ABSTRACT
The CC chemokine receptor 6 (CCR6) is a potential target for chronic inflammatory diseases. Previously, we reported an active CCR6 structure in complex with its cognate chemokine CCL20, revealing the molecular basis of CCR6 activation. Here, we present two inactive CCR6 structures in ternary complexes with different allosteric antagonists, CCR6/SQA1/OXM1 and CCR6/SQA1/OXM2. The oxomorpholine analogues, OXM1 and OXM2 are highly selective CCR6 antagonists which bind to an extracellular pocket and disrupt the receptor activation network. An energetically favoured U-shaped conformation in solution that resembles the bound form is observed for the active analogues. SQA1 is a squaramide derivative with close-in analogues reported as antagonists of chemokine receptors including CCR6. SQA1 binds to an intracellular pocket which overlaps with the G protein site, stabilizing a closed pocket that is a hallmark of inactive GPCRs. Minimal communication between the two allosteric pockets is observed, in contrast to the prevalent allosteric cooperativity model of GPCRs. This work highlights the versatility of GPCR antagonism by small molecules, complementing previous knowledge of CCR6 activation, and sheds light on drug discovery targeting CCR6.
Subject(s)
Receptors, CCR6 , Receptors, CCR6/metabolism , Receptors, CCR6/chemistry , Humans , Allosteric Regulation/drug effects , Allosteric Site , Protein Binding , Binding Sites , Models, Molecular , Crystallography, X-RayABSTRACT
HIV-1 protease is an important target for pharmaceutical intervention in HIV infection. Extensive structure-based drug design led to darunavir becoming a key chemotherapeutic agent. We replaced the aniline group of darunavir with a benzoxaborolone to form BOL-darunavir. This analogue has the same potency as darunavir as an inhibitor of catalysis by wild-type HIV-1 protease and, unlike darunavir, does not lose potency as an inhibitor of the common D30N variant. Moreover, BOL-darunavir is much more stable to oxidation than is a simple phenylboronic acid analogue of darunavir. X-ray crystallography revealed an extensive network of hydrogen bonds between the enzyme and benzoxaborolone moiety, including a novel direct hydrogen bond from a main-chain nitrogen to the carbonyl oxygen of the benzoxaborolone moiety that displaces a water molecule. These data highlight the utility of benzoxaborolone as a pharmacophore.
ABSTRACT
There is an unmet need for monoclonal antibodies (mAbs) for prevention or as adjunctive treatment of herpes simplex virus (HSV) disease. Most vaccine and mAb efforts focus on neutralizing antibodies, but for HSV this strategy has proven ineffective. Preclinical studies with a candidate HSV vaccine strain, ΔgD-2, demonstrated that non-neutralizing antibodies that activate Fcγ receptors (FcγRs) to mediate antibody-dependent cellular cytotoxicity (ADCC) provide active and passive protection against HSV-1 and HSV-2. We hypothesized that this vaccine provides a tool to identify and characterize protective mAbs. We isolated HSV-specific mAbs from germinal center and memory B cells and bone marrow plasmacytes of ΔgD-2-vaccinated mice and evaluated these mAbs for binding, neutralizing, and FcγR-activating activity and for protective efficacy in mice. The most potent protective mAb, BMPC-23, was not neutralizing but activated murine FcγRIV, a biomarker of ADCC. The cryo-electron microscopic structure of the Fab-glycoprotein B (gB) assembly identified domain IV of gB as the epitope. A single dose of BMPC-23 administered 24 hours before or after viral challenge provided significant protection when configured as mouse IgG2c and protected mice expressing human FcγRIII when engineered as a human IgG1. These results highlight the importance of FcR-activating antibodies in protecting against HSV.
