Native Mass Spectrometry Dissects the Structural Dynamics of an Allosteric Heterodimer of SARS-CoV-2 Nonstructural Proteins.

Structure-based drug design, which relies on precise understanding of the target protein and its interaction with the drug candidate, is dramatically expedited by advances in computational methods for candidate prediction. Yet, the accuracy needs to be improved with more structural data from high throughput experiments, which are challenging to generate, especially for dynamic and weak associations. Herein, we applied native mass spectrometry (native MS) to rapidly characterize ligand binding of an allosteric heterodimeric complex of SARS-CoV-2 nonstructural proteins (nsp) nsp10 and nsp16 (nsp10/16), a complex essential for virus survival in the host and thus a desirable drug target. Native MS showed that the dimer is in equilibrium with monomeric states in solution. Consistent with the literature, well characterized small cosubstrate, RNA substrate, and product bind with high specificity and affinity to the dimer but not the free monomers. Unsuccessfully designed ligands bind indiscriminately to all forms. Using neutral gas collision, the nsp16 monomer with bound cosubstrate can be released from the holo dimer complex, confirming the binding to nsp16 as revealed by the crystal structure. However, we observed an unusual migration of the endogenous zinc ions bound to nsp10 to nsp16 after collisional dissociation. The metal migration can be suppressed by using surface collision with reduced precursor charge states, which presumably resulted in minimal gas-phase structural rearrangement and highlighted the importance of complementary techniques. With minimal sample input (∼µg), native MS can rapidly detect ligand binding affinities and locations in dynamic multisubunit protein complexes, demonstrating the potential of an "all-in-one" native MS assay for rapid structural profiling of protein-to-AI-based compound systems to expedite drug discovery.

Self-Sorting Collagen Heterotrimers.

Nature uses elaborate methods to control protein assembly, including that of heterotrimeric collagen. Here, we established design principles for the composition and register-selective assembly of synthetic collagen heterotrimers. The assembly code enabled the self-sorting of eight different strands into three─out of 512 possible─triple helices via complementary (4S)-aminoproline and aspartate residues. Native ESI-MS corroborated the specific assembly into coexisting heterotrimers.

PIP aquaporin pH-sensing is regulated by the length and charge of the C-terminal region.

Plant PIP aquaporins play a central role in controlling plant water status. The current structural model for PIP pH-gating states that the main pH sensor is located in loopD and that all the mobile cytosolic elements participate in a complex interaction network that ensures the closed structure. However, the precise participation of the last part of the C-terminal domain (CT) in PIP pH gating remains unknown. This last part has not been resolved in PIP crystal structures and is a key difference between PIP1 and PIP2 paralogues. Here, by a combined experimental and computational approach, we provide data about the role of CT in pH gating of Beta vulgaris PIP. We demonstrate that the length of CT and the positive charge located among its last residues modulate the pH at which the open/closed transition occurs. We also postulate a molecular-based mechanism for the differential pH sensing in PIP homo- or heterotetramers by performing atomistic molecular dynamics simulations (MDS) on complete models of PIP tetramers. Our findings show that the last part of CT can affect the environment of loopD pH sensors in the closed state. Results presented herein contribute to the understanding of how the characteristics of CT in PIP channels play a crucial role in determining the pH at which water transport through these channels is blocked, highlighting the relevance of the differentially conserved very last residues in PIP1 and PIP2 paralogues.

Molecular architecture of black widow spider neurotoxins.

Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, ß, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca2+-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small ß-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca2+ ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.

The phage defence island of a multidrug resistant plasmid uses both BREX and type IV restriction for complementary protection from viruses.

Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in 'defence islands', providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.

Influence of the macromolecular crowder alginate in the fibrillar organization of the functional amyloid FapC from Pseudomonas aeruginosa.

Bacterial biofilms are an alternative lifestyle in which communities of bacteria are embedded in an extracellular matrix manly composed by polysaccharides, nucleic acids and proteins, being the hallmark of bacterial survival in a variety of ecological niches. Amyloid fibrils are one of the proteinaceous components of such extracellular crowded environments. FapC is the main component of the functional amyloid recently discovered in Pseudomonas species, including the opportunistic pathogen P. aeruginosa, which is a major cause of nosocomial infections and contamination of medical devices. Considering that several functional roles have been attributed to this bacterial amyloid, FapC emerged as a novel target to control Pseudomonas biofilm formation and to design new treatments against chronic infections. In this study, we used complementary biophysical techniques to evaluate conformational signatures of FapC amyloids formed in the presence of alginate, the major exopolysaccharide associated with the mucoid phenotype of P. aeruginosa strains isolated from cystic fibrosis patients. We found that the this naturally occurring macromolecular crowder leads to morphological similar yet polymorphic FapC fibrils, highlighting the importance of considering the complexity of the extracellular matrix in order to improve our understanding of microbial functional amyloids.

