Biology subjects

Aggarwal, A.

Publications and source records attributed to Aggarwal, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Targeting an Olfactory Receptor Mitigates High Residual Platelet Reactivity and Arterial Thrombosis Through Actin Cytoskeleton Depolymerization

BackgroundDespite antiplatelet therapy, some patients remain at high risk for ischemic events due to medication non-responsiveness or High Residual Platelet Reactivity (HRPR). Our goal was to target an orphan G-Protein-Coupled Receptor (GPCR) on platelets belonging to the olfactory receptor family as a new antithrombotic strategy. MethodsUsing an engineered reporter cell line expressing Olfactory Receptor 2L13 (OR2L13) that was recently identified as an orphan GPCR to limit platelet reactivity, we performed a high-throughput screen (HST) of non-odorant bioactive compounds with counter-screen validation, followed by studies to determine changes in platelet function in healthy subjects and in patients with coronary artery disease and peripheral artery disease. Phospho-proteomics identified key signal transduction pathways, and a variety of ex vivo assays and in vivo functional studies examined the impact of an identified non-odorant compound on platelet signaling, platelet biomechanics, and thrombosis. ResultsWe identified 6 ligands specific for OR2L13 that function as receptor agonists, leading to the suppression of platelet aggregation and -granule exocytosis via P2Y12, PAR1, Thromboxane Receptor (TxR) and glycoprotein VI (GPVI) receptors, suggesting involvement of common downstream mediator. The lead OR2L13 agonist identified phosphorylates heat shock protein 27 (HSP27) and depolymerizes the platelet filamentous actin cytoskeleton which was further confirmed in a clot retraction assay (CCF0054500 clot area 70.6 vs. vehicle clot area 5.2, P<0.0001). This anti-thrombotic effect of the OR2L13 agonist was reversed by a HSP27 inhibitor (clot area 3.6, P<0.0001). In a murine cremaster arterial injury model, platelet accumulation at the injury site was reduced by 88.9% by the lead OR2L13 agonist compared to vehicle (P<0.0003) without altering fibrin generation in vivo, or interfering with the coagulation cascade, and without impairing the protective mechanism of platelet hemostasis. ConclusionWe describe and characterize the first non-olfactory tool to target an olfactory receptor for the purpose of inhibiting platelet activation and thrombosis through downstream HSP27 in a comprehensive investigation using a first-of-its kind platelet inhibitor targeting an orphan platelet GPCR.

physiology↗

bioRxiv2024

Mitochondrial calcium uniporter regulates human fibroblast-like synoviocytes invasion via altering mitochondrial dynamics and dictates rheumatoid arthritis pathogenesis

Rheumatoid arthritis (RA) is characterized by the aggressive migration and invasion of fibroblast-like synoviocytes (FLS) into cartilage and bone, a process that is significantly influenced by mitochondrial calcium uptake. This study highlights the critical role of mitochondrial calcium uniporter (MCU) in regulating FLS migration and mitochondrial dynamics, and its potential as a therapeutic target in RA. Notably, RA-FLS exhibited increased MCU expression and mitochondrial dysfunction compared to controls. Treatment with Ru360, a potent MCU inhibitor significantly reduced RA-FLS migration, calcium influx, and mitochondrial reactive oxygen species (ROS) levels, while restoring mitochondrial morphology and enhancing ATP production. Further analysis of MCU complex expression revealed elevated levels of MCU and other regulatory subunits (EMRE, MICU1, MICU2) in RA-FLS compared to controls, indicating mitochondrial dysfunction in RA. Mechanistically, MCU inhibition altered gene expression related to cytoskeletal dynamics, focal adhesion, and metabolic pathways. In human RA-FLS, MCU inhibition suppressed migration and invasion both in vitro and in vivo. The interaction between MCU and Miro1, essential for mitochondrial transport, was validated, and its disruption impaired FLS migration. Our findings highlight MCU as a promising therapeutic target to inhibit FLS migration and ameliorate RA progression.