Subject(s)
Herpes Simplex , Herpesvirus 1, Human , Animals , Humans , Mice , Antibodies, Neutralizing , Herpes Simplex/prevention & control , Antibodies, Viral , Glycoproteins , Antibodies, Monoclonal , Viral Envelope Proteins/geneticsABSTRACT
The emergence of the antigenically distinct and highly transmissible Omicron variant highlights the possibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune escape due to viral evolution. This continued evolution, along with the possible introduction of new sarbecoviruses from zoonotic reservoirs, may evade host immunity elicited by current SARS-CoV-2 vaccines. Identifying cross-reactive antibodies and defining their epitope(s) can provide templates for rational immunogen design strategies for next-generation vaccines. Here, we characterize the receptor-binding-domain-directed, cross-reactive humoral repertoire across 10 human vaccinated donors. We identify cross-reactive antibodies from diverse gene rearrangements targeting two conserved receptor-binding domain epitopes. An engineered immunogen enriches antibody responses to one of these conserved epitopes in mice with pre-existing SARS-CoV-2 immunity; elicited responses neutralize SARS-CoV-2, variants, and related sarbecoviruses. These data show how immune focusing to a conserved epitope targeted by human cross-reactive antibodies may guide pan-sarbecovirus vaccine development, providing a template for identifying such epitopes and translating to immunogen design.
Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Humans , Mice , Epitopes/genetics , SARS-CoV-2/genetics , COVID-19 Vaccines , COVID-19/prevention & control , AntibodiesABSTRACT
Pathogens evade host humoral responses by accumulating mutations in surface antigens. While variable, there are conserved regions that cannot mutate without compromising fitness. Antibodies targeting these conserved epitopes are often broadly protective but remain minor components of the repertoire. Rational immunogen design leverages a structural understanding of viral antigens to modulate humoral responses to favor these responses. Here, we report an epitope-enriched immunogen presenting a higher copy number of the influenza hemagglutinin (HA) receptor-binding site (RBS) epitope relative to other B cell epitopes. Immunization in a partially humanized murine model imprinted with an H1 influenza shows H1-specific serum and >99% H1-specific B cells being RBS-directed. Single B cell analyses show a genetically restricted response that structural analysis defines as RBS-directed antibodies engaging the RBS with germline-encoded contacts. These data show how epitope enrichment expands B cell responses toward conserved epitopes and advances immunogen design approaches for next-generation viral vaccines.
Subject(s)
Influenza Vaccines , Influenza, Human , Humans , Mice , Animals , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Antibodies, Viral , Epitopes, B-LymphocyteABSTRACT
Neutralizing antibodies that recognize the SARS-CoV-2 spike glycoprotein are the principal host defense against viral invasion. Variants of SARS-CoV-2 bear mutations that allow escape from neutralization by many human antibodies, especially those in widely distributed ("public") classes. Identifying antibodies that neutralize these variants of concern and determining their prevalence are important goals for understanding immune protection. To determine the Delta and Omicron BA.1 variant specificity of B cell repertoires established by an initial Wuhan strain infection, we measured neutralization potencies of 73 antibodies from an unbiased survey of the early memory B cell response. Antibodies recognizing each of three previously defined epitopic regions on the spike receptor binding domain (RBD) varied in neutralization potency and variant-escape resistance. The ACE2 binding surface ("RBD-2") harbored the binding sites of neutralizing antibodies with the highest potency but with the greatest sensitivity to viral escape; two other epitopic regions on the RBD ("RBD-1" and "RBD-3") bound antibodies of more modest potency but greater breadth. The structures of several Fab:spike complexes that neutralized all five variants of concern tested, including one Fab each from the RBD-1, -2, and -3 clusters, illustrated the determinants of broad neutralization and showed that B cell repertoires can have specificities that avoid immune escape driven by public antibodies. The structure of the RBD-2 binding, broad neutralizer shows why it retains neutralizing activity for Omicron BA.1, unlike most others in the same public class. Our results correlate with real-world data on vaccine efficacy, which indicate mitigation of disease caused by Omicron BA.1.
Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing/chemistry , Antibodies, Viral , Humans , Neutralization Tests , SARS-CoV-2/geneticsABSTRACT
Eliciting antibodies to surface-exposed viral glycoproteins can generate protective responses that control and prevent future infections. Targeting conserved sites may reduce the likelihood of viral escape and limit the spread of related viruses with pandemic potential. Here we leverage rational immunogen design to focus humoral responses on conserved epitopes. Using glycan engineering and epitope scaffolding in boosting immunogens, we focus murine serum antibody responses to conserved receptor binding motif (RBM) and receptor binding domain (RBD) epitopes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike imprinting. Although all engineered immunogens elicit a robust SARS-CoV-2-neutralizing serum response, RBM-focusing immunogens exhibit increased potency against related sarbecoviruses, SARS-CoV, WIV1-CoV, RaTG13-CoV, and SHC014-CoV; structural characterization of representative antibodies defines a conserved epitope. RBM-focused sera confer protection against SARS-CoV-2 challenge. Thus, RBM focusing is a promising strategy to elicit breadth across emerging sarbecoviruses without compromising SARS-CoV-2 protection. These engineering strategies are adaptable to other viral glycoproteins for targeting conserved epitopes.
Subject(s)
COVID-19 , Viral Envelope Proteins , Animals , Mice , Mice, Inbred BALB C , SARS-CoV-2 , Spike Glycoprotein, CoronavirusABSTRACT
BACKGROUND: Targeting RNA is a promising yet underdeveloped modality for the selective killing of cells infected with HIV-1. The secretory ribonucleases (RNases) found in vertebrates have cytotoxic ribonucleolytic activity that is kept in check by a cytosolic ribonuclease inhibitor protein, RI. METHODS: We engineered amino acid substitutions that enable human RNase 1 to evade RI upon its cyclization into a zymogen that is activated by the HIV-1 protease. In effect, the zymogen has an HIV-1 protease cleavage site between the termini of the wild-type enzyme, thereby positioning a cleavable linker over the active site that blocks access to a substrate. RESULTS: The amino acid substitutions in RNase 1 diminish its affinity for RI by 106-fold and confer high toxicity for T-cell leukemia cells. Pretreating these cells with the zymogen leads to a substantial drop in their viability upon HIV-1 infection, indicating specific toxicity toward infected cells. CONCLUSIONS: These data demonstrate the utility of ribonuclease zymogens as biologic prodrugs.
Subject(s)
HIV Infections , HIV-1 , Animals , Enzyme Precursors/genetics , HIV Infections/drug therapy , HIV-1/genetics , Humans , Models, Molecular , Ribonucleases/geneticsABSTRACT
The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease (COVID-19), is an ideal target for pharmaceutical inhibition. Mpro is conserved among coronaviruses and distinct from human proteases. Viral replication depends on the cleavage of the viral polyprotein at multiple sites. We present crystal structures of SARS-CoV-2 Mpro bound to two viral substrate peptides. The structures show how Mpro recognizes distinct substrates and how subtle changes in substrate accommodation can drive large changes in catalytic efficiency. One peptide, constituting the junction between viral nonstructural proteins 8 and 9 (nsp8/9), has P1' and P2' residues that are unique among the SARS-CoV-2 Mpro cleavage sites but conserved among homologous junctions in coronaviruses. Mpro cleaves nsp8/9 inefficiently, and amino acid substitutions at P1' or P2' can enhance catalysis. Visualization of Mpro with intact substrates provides new templates for antiviral drug design and suggests that the coronavirus lifecycle selects for finely tuned substrate-dependent catalytic parameters.
Subject(s)
COVID-19 , Coronavirus 3C Proteases/metabolism , SARS-CoV-2 , Antiviral Agents , Humans , Peptide Hydrolases , Viral Nonstructural ProteinsABSTRACT
Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded antibodies from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found seven major antibody competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of antibody-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. Although emerging SARS-CoV-2 variants of concern escaped binding by many members of the groups associated with the most potent neutralizing activity, some antibodies in each of those groups retained affinity-suggesting that otherwise redundant components of a primary immune response are important for durable protection from evolving pathogens. Our results furnish a global atlas of S-specific memory B cell repertoires and illustrate properties driving viral escape and conferring robustness against emerging variants.