Studies towards the Design and Synthesis of Novel 1,5-Diaryl-1H-imidazole-4-carboxylic Acids and 1,5-Diaryl-1H-imidazole-4-carbohydrazides as Host LEDGF/p75 and HIV-1 Integrase Interaction Inhibitors.

Two targeted sets of novel 1,5-diaryl-1H-imidazole-4-carboxylic acids 10 and carbohydrazides 11 were designed and synthesized from their corresponding ester intermediates 17, which were prepared via cycloaddition of ethyl isocyanoacetate 16 and diarylimidoyl chlorides 15. Evaluation of these new target scaffolds in the AlphaScreenTM HIV-1 IN-LEDGF/p75 inhibition assay identified seventeen compounds exceeding the pre-defined 50% inhibitory threshold at 100 µM concentration. Further evaluation of these compounds in the HIV-1 IN strand transfer assay at 100 µM showed that none of the compounds (with the exception of 10a, 10l, and 11k, with marginal inhibitory percentages) were actively bound to the active site, indicating that they are selectively binding to the LEDGF/p75-binding pocket. In a cell-based HIV-1 antiviral assay, compounds 11a, 11b, 11g, and 11h exhibited moderate antiviral percentage inhibition of 33-45% with cytotoxicity (CC50) values of >200 µM, 158.4 µM, >200 µM, and 50.4 µM, respectively. The antiviral inhibitory activity displayed by 11h was attributed to its toxicity. Upon further validation of their ability to induce multimerization in a Western blot gel assay, compounds 11a, 11b, and 11h appeared to increase higher-order forms of IN.

Oral Administration of Resveratrol-Selenium-Peptide Nanocomposites Alleviates Alzheimer's Disease-like Pathogenesis by Inhibiting Aß Aggregation and Regulating Gut Microbiota.

Alzheimer's disease (AD) is a neurodegenerative disease associated with amyloid-ß (Aß) deposition, leading to neurotoxicity (oxidative stress and neuroinflammation) and gut microbiota imbalance. Resveratrol (Res) has neuroprotective properties, but its bioavailability in vivo is very low. Herein, we developed a small Res-selenium-peptide nanocomposite to enable the application of Res for eliminating Aß aggregate-induced neurotoxicity and mitigating gut microbiota disorder in aluminum chloride (AlCl3) and d-galactose(d-gal)-induced AD model mice. Res functional selenium nanoparticles (Res@SeNPs) (8 ± 0.34 nm) were prepared first, after which the surface of Res@SeNPs was decorated with a blood-brain barrier transport peptide (TGN peptide) to generate Res-selenium-peptide nanocomposites (TGN-Res@SeNPs) (14 ± 0.12 nm). Oral administration of TGN-Res@SeNPs improves cognitive disorder through (1) interacting with Aß and decreasing Aß aggregation, effectively inhibiting Aß deposition in the hippocampus; (2) decreasing Aß-induced reactive oxygen species (ROS) and increasing activity of antioxidation enzymes in PC12 cells and in vivo; (3) down-regulating Aß-induced neuroinflammation via the nuclear factor kappa B/mitogen-activated protein kinase/Akt signal pathway in BV-2 cells and in vivo; and (4) alleviating gut microbiota disorder, particularly with respect to oxidative stress and inflammatory-related bacteria such as Alistipes, Helicobacter, Rikenella, Desulfovibrio, and Faecalibaculum. Thus, we anticipate that Res-selenium-peptide nanocomposites will offer a new potential strategy for the treatment of AD.

NMR Spectroscopy of the Main Protease of SARS-CoV-2 and Fragment-Based Screening Identify Three Protein Hotspots and an Antiviral Fragment.

The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.

Identification of new IL-7Rα small-molecule agonists: a multi-computational approach.

Interleukin 7 (IL-7) is an essential cytokine that acts as a potent growth factor of T-cells and supports the growth of B-cell precursors. IL-7 binds to a heterodimeric receptor consisting of an IL-7 receptor alpha (IL-7Rα) and the common gamma chain receptor (γc) which is shared with IL-2, IL-4, IL-9, IL-15 and IL-21. The discovery of small-molecule agonists of cytokines would be of great pharmaceutical interest with the increasing scientific rationale. In this study, a series of molecular modelling methods, including field-based pharmacophore virtual screening, protein-protein docking and molecular dynamics simulations, led to the identification of two compounds (i.e. 1 and 2) of different classes that exhibit enhanced agonistic effects by activating the IL-7 signalling cascade. One of these compounds was selected as a hit and represents the first small-molecule agonist of IL-7Rα with single-digit micromolar activity. Moreover, the prediction model of the active compound to the IL-7Rα/γc interaction complex provides insight into the binding of a small-molecule agonist to its receptor.

Molecular noise filtering in the ß-adrenergic signaling network by phospholamban pentamers.

Phospholamban (PLN) is an important regulator of cardiac calcium handling due to its ability to inhibit the calcium ATPase SERCA. ß-Adrenergic stimulation reverses SERCA inhibition via PLN phosphorylation and facilitates fast calcium reuptake. PLN also forms pentamers whose physiological significance has remained elusive. Using mathematical modeling combined with biochemical and cell biological experiments, we show that pentamers regulate both the dynamics and steady-state levels of monomer phosphorylation. Substrate competition by pentamers and a feed-forward loop involving inhibitor-1 can delay monomer phosphorylation by protein kinase A (PKA), whereas cooperative pentamer dephosphorylation enables bistable PLN steady-state phosphorylation. Simulations show that phosphorylation delay and bistability act as complementary filters that reduce the effect of random fluctuations in PKA activity, thereby ensuring consistent monomer phosphorylation and SERCA activity despite noisy upstream signals. Preliminary analyses suggest that the PLN mutation R14del could impair noise filtering, offering a new perspective on how this mutation causes cardiac arrhythmias.

Multifunctional Selenium Quantum Dots for the Treatment of Alzheimer's Disease by Reducing Aß-Neurotoxicity and Oxidative Stress and Alleviate Neuroinflammation.

At present, the complex pathogenesis, the difficult-to-overcome blood-brain barrier (BBB), the development of the disease course which cannot be prevented, and other problems are serious challenges in the treatment of Alzheimer's disease (AD). In order to enhance the therapeutic effect of drugs through BBB, we synthesized simple and easy-to-obtain selenium quantum dots (SeQDs), with a multitarget therapeutic effect. This new type of SeQDs has an ultrasmall size and can quickly penetrate the BBB. According to the fluorescence characteristics of SeQDs, we can diagnose and track AD. The experimental results show that SeQDs have strong free-radical scavenging activity, protect cells from oxidative stress induced by different stimuli, and show broad-spectrum antioxidant activity. The SeQDs can not only effectively inhibit Aß aggregation and significantly reduce Aß-mediated cytotoxicity, thus preventing AD cascade reaction, but also effectively reduce tau protein phosphorylation by down-regulating PHF1 and CP13 and further reduce oxidative stress, restore mitochondrial functions, and maintain nerve cell stability and protect nerve cells from oxidative stress. In vivo studies demonstrate that SeQDs can continuously accumulate in the brain after rapid passage of BBB and can quickly alleviate AD, significantly improve the memory impairment of AD mice, and improve their learning and memory ability. Therefore, the use of SeQDs in the treatment of AD has great advantages compared with traditional single-target drugs and provides a new direction for the combination of prevention and treatment of neurodegenerative diseases.

Cooperative inhibition of SNARE-mediated vesicle fusion by α-synuclein monomers and oligomers.

The primary hallmark of Parkinson's disease (PD) is the generation of Lewy bodies of which major component is α-synuclein (α-Syn). Because of increasing evidence of the fundamental roles of α-Syn oligomers in disease progression, α-Syn oligomers have become potential targets for therapeutic interventions for PD. One of the potential toxicities of α-Syn oligomers is their inhibition of SNARE-mediated vesicle fusion by specifically interacting with vesicle-SNARE protein synaptobrevin-2 (Syb2), which hampers dopamine release. Here, we show that α-Syn monomers and oligomers cooperatively inhibit neuronal SNARE-mediated vesicle fusion. α-Syn monomers at submicromolar concentrations increase the fusion inhibition by α-Syn oligomers. This cooperative pathological effect stems from the synergically enhanced vesicle clustering. Based on this cooperative inhibition mechanism, we reverse the fusion inhibitory effect of α-Syn oligomers using small peptide fragments. The small peptide fragments, derivatives of α-Syn, block the binding of α-Syn oligomers to Syb2 and dramatically reverse the toxicity of α-Syn oligomers in vesicle fusion. Our findings demonstrate a new strategy for therapeutic intervention in PD and related diseases based on this specific interaction of α-Syn.

Plant-based, adjuvant-free, potent multivalent vaccines for avian influenza virus via Lactococcus surface display.

Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHAH5N6 + H9N2 BLP or a combination of tHAH5N6 BLP and tHAH9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.

Crystal structure of timothy grass allergen Phl p 12.0101 reveals an unusual profilin dimer.

Timothy grass pollen is a source of potent allergens. Among them, Phl p 1 and Phl p 5 are thought to be the most important, as a majority of timothy grass-allergic individuals have IgE antibodies directed against these two allergens. The profilin from timothy grass (Phl p 12) has been registered as a minor allergen, with up to 35% of individuals in populations of grass pollen allergic patients showing IgE binding to Phl p 12. Profilins are primarily minor allergens and are known for a high likelihood of co-sensitization as well as cross-reactivity situations caused by their sequence and structure similarity. The crystal structure of Phl p 12.0101 was determined and it revealed that this allergen may form an unusual dimer not previously observed among any profilins. For example, the Phl p 12 dimer has a completely different geometry and interface when compared with the latex profilin (Hev b 8) dimer that has its crystal structure determined. The structure of Phl p 12.0101 is described in the context of allergenic sensitization and allergy diagnostics. Moreover, the structure of the Phl p 12.0101 dimer is discussed, taking into account the production of recombinant allergens and their storage.

A Non-immunogenic Bivalent d-Protein Potently Inhibits Retinal Vascularization and Tumor Growth.

We report a general approach to engineering multivalent d-proteins with antibody-like activities in vivo. Mirror-image phage display and structure-guided design were utilized to create a d-protein that uses receptor mimicry to antagonize vascular endothelial growth factor A (VEGF-A). Selections against the d-protein form of VEGF-A using phage-displayed libraries of two different domain scaffolds yielded two proteins that bound distinct receptor interaction sites on VEGF-A. X-ray crystal structures of the d-protein/VEGF-A complexes were used to guide affinity maturation and to construct a heterodimeric d-protein VEGF-A antagonist with picomolar activity. The d-protein VEGF-A antagonist prevented vascular leakage in a rabbit eye model of wet age-related macular degeneration and slowed tumor growth in the MC38 syngeneic mouse tumor model with efficacies comparable to those of approved antibody drugs, and in contrast with antibodies, the d-protein was non-immunogenic during treatment and following subcutaneous immunizations.

A Multipronged Screening Approach Targeting Inhibition of ETV6 PNT Domain Polymerization.

ETV6 is an ETS family transcriptional repressor for which head-to-tail polymerization of its PNT domain facilitates cooperative binding to DNA by its ETS domain. Chromosomal translocations frequently fuse the ETV6 PNT domain to one of several protein tyrosine kinases. The resulting chimeric oncoproteins undergo ligand-independent self-association, autophosphorylation, and aberrant stimulation of downstream signaling pathways, leading to a variety of cancers. Currently, no small-molecule inhibitors of ETV6 PNT domain polymerization are known and no assays targeting PNT domain polymerization have been described. In this study, we developed complementary experimental and computational approaches for identifying such inhibitory compounds. One mammalian cellular approach utilized a mutant PNT domain heterodimer system covalently attached to split Gaussia luciferase fragments. In this protein-fragment complementation assay, inhibition of PNT domain heterodimerization reduces luminescence. A yeast assay took advantage of activation of the reporter HIS3 gene upon heterodimerization of mutant PNT domains fused to DNA-binding and transactivation domains. In this two-hybrid screen, inhibition of PNT domain heterodimerization prevents cell growth in medium lacking histidine. The Bristol University Docking Engine (BUDE) was used to identify virtual ligands from the ZINC8 library predicted to bind the PNT domain polymerization interfaces. More than 75 hits from these three assays were tested by nuclear magnetic resonance spectroscopy for binding to the purified ETV6 PNT domain. Although none were found to bind, the lessons learned from this study may facilitate future approaches for developing therapeutics that act against ETV6 oncoproteins by disrupting PNT domain polymerization.

Anemoside B4 Protects against Acute Lung Injury by Attenuating Inflammation through Blocking NLRP3 Inflammasome Activation and TLR4 Dimerization.

Acute lung injury (ALI) is an acute inflammatory process in the lung parenchyma. Anemoside B4 (B4) was isolated from Pulsatilla, a plant-based drug against inflammation and commonly applied in traditional Chinese medicine. However, the anti-inflammatory effect and the mechanisms of B4 are not clear. In this study, we explored the potential mechanisms and anti-inflammatory activity of B4 both in vitro and in vivo. The results indicated that B4 suppressed the expression of iNOS, COX-2, NLRP3, caspase-1, and IL-1ß. The ELISA assay results showed that B4 significantly restrained the release of inflammatory cytokines like TNF-α, IL-6, and IL-1ß in macrophage cells. In addition, B4 rescued mitochondrial membrane potential (MMP) loss in (lipopolysaccharide) LPS plus ATP stimulated macrophage cells. Co-IP and molecular docking results illustrated that B4 disrupted the dimerization of TLR4. For in vivo results, B4 exhibited a protective effect on LPS and bleomycin- (BLM-) induced ALI in mice through suppressing the lesions of lung tissues, the release of inflammatory cytokines, and the levels of white blood cells, neutrophils, and lymphoid cells in the blood. Collectively, B4 has a protective effect on ALI via blocking TLR4 dimerization and NLRP3 inflammasome activation, suggesting that B4 is a potential agent for the treatment of ALI.

Identification of novel inhibitors of the ABC transporter BmrA.

The resistance of microbes to commonly used antibiotics has become a worldwide health problem. A major underlying mechanism of microbial antibiotic resistance is the export of drugs from bacterial cells. Drug efflux is mediated through the action of multidrug resistance efflux pumps located in the bacterial cell membranes. The critical role of bacterial efflux pumps in antibiotic resistance has directed research efforts to the identification of novel efflux pump inhibitors that can be used alongside antibiotics in clinical settings. Here, we aimed to find potential inhibitors of the archetypical ATP-binding cassette (ABC) efflux pump BmrA of Bacillus subtilis via virtual screening of the Mu.Ta.Lig. Chemotheca small molecule library. Molecular docking calculations targeting the nucleotide-binding domain of BmrA were performed using AutoDock Vina. Following a further drug-likeness filtering step based on Lipinski's Rule of Five, top 25 scorers were identified. These ligands were then clustered into separate groups based on their contact patterns with the BmrA nucleotide-binding domain. Six ligands with distinct contact patterns were used for further in vitro inhibition assays based on intracellular ethidium bromide accumulation. Using this methodology, we identified two novel inhibitors of BmrA from the Chemotheca small molecule library.

VZHE-039, a novel antisickling agent that prevents erythrocyte sickling under both hypoxic and anoxic conditions.

Sickle cell disease (SCD) results from a hemoglobin (Hb) mutation ßGlu6 → ßVal6 that changes normal Hb (HbA) into sickle Hb (HbS). Under hypoxia, HbS polymerizes into rigid fibers, causing red blood cells (RBCs) to sickle; leading to numerous adverse pathological effects. The RBC sickling is made worse by the low oxygen (O2) affinity of HbS, due to elevated intra-RBC concentrations of the natural Hb effector, 2,3-diphosphoglycerate. This has prompted the development of Hb modifiers, such as aromatic aldehydes, with the intent of increasing Hb affinity for O2 with subsequent prevention of RBC sickling. One such molecule, Voxelotor was recently approved by U.S. FDA to treat SCD. Here we report results of a novel aromatic aldehyde, VZHE-039, that mimics both the O2-dependent and O2-independent antisickling properties of fetal hemoglobin. The latter mechanism of action-as elucidated through crystallographic and biological studies-is likely due to disruption of key intermolecular contacts necessary for stable HbS polymer formation. This dual antisickling mechanism, in addition to VZHE-039 metabolic stability, has translated into significantly enhanced and sustained pharmacologic activities. Finally, VZHE-039 showed no significant inhibition of several CYPs, demonstrated efficient RBC partitioning and high membrane permeability, and is not an efflux transporter (P-gp) substrate.