De novo design of drug-binding proteins with predictable binding energy and specificity.

The de novo design of small molecule-binding proteins has seen exciting recent progress; however, high-affinity binding and tunable specificity typically require laborious screening and optimization after computational design. We developed a computational procedure to design a protein that recognizes a common pharmacophore in a series of poly(ADP-ribose) polymerase-1 inhibitors. One of three designed proteins bound different inhibitors with affinities ranging from <5 nM to low micromolar. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free energy calculations performed directly on the designed models were in excellent agreement with the experimentally measured affinities. We conclude that de novo design of high-affinity small molecule-binding proteins with tuned interaction energies is feasible entirely from computation.

De novo-designed transmembrane proteins bind and regulate a cytokine receptor.

Transmembrane (TM) domains as simple as a single span can perform complex biological functions using entirely lipid-embedded chemical features. Computational design has the potential to generate custom tool molecules directly targeting membrane proteins at their functional TM regions. Thus far, designed TM domain-targeting agents have been limited to mimicking the binding modes and motifs of natural TM interaction partners. Here, we demonstrate the design of de novo TM proteins targeting the erythropoietin receptor (EpoR) TM domain in a custom binding topology competitive with receptor homodimerization. The TM proteins expressed in mammalian cells complex with EpoR and inhibit erythropoietin-induced cell proliferation. In vitro, the synthetic TM domain complex outcompetes EpoR homodimerization. Structural characterization reveals that the complex involves the intended amino acids and agrees with our designed molecular model of antiparallel TM helices at 1:1 stoichiometry. Thus, membrane protein TM regions can now be targeted in custom-designed topologies.

De novo Design of Peptides that Bind Specific Conformers of α-Synuclein.

Insoluble amyloids rich in cross-ß fibrils are observed in a number of neurodegenerative diseases. Depending on the clinicopathology, the amyloids can adopt distinct supramolecular assemblies, termed conformational strains. However, rapid methods to study amyloid in a conformationally specific manner are lacking. We introduce a novel computational method for de novo design of peptides that tile the surface of α-synuclein fibrils in a conformationally specific manner. Our method begins by identifying surfaces that are unique to the conformational strain of interest, which becomes a "target backbone" for the design of a peptide binder. Next, we interrogate structures in the PDB database with high geometric complementarity to the target. Then, we identify secondary structural motifs that interact with this target backbone in a favorable, highly occurring geometry. This method produces monomeric helical motifs with a favorable geometry for interaction with the strands of the underlying amyloid. Each motif is then symmetrically replicated to form a monolayer that tiles the amyloid surface. Finally, amino acid sequences of the peptide binders are computed to provide a sequence with high geometric and physicochemical complementarity to the target amyloid. This method was applied to a conformational strain of α-synuclein fibrils, resulting in a peptide with high specificity for the target relative to other amyloids formed by α-synuclein, tau, or Aß40. This designed peptide also markedly slowed the formation of α-synuclein amyloids. Overall, this method offers a new tool for examining conformational strains of amyloid proteins.

Hexamethylene amiloride binds the SARS-CoV-2 envelope protein at the protein-lipid interface.

The SARS-CoV-2 envelope (E) protein forms a five-helix bundle in lipid bilayers whose cation-conducting activity is associated with the inflammatory response and respiratory distress symptoms of COVID-19. E channel activity is inhibited by the drug 5-(N,N-hexamethylene) amiloride (HMA). However, the binding site of HMA in E has not been determined. Here we use solid-state NMR to measure distances between HMA and the E transmembrane domain (ETM) in lipid bilayers. 13 C, 15 N-labeled HMA is combined with fluorinated or 13 C-labeled ETM. Conversely, fluorinated HMA is combined with 13 C, 15 N-labeled ETM. These orthogonal isotopic labeling patterns allow us to conduct dipolar recoupling NMR experiments to determine the HMA binding stoichiometry to ETM as well as HMA-protein distances. We find that HMA binds ETM with a stoichiometry of one drug per pentamer. Unexpectedly, the bound HMA is not centrally located within the channel pore, but lies on the lipid-facing surface in the middle of the TM domain. This result suggests that HMA may inhibit the E channel activity by interfering with the gating function of an aromatic network. These distance data are obtained under much lower drug concentrations than in previous chemical shift perturbation data, which showed the largest perturbation for N-terminal residues. This difference suggests that HMA has higher affinity for the protein-lipid interface than the channel pore. These results give insight into the inhibition mechanism of HMA for SARS-CoV-2 E.

Patterning and folding of intestinal villi by active mesenchymal dewetting.

Tissue folding generates structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, the numerous finger-like protrusions that are essential for nutrient absorption. However, the molecular and mechanical mechanisms driving the initiation and morphogenesis of villi remain a matter of debate. Here, we identify an active mechanical mechanism that simultaneously patterns and folds intestinal villi. We find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. At the cell-level, this occurs through a process dependent upon matrix metalloproteinase-mediated tissue fluidization and altered cell-ECM adhesion. By combining computational models with in vivo experiments, we reveal these cellular features manifest at the tissue-level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active de-wetting of a thin liquid film.

A host defense peptide mimetic, brilacidin, potentiates caspofungin antifungal activity against human pathogenic fungi.

Fungal infections cause more than 1.5 million deaths a year. Due to emerging antifungal drug resistance, novel strategies are urgently needed to combat life-threatening fungal diseases. Here, we identify the host defense peptide mimetic, brilacidin (BRI) as a synergizer with caspofungin (CAS) against CAS-sensitive and CAS-resistant isolates of Aspergillus fumigatus, Candida albicans, C. auris, and CAS-intrinsically resistant Cryptococcus neoformans. BRI also potentiates azoles against A. fumigatus and several Mucorales fungi. BRI acts in A. fumigatus by affecting cell wall integrity pathway and cell membrane potential. BRI combined with CAS significantly clears A. fumigatus lung infection in an immunosuppressed murine model of invasive pulmonary aspergillosis. BRI alone also decreases A. fumigatus fungal burden and ablates disease development in a murine model of fungal keratitis. Our results indicate that combinations of BRI and antifungal drugs in clinical use are likely to improve the treatment outcome of aspergillosis and other fungal infections.

Designed Transmembrane Proteins Inhibit the Erythropoietin Receptor in a Custom Binding Topology.

Transmembrane (TM) domains as simple as a single span can perform complex biological functions using entirely lipid-embedded chemical features. Computational design has potential to generate custom tool molecules directly targeting membrane proteins at their functional TM regions. Thus far, designed TM domain-targeting agents have been limited to mimicking binding modes and motifs of natural TM interaction partners. Here, we demonstrate the design of de novo TM proteins targeting the erythropoietin receptor (EpoR) TM domain in a custom binding topology competitive with receptor homodimerization. The TM proteins expressed in mammalian cells complex with EpoR and inhibit erythropoietin-induced cell proliferation. In vitro, the synthetic TM domain complex outcompetes EpoR homodimerization. Structural characterization reveals that the complex involves the intended amino acids and agrees with our designed molecular model of antiparallel TM helices at 1:1 stoichiometry. Thus, membrane protein TM regions can now be targeted in custom designed topologies.

De novo design of drug-binding proteins with predictable binding energy and specificity.

The de novo design of small-molecule-binding proteins has seen exciting recent progress; however, the ability to achieve exquisite affinity for binding small molecules while tuning specificity has not yet been demonstrated directly from computation. Here, we develop a computational procedure that results in the highest affinity binders to date with predetermined relative affinities, targeting a series of PARP1 inhibitors. Two of four designed proteins bound with affinities ranging from < 5 nM to low µM, in a predictable manner. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free-energy calculations performed directly on the designed models are in excellent agreement with the experimentally measured affinities, suggesting that the de novo design of small-molecule-