ABSTRACT
Despite their desirable attributes, boronic acids have had a minimal impact in biological contexts. A significant problem has been their oxidative instability. At physiological pH, phenylboronic acid and its boronate esters are oxidized by reactive oxygen species at rates comparable to those of thiols. After considering the mechanism and kinetics of the oxidation reaction, we reasoned that diminishing electron density on boron could enhance oxidative stability. We found that a boralactone, in which a carboxyl group serves as an intramolecular ligand for the boron, increases stability by 104-fold. Computational analyses revealed that the resistance to oxidation arises from diminished stabilization of the p orbital of boron that develops in the rate-limiting transition state of the oxidation reaction. Like simple boronic acids and boronate esters, a boralactone binds covalently and reversibly to 1,2-diols such as those in saccharides. The kinetic stability of its complexes is, however, at least 20-fold greater. A boralactone also binds covalently to a serine side chain in a protein. These attributes confer unprecedented utility upon boralactones in the realms of chemical biology and medicinal chemistry.
Subject(s)
Boron/chemistry , Boronic Acids/chemistry , Hydrogen-Ion Concentration , Oxidation-ReductionABSTRACT
Eliciting antibodies to surface-exposed viral glycoproteins can lead to protective responses that ultimately control and prevent future infections. Targeting functionally conserved epitopes may help reduce the likelihood of viral escape and aid in preventing the spread of related viruses with pandemic potential. One such functionally conserved viral epitope is the site to which a receptor must bind to facilitate viral entry. Here, we leveraged rational immunogen design strategies to focus humoral responses to the receptor binding motif (RBM) on the SARS-CoV-2 spike. Using glycan engineering and epitope scaffolding, we find an improved targeting of the serum response to the RBM in context of SARS-CoV-2 spike imprinting. Furthermore, we observed a robust SARS-CoV-2-neutralizing serum response with increased potency against related sarbecoviruses, SARS-CoV, WIV1-CoV, RaTG13-CoV, and SHC014-CoV. Thus, RBM focusing is a promising strategy to elicit breadth across emerging sarbecoviruses and represents an adaptable design approach for targeting conserved epitopes on other viral glycoproteins. ONE SENTENCE SUMMARY: SARS-CoV-2 immune focusing with engineered immunogens.
ABSTRACT
Palladium oxidative addition complexes (OACs) are traditionally accessed by treating an aryl halide-containing substrate with a palladium(0) source. Here, a new strategy to selectively prepare stable OACs from amino groups on native proteins is presented. The approach relies on an amine-selective acylation reaction that occurs without modification of a preformed palladium(II)-aryl group. Once transferred onto a protein substrate, the palladium(II)-aryl group facilitates conjugation by undergoing reaction with a second, cysteine-containing protein. This operationally simple method is applicable to native, nonengineered enzymes as well as antibodies and is carried out in an aqueous setting and open to air. The resulting Pd-protein OACs are stable, storable reagents that retain biological activity and can be used to achieve protein-protein cross-coupling at nanomolar concentrations within hours.
Subject(s)
Amines/chemistry , Palladium/chemistry , Proteins/chemistry , Acylation , Kinetics , Oxidation-ReductionABSTRACT
Recognition of distinct glycans is central to biology, and lectins mediate this function. Lectin glycan preferences are usually centered on specific monosaccharides. In contrast, human intelectin-1 (hItln-1, also known as Omentin-1) is a soluble lectin that binds a range of microbial sugars, including ß-d-galactofuranose (ß-Galf), d-glycerol 1-phosphate, d-glycero-d-talo-oct-2-ulosonic acid (KO), and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). Though these saccharides differ dramatically in structure, they share a common feature-an exocyclic vicinal diol. How and whether such a small fragment is sufficient for recognition was unclear. We tested several glycans with this epitope and found that l-glycero-α-d-manno-heptose and d-glycero-α-d-manno-heptose possess the critical diol motif yet bind weakly. To better understand hItln-1 recognition, we determined the structure of the hItln-1·KO complex using X-ray crystallography, and our 1.59 Å resolution structure enabled unambiguous assignment of the bound KO conformation. This carbohydrate conformation was present in >97% of the KDO/KO structures in the Protein Data Bank. Bioinformatic analysis revealed that KO and KDO adopt a common conformation, while heptoses prefer different conformers. The preferred conformers of KO and KDO favor hItln-1 engagement, but those of the heptoses do not. Natural bond orbital (NBO) calculations suggest these observed conformations, including the side chain orientations, are stabilized by not only steric but also stereoelectronic effects. Thus, our data highlight a role for stereoelectronic effects in dictating the specificity of glycan recognition by proteins. Finally, our finding that hItln-1 avoids binding prevalent glycans with a terminal 1,2-diol (e.g., N-acetyl-neuraminic acid and l-glycero-α-d-manno-heptose) suggests the lectin has evolved to recognize distinct bacterial species.
Subject(s)
Polysaccharides/chemistry , Carbohydrate Conformation , Crystallography, X-Ray , Protein Binding , StereoisomerismABSTRACT
Adenosine and uridine 5'-tetra- and 5'-pentaphosphates were synthesized from an activated tetrametaphosphate ([PPN]2[P4O11], [PPN]2[1], PPN = bis(triphenylphosphine)iminium) and subsequently tested for inhibition of the enzymatic activity of ribonuclease A (RNase A). Reagent [PPN]2[1] reacts with unprotected uridine and adenosine in the presence of a base under anhydrous conditions to give nucleoside tetrametaphosphates. Ring opening of these intermediates with tetrabutylammonium hydroxide ([TBA][OH]) yields adenosine and uridine tetraphosphates (p4A, p4U) in 92% and 85% yields, respectively, from the starting nucleoside. Treatment of ([PPN]2[1]) with AMP or UMP yields nucleoside-monophosphate tetrametaphosphates (cp4pA, cp4pU) having limited aqueous stability. Ring opening of these ultraphosphates with [TBA][OH] yields p5A and p5U in 58% and 70% yield from AMP and UMP, respectively. We characterized inorganic and nucleoside-conjugated linear and cyclic oligophosphates as competitive inhibitors of RNase A. Increasing the chain length in both linear and cyclic inorganic oligophosphates resulted in improved binding affinity. Increasing the length of oligophosphates on the 5' position of adenosine beyond three had a deleterious effect on binding. Conversely, uridine nucleotides bearing 5' oligophosphates saw progressive increases in binding with chain length. We solved X-ray cocrystal structures of the highest affinity binders from several classes. The terminal phosphate of p5A binds in the P1 enzymic subsite and forces the oligophosphate to adopt a convoluted conformation, while the oligophosphate of p5U binds in several extended conformations, targeting multiple cationic regions of the active-site cleft.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Nucleosides/chemistry , Nucleosides/pharmacology , Ribonuclease, Pancreatic/antagonists & inhibitors , Animals , Catalytic Domain/drug effects , Cattle , Models, Molecular , Polyphosphates/chemistry , Polyphosphates/pharmacology , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/metabolismABSTRACT
The endogenous production of enzymes as zymogens provides a means to control catalytic activities. Here, we describe the heterologous production of ribonuclease 1 (RNase 1), which is the most prevalent secretory ribonuclease in humans, as a zymogen. In folded RNase 1, the N and C termini flank the enzymic active site. By using intein-mediated cis-splicing, we created circular proteins in which access to the active site of RNase 1 is obstructed by an amino-acid sequence that is recognized by the HIV-1 protease. Installing a sequence that does not perturb the RNase 1 fold led to only modest inactivation. In contrast, the ancillary truncation of residues from each terminus led to a substantial decrease in the catalytic activity of the zymogen with the maintenance of thermostability. For optimized zymogens, activation by HIV-1 protease led to a > 104 -fold increase in ribonucleolytic activity at a rate comparable to that for the cleavage of endogenous viral substrates. Molecular modeling indicated that these zymogens are inactivated by conformational distortion in addition to substrate occlusion. Because protease levels are elevated in many disease states and ribonucleolytic activity can be cytotoxic, RNase 1 zymogens have potential as generalizable prodrugs.
Subject(s)
Enzyme Precursors/chemistry , Enzyme Precursors/genetics , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/genetics , Amino Acid Sequence , Catalytic Domain , Enzyme Precursors/metabolism , Enzyme Stability , HIV Protease/metabolism , Humans , Inteins , Models, Molecular , Protein Conformation , Protein Engineering/methods , Ribonuclease, Pancreatic/metabolism , ThermodynamicsABSTRACT
Aspartic proteases regulate many biological processes and are prominent targets for therapeutic intervention. Structural studies have captured intermediates along the reaction pathway, including the Michaelis complex and tetrahedral intermediate. Using a Ramachandran analysis of these structures, we discovered that residues occupying the P1 and P1' positions (which flank the scissile peptide bond) adopt the dihedral angle of an inverse γ-turn and polyproline type-II helix, respectively. Computational analyses reveal that the polyproline type-II helix engenders an nâπ* interaction in which the oxygen of the scissile peptide bond is the donor. This interaction stabilizes the negative charge that develops in the tetrahedral intermediate, much like the oxyanion hole of serine proteases. The inverse γ-turn serves to twist the scissile peptide bond, vacating the carbonyl π* orbital and facilitating its hydration. These previously unappreciated interactions entail a form of substrate-assisted catalysis and offer opportunities for drug design.
ABSTRACT
Boronic acids have been typecast as moieties for covalent complexation and are employed only rarely as agents for non-covalent recognition. By exploiting the profuse ability of a boronic acid group to form hydrogen bonds, we have developed an inhibitor of HIV-1 protease with extraordinary affinity. Specifically, we find that replacing an aniline moiety in darunavir with a phenylboronic acid leads to 20-fold greater affinity for the protease. X-ray crystallography demonstrates that the boronic acid group participates in three hydrogen bonds, more than the amino group of darunavir or any other analog. Importantly, the boronic acid maintains its hydrogen bonds and its affinity for the drug-resistant D30N variant of HIV-1 protease. The BOH···OC hydrogen bonds between the boronic acid hydroxy group and Asp30 (or Asn30) of the protease are short ( rO···O = 2.2 Å), and density functional theory analysis reveals a high degree of covalency. These data highlight the utility of boronic acids as versatile functional groups in the design of small-molecule ligands.
Subject(s)
Boronic Acids/pharmacology , HIV Protease Inhibitors/pharmacology , HIV Protease/metabolism , Boronic Acids/chemistry , Dose-Response Relationship, Drug , HIV Protease Inhibitors/chemistry , Molecular Structure , Structure-Activity RelationshipABSTRACT
Crystal structures of inactive variants of HIV-1 protease bound to peptides have revealed how the enzyme recognizes its endogenous substrates. The best of the known substrates is, however, a nonnatural substrate that was identified by directed evolution. The crystal structure of the complex between this substrate and the D25N variant of the protease is reported at a resolution of 1.1â Å. The structure has several unprecedented features, especially the formation of additional hydrogen bonds between the enzyme and the substrate. This work expands the understanding of molecular recognition by HIV-1 protease and informs the design of new substrates and inhibitors.
Subject(s)
HIV Protease/chemistry , Peptide Library , Binding Sites , Crystallography, X-Ray , Directed Molecular Evolution , Genetic Variation/genetics , Humans , Hydrogen Bonding , Peptides/chemistry , Protein Binding , Substrate SpecificityABSTRACT
Transthyretin (TTR) is a homotetrameric protein. Its dissociation into monomers leads to the formation of fibrils that underlie human amyloidogenic diseases. The binding of small molecules to the thyroxin-binding sites in TTR stabilizes the homotetramer and attenuates TTR amyloidosis. Herein, we report on boronic acid-substituted stilbenes that limit TTR amyloidosis in vitro. Assays of affinity for TTR and inhibition of its tendency to form fibrils were coupled with X-ray crystallographic analysis of nine TTR·ligand complexes. The ensuing structure-function data led to a symmetrical diboronic acid that forms a boronic ester reversibly with serine 117. This diboronic acid inhibits fibril formation by both wild-type TTR and a common disease-related variant, V30M TTR, as effectively as does tafamidis, a small-molecule drug used to treat TTR-related amyloidosis in the clinic. These findings establish a new modality for covalent inhibition of fibril formation and illuminate a path for future optimization.