immunology↗

bioRxiv2024

A novel variant in ADPRS disrupts ARH3 stability and subcellular localization in childrenwith neurodegeneration and respiratory failure

PurposeADP-ribosylation is a post-translational modification involving the transfer of one or more ADP-ribose units from NAD+ to target proteins. Dysregulation of ADP-ribosylation is implicated in neurodegenerative diseases. Here we report a novel homozygous variant in the ADPRS gene (c.545A>G, p.His182Arg) encoding the mono(ADP-ribosyl) hydrolase ARH3 found in 2 patients with childhood-onset neurodegeneration with stress-induced ataxia and seizures (CONDSIAS). MethodsGenetic testing via exome sequencing was used to identify the underlying disease cause in two siblings with developmental delay, seizures, progressive muscle weakness, and respiratory failure following an episodic course. Studies in a cell culture model uncover biochemical and cellular consequences of the identified genetic change. ResultsThe ARH3H182R variant affects a highly conserved residue in the active site of ARH3, leading to protein instability, degradation, and reduced expression. ARH3H182R additionally fails to localize to the nucleus. The combination of reduced expression and mislocalization of ARH3H182R resulted in accumulation of mono-ADP ribosylated species in cells. ConclusionsThe childrens clinical course combined with the biochemical characterization of their genetic variant develops our understanding of the pathogenic mechanisms driving CONDSIAS and highlights a critical role for ARH3-regulated ADP ribosylation in nervous system integrity.

molecular biology↗

bioRxiv2024

Burkholderia cenocepacia epigenetic regulator M.BceJIV simultaneously engages two DNA recognition sequences for methylation

Burkholderia cenocepacia is an opportunistic and infective bacterium containing an orphan DNA methyltransferase (M.BceJIV) with roles in regulating gene expression and motility of the bacterium. M.BceJIV recognizes a GTWWAC motif (where W can be an adenine or a thymine) and methylates the N6 of the adenine at the fifth base position (GTWWAC). Here, we present a high-resolution crystal structure of M.BceJIV/DNA/sinefungin ternary complex and allied biochemical, computational, and thermodynamic analyses. Remarkably, the structure shows not one, but two DNA substrates bound to the M.BceJIV dimer, wherein each monomer contributes to the recognition of two recognition sequences. This unexpected mode of DNA binding and methylation has not been observed previously and sets a new precedent for a DNA methyltransferase. We also show that methylation at two recognition sequences occurs independently, and that GTWWAC motifs are enriched in intergenic regions of a strain of B. cenocepacias genome. We further computationally assess the interactions underlying the affinities of different ligands (SAM, SAH, and sinefungin) for M.BceJIV, as a step towards developing selective inhibitors for limiting B. cenocepacia infection.

biophysics↗

bioRxiv2023

Machine Learning Elucidates Design Features of Plasmid DNA Lipid Nanoparticles for Cell Type-Preferential Transfection

For cell and gene therapies to become more broadly accessible, it is critical to develop and optimize non-viral cell type-preferential gene carriers such as lipid nanoparticles (LNPs). Despite the effectiveness of high throughput screening (HTS) approaches in expediting LNP discovery, they are often costly, labor-intensive, and often do not provide actionable LNP design rules that focus screening efforts on the most relevant chemical and formulation parameters. Here we employed a machine learning (ML) workflow using well-curated plasmid DNA LNP transfection datasets across six cell types to maximize chemical insights from HTS studies and has achieved predictions with 5-9% error on average depending on cell type. By applying Shapley additive explanations to our ML models, we unveiled composition-function relationships dictating cell type-preferential LNP transfection efficiency. Notably, we identified consistent LNP composition parameters that enhance in vitro transfection efficiency across diverse cell types, such as ionizable to helper lipid ratios near 1:1 or 10:1 and the incorporation of cationic/zwitterionic helper lipids. In addition, several parameters were found to modulate cell type-preferentiality, including the ionizable and helper lipid total molar percentage, N/P ratio, cholesterol to PEGylated lipid ratio, and the chemical identity of the helper lipid. This study leverages HTS of compositionally diverse LNP libraries and ML analysis to understand the interactions between lipid components in LNP formulations; and offers fundamental insights that contribute to the establishment of unique sets of LNP compositions tailored for cell type-preferential transfection.

bioengineering↗

bioRxiv2023

A blue-shifted genetically encoded Ca2+ indicator with enhanced two-photon absorption

SignificanceGenetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy. AimWe describe the development and applications of T-GECO1 - a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1. ApproachWe used protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices. ResultsThe Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant. ConclusionT-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

neuroscience↗

bioRxiv2023

TMPRSS2 activation of Omicron lineage Spike glycoproteins is regulated by TMPRSS2 cleavage of ACE2

Continued high-level spread of SARS-CoV-2 has enabled an accumulation of changes within the Spike glycoprotein, leading to resistance to neutralising antibodies and concomitant changes to entry requirements that increased viral transmission fitness. Herein, we demonstrate a significant change in angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) dependent entry by primary SARS-CoV-2 isolates that occurred upon arrival of Omicron lineages. Mechanistically we show this shift to be a function of two distinct ACE2 pools based on TMPRS22 association with the ACE2 Collectrin-Like Domain (CLD). In engineered cells overexpressing ACE2 and TMPRSS2, ACE2/TMPRSS2 complexes led to either augmentation or attenuation of viral infectivity of pre-Omicron and Omicron lineages, respectively. Mutagenesis of the ACE2-CLD TMPRSS2 cleavage site in ACE2 restored infectivity across all Omicron lineages through enabling ACE2 binding that facilitated TMPRSS2 activation of viral fusion. Our data supports the evolution of Omicron lineages towards the use of ACE2 unable to form complexes with TMPRSS2 and consistent with ACE2 structure and function as a chaperone for many tissue specific amino acid transport proteins. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/558930v4_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1c86cf0org.highwire.dtl.DTLVardef@16815feorg.highwire.dtl.DTLVardef@7be9e1org.highwire.dtl.DTLVardef@137de1c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO ACE2-TMPRSS2 pool model and evolution of SARS-CoV-2 tropism.A. &-B. ACE2-TMPRSS2 pool model to reconcile the evolving entry requirements of SARS-CoV-2 and changes in viral tropism in vivo. A. Both SARS-CoV-1 and early SARS-CoV-2 (pre-Omicron) lineages have molecular dual tropism, with efficient entry when ACE2 (blue protein) can form complexes with TMPRSS2 (green protein) and in settings where TMPRSS2 is excluded from ACE2 (C4-ACE2 CLD). In both settings ACE2 initially engages SARS-CoV-2 spike and fusion is then triggered through TMPRSS2 cleavage of the Spike S2 domain B. Over time, the dual tropism for two distinct pools of ACE2 (with and without TMPRSS2) has been lost, with consolidation towards ACE2 where TMPRSS2 is no longer in a complex. With the arrival of Omicron lineages, ACE2-TMPRSS2 complexes could no longer enable efficient Spike S2 cleavage and fusogenic activation (TMPRSS2 "off" confirmation"). Rather, only TMPRSS2 uncoupled from ACE2 could facilitate the latter cleavage of S2. C. Over time this has further consolidated over generations of omicron lineages from 2022 lineages (BA.1, BA.2 and BA.5) through to 2023 lineages (XBB.1.5) and now in 2024 JN.1 lineages such as KP.3. Overall, this supports the initial molecular tropism of early SARS-CoV-2 clades to be similar to that observed for SARS-CoV-1, with dual tropism across both ACE2 pools and replication proceeding in tissues where ACE2-TMPRSS2 complexes would be prevalent (e.g. Lung). The evolution away from ACE2-TMPRSS2 complexes towards ACE2 where TMPRSS2 is structurally uncoupled (e.g. ACE2 as a chaperone for solute carriers SLCA619 or SLCA620) is consistent selection of this ACE2 pool in a manner that has sustained transmission fitness within the human population. C_FIG

microbiology↗

bioRxiv2023

A 3D microtumour system that faithfully represents ovarian cancer minimal residual disease

BackgroundBulk cancer and minimal residual disease (MRD) are characterised by different molecular drivers and therefore necessitate different therapeutic strategies. However, there are currently no 3D models that can faithfully recapitulate MRD ex vivo for therapy development. MethodsA microfluidic technique was implemented to construct 3D microtumours, in which tumour cells, either by themselves or with fibroblasts, were encapsulated in viscous hydrogels. The 3D microtumours were analysed for their response to first-line chemotherapeutics and characterised through RNA-Seq, by comparing them to both 2D cultures and clinical samples. ResultsOur microfluidic platform guarantees the fabrication of 3D microtumours of tailorable size and cell content, which recreate key features of tumours such as hypoxia, characteristic organization of the cytoskeleton and a dose-response to chemotherapeutics close to the physiological range. The 3D microtumours were also used to examine non-genetic heterogeneity in ovarian cancer and could fully reflect the recently described "Oxford Classic" five molecular signatures. The gene expression profile of 3D microtumours following chemotherapy treatment closely resembled that of MRD in ovarian cancer patients, showing the upregulation of genes involved in fatty acid metabolism. We demonstrate that these 3D microtumours are ideal for drug development by showing how they support the identification of a promising inhibitor of fatty acid oxidation, perhexiline, which specifically targets chemotherapy-resistant MRD ovarian cancer cells and not bulk cancer cells. ConclusionWe have obtained the first 3D model of ovarian cancer MRD by using microtumours generated through microfluidics. This system is ideal for high-throughput drug screening and, given its versatility, it can be readily extended to additional types of cancer, as well as accommodate multiple cell types to generate complex tumour microenvironments.

cancer biology↗

bioRxiv2023

Glycoprotein VI is Critical for the Detection and Progression of Abdominal Aortic Aneurysms

A common feature in patients with abdominal aortic aneurysms (AAA) is the formation of a nonocclusive intraluminal thrombus (ILT) in regions of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation impacts the pathogenesis of AAA. Using RNA-sequencing, we identify that the platelet-associated transcripts are significantly enriched in the ILT compared to the adjacent aneurysm wall and healthy control aortas. We found that the platelet specific receptor glycoprotein VI (GPVI) is among the top enriched genes in AAA ILT and is increased on the platelet surface of AAA patients. Examination of a specific indicator of platelet activity, soluble GPVI (sGPVI), in two independent AAA patient cohorts is highly predictive of a AAA diagnosis and associates more strongly with aneurysm growth rate when compared to D-dimer in humans. Finally, intervention with the anti-GPVI antibody (J) in mice with established aneurysms blunted the progression of AAA in two independent mouse models. In conclusion, we show that levels of sGPVI in humans can predict a diagnosis of AAA and AAA growth rate, which may be critical in the identification of high-risk patients. We also identify GPVI as a novel platelet-specific AAA therapeutic target, with minimal risk of adverse bleeding complications, where none currently exist. KEY POINTSO_LISoluble glycoprotein VI, which is a platelet-derived blood biomarker, predicts a diagnosis of AAA, with high sensitivity and specificity in distinguishing patients with fast from slow-growing AAA. C_LIO_LIBlockade of glycoprotein VI in mice with established aneurysms reduces AAA progression and mortality, indicating therapeutic potential. C_LI

pathology↗

bioRxiv2023

An engineered biosensor enables dynamic aspartate measurements in living cells

Intracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a GFP-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by mass spectrometry and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high throughput applications of variables that affect aspartate levels.

biochemistry↗

bioRxiv2023

Dysregulated Platelet Function and Thrombosis in Patients with Post-Acute Sequelae of COVID-19

BackgroundPost-acute sequelae of COVID-19 (PASC), also referred as Long-COVID, sometimes follows COVID-19, a disease caused by SARS-CoV-2. While SARS-CoV-2 is well-known to promote a prothrombotic state, less is known about the thrombosis risk in PASC. AimOur objective was to evaluate the platelet function and thrombotic potential in patients following recovery from SARS-CoV-2 with clear symptoms of PASC. MethodsPASC patients and matched healthy controls were enrolled in the study on average 15 months after documented SARS-CoV-2 infection. Platelet activation was evaluated by Light Transmission Aggregometry (LTA) and flow cytometry in response to platelet surface receptor agonists. Thrombosis in platelet-deplete plasma was evaluated by Factor Xa activity. A microfluidics system assessed thrombosis in whole blood under shear stress conditions. ResultsA mild increase in platelet aggregation in PASC patients through the thromboxane receptor was observed and platelet activation through the glycoprotein VI (GPVI) receptor was decreased in PASC patients compared to age- and sex-matched healthy controls. Thrombosis under shear conditions as well as Factor Xa activity were reduced in PASC patients. Plasma from PASC patients was an extremely potent activator of washed, healthy platelets - a phenomenon not observed when stimulating healthy platelets after incubation with plasma from healthy individuals. ConclusionsPASC patients show dysregulated responses in platelets and coagulation in plasma, likely caused by a circulating molecule that promotes thrombosis. A hitherto undescribed protective response appears to exists in PASC patients to counterbalance ongoing thrombosis that is common to SARS-CoV-2 infection.

pharmacology and toxicology↗

bioRxiv2023

Neural assemblies coordinated by cortical waves are associated with waking and hallucinatory brain states

The relationship between sensory stimuli and perceptions is brain-state dependent: in wakefulness stimuli evoke perceptions; under anesthesia perceptions are abolished; during dreaming and in dissociated states, percepts are internally generated. Here, we exploit this state dependence to identify brain activity associated with internally generated or stimulus-evoked perception. In awake mice, visual stimuli phase reset spontaneous cortical waves to elicit 3-6 Hz feedback traveling waves. These stimulus-evoked waves traverse the cortex and entrain visual and parietal neurons. Under anesthesia and during ketamine-induced dissociation, visual stimuli do not disrupt spontaneous waves. Uniquely in the dissociated state, spontaneous waves traverse the cortex caudally and entrain visual and parietal neurons, akin to stimulus-evoked waves in wakefulness. Thus, coordinated neuronal assemblies orchestrated by traveling cortical waves emerge in states in which perception can manifest. The awake state is privileged in that this coordination is elicited by specifically by external visual stimuli.

neuroscience↗

bioRxiv2023

Migration and establishment of progenitor pool of melanocytes is governed by SEMA3E-PLXND1 signaling

Vertebrate pigmentation is an outcome of an interplay of several signaling pathways that result in immense diversity of pigment patterns observed across the animal kingdom. Transitory nature of these signaling events impedes deciphering pathways that control migration and establishment of melanocyte stem cells (McSC), necessary for pigment patterning. Using zebrafish and cultured mammalian melanocytes, we uncover a hitherto unknown role for Plexin D1 signaling. This pathway directs migration by F-actin modulation and further dictates subsequent functional states of melanocytes through a transcriptional response. In zebrafish, abrogation of PLXND1 derails melanocyte migration and reduces mid-line melanophores emerging from the regeneration competent McSC pool. In cultured melanocytes, activation of PLXND1 by the ligand semaphorin 3E reduces the velocity of migration, influences directional correlation, and promotes movement towards positive cues such as SCF. PLXND1 activation results in EGFR signaling necessary for McSC establishment, and induces GNAS, an effector of MC1R pathway involved in melanocyte maturation. Identification of this long-range secreted negative chemotactic signaling provides a missing player and enriches reaction diffusion model for pigment patterning.

developmental biology↗

bioRxiv2023

P-Selectin promotes SARS-CoV-2 interactions with platelets and the endothelium

COVID-19 causes a clinical spectrum of acute and chronic illness and host / virus interactions are not completely understood1,2. To identify host factors that can influence SARS-CoV-2 infection, we screened the human genome for genes that, when upregulated, alter the outcome of authentic SARS-CoV-2 infection. From this, we identify 34 new genes that can alter the course of infection, including the innate immune receptor P-selectin, which we show is a novel SARS-CoV-2 spike receptor. At the cellular level expression of P-selectin does not confer tropism for SARS-CoV-2, instead it acts to suppress infection. More broadly, P-selectin can also promote binding to SARS-CoV-2 variants, SARS-CoV-1 and MERS, acting as a general spike receptor for highly pathogenic coronaviruses. P-selectin is expressed on platelets and endothelium3, and we confirm SARS-CoV-2 spike interactions with these cells are P-selectin-dependent and can occur under shear flow conditions. In vivo, authentic SARS-CoV-2 uses P-selectin to home to airway capillary beds where the virus interacts with the endothelium and platelets, and blocking this interaction can clear vascular-associated SARS-CoV-2 from the lung. Together we show for the first time that coronaviruses can use the leukocyte recruitment system to control tissue localization, and this fundamental insight may help us understand and control highly pathogenic coronavirus disease progression.

immunology↗

bioRxiv2022

Improved genetically encoded fluorescent biosensors for monitoring of intra- and extracellular L-lactate

O_SCPLOWLC_SCPLOW-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. To enable investigations of both the inter- and intra-cellular dynamics of O_SCPLOWLC_SCPLOW-Lactate, we develop a second-generation green fluorescent extracellular O_SCPLOWLC_SCPLOW-Lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular O_SCPLOWLC_SCPLOW-Lactate biosensor, designated R-iLACCO1. Compared to the first-generation eLACCO1.1 ({Delta}F/F = 1.5 in cultured neurons), eLACCO2.1 exhibits better membrane localization and fluorescence response ({Delta}F/F = 8.1 in cultured neurons) with faster response kinetics to extracellular O_SCPLOWLC_SCPLOW-Lactate on the surface of live mammalian cells. R-iLACCO1 and its affinity variants exhibit large fluorescence responses to changes in O_SCPLOWLC_SCPLOW-Lactate concentration in vitro ({Delta}F/F = 15 to 22) and in live mammalian cells ({Delta}F/F = 5.5 to 11). We demonstrate that these biosensors enable cellular-resolution imaging of extracellular and intracellular O_SCPLOWLC_SCPLOW-Lactate in cultured mammalian cells.

molecular biology↗

bioRxiv2022

A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used mRNA display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor binding domain, N-terminal domain and S2 region, distal to the ACE2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target. Significance statementThis study reports on the discovery of a macrocyclic peptide that is able to inhibit SARS-CoV-2 infection by exploiting a new vulnerable site in the spike glycoprotein. This region is highly conserved across SARS-CoV-2 variants and the subgenus sarbecovirus. Due to the inaccessability and mutational contraint of this site, it is anticipated to be resistant to the development of resistance through antibody selective pressure. In addition to the discovery of a new molecule for development of potential new peptide or biomolecule therapeutics, the discovery of this broadly active conserved site can also stimulate a new direction of drug development, which together may prevent future outbreaks of related viruses.

biochemistry↗

bioRxiv2022

Enhanced stability of the SARS CoV-2 spike glycoprotein trimer following modification of an alanine cavity in the protein core.

The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity formed by Ala1016 and Ala1020 that form part of the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. The trimer stabilizing effects of filling the Ala1016/Ala1020 cavity was linked to improved S glycoprotein membrane fusion function. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited very high titers of neutralizing antibodies to ancestral and Delta-derived viruses (1/2,700-1/5,110), while neutralization titer was somewhat reduced with Omicron BA.1 (1/210-1,1744). The antigens elicited antibody specificities that could compete with ACE2-Fc for binding to the receptor-binding motif (RBM) and NAbs directed to key neutralization epitopes within the receptor-binding domain (RBD), N-terminal domain (NTD) and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S ectodomain trimers in the absence of an external trimerization motif (T4 foldon). The VI mutation represents a method for producing an intrinsically stable trimeric S ectodomain glycoprotein vaccine in the absence of a foreign trimerization tag. AUTHOR SUMMARYFirst-generation SARS CoV-2 vaccines that generate immune responses to ancestral Spike glycoprotein sequences have averted at least 14.4 million deaths, but their effectiveness against the recently emerged Omicron lineages is reduced. The updating of booster vaccines with variant Spike sequences are therefore likely required to maintain immunity as the pandemic continues to evolve. The Spike is a trimeric integral membrane protein with a membrane spanning sequence at its C-terminus. The Spike protein-based vaccine that is currently licensed for human use is produced by a complex process that reconstitutes the Spike in an artificial membrane. Alternatively, production of the Spike trimer as a soluble protein generally requires replacement of the membrane spanning sequence with a foreign often highly immunogenic trimerization motif that can complicate clinical advancement. We used systematic structure-directed mutagenesis coupled with functional studies to identify an alternative stabilization approach that negates the requirement for an external trimerization motif or membrane-spanning sequence. The replacement of 2 alanine residues that form a cavity in the core of the Spike trimer with bulkier hydrophobic residues resulted in increased Spike thermal stability. Thermostable Spike mutants retained major conserved neutralizing antibody epitopes and the ability to elicit broad and potent neutralizing antibody responses. One such mutation, referred to as VI, enabled the production of intrinsically stable Omicron variant Spike ectodomain trimers in the absence of an external trimerization motif. The VI mutation potentially enables a simplified method for producing a stable trimeric S ectodomain glycoprotein vaccine.

microbiology↗

bioRxiv2022

Cellular, Molecular, and Enzymatic Signatures ofThrombi are Vascular Bed-Dependent

BackgroundThe contribution of arterial and venous thrombi to vascular remodeling is unclear. While catheter-extraction of thrombus in cerebrovascular accident (CVA) is time-sensitive, similar urgency is rare in managing venous thromboembolism (VTE). ObjectivesOur goal was to determine molecular cellular signatures of thrombus extracted by catheter from various vascular beds to gain insight into vascular remodeling. MethodsTwenty-five patients underwent catheter-directed thrombectomy (CDT), 13 for acute CVA, 8 for pulmonary embolism (PE), and 4 for deep vein thrombosis (DVT). Protein and RNA extracted from thrombus was evaluated by immunoblotting and sequencing, respectively. Thrombus-derived enzymes for which substrate is present in the blood vessel wall were examined for enzymatic activity. ResultsTime from symptom onset to thrombus extraction was 7.7 {+/-} 1.9 hours for CVA and 109 {+/-} 55 hours for VTE. Protein concentration, white blood cell and red blood cell content were all greater in venous compared with arterial thrombus while platelet content was similar. Both venous and arterial thrombus contained multiple Matrix Metalloproteinase (MMP) isoforms. MMP9 specific activity was greater in venous than in arterial thrombus (57 {+/-} 6 ng/mL.g protein-1 vs. 24 {+/-} 8 ng/mL.g protein-1, P=0.0051). ConclusionsArterial and venous thrombus have dissimilar phenotypes, each with biologically-active enzymes known to remodel blood vessels, and enzymatic activity proportional to the white blood cell content which increases with thrombus age. These data suggest a mechanistically-important role for early CDT to avoid the consequences of irreversible vascular remodeling. Condensed AbstractEmergent extraction of acute thrombus from arterial vascular beds restores limb and end-organ perfusion and is widely-accepted to be the standard of care. Extraction of thrombus from venous vascular beds, however, is rarely considered urgent, even though many patients subsequently develop debilitating symptoms. By capitalizing on privileged thrombus extracted from multiple vascular beds, we gained mechanistic insight regarding the cellular composition and cell-derived enzymes secreted from thrombus that may remodel the vessel wall. This study shows thrombi are biologically-active entities, continuously recruiting circulating cells that secrete enzymes both proportional to thrombus age and the time of patient presentation.

biochemistry↗