA) increased [~]200-fold in infected-untreated cultures (at three weeks post infection), but levels were comparable to those of uninfected cultures in the presence of PC-876, a RSV-replication inhibitor, demonstrating that an efficient antiviral treatment inhibits virus induced inflammation in this model. Overall, HuAEC offer a robust and physiologically relevant model to study RSV replication and to assess the efficacy of antiviral compounds.

microbiology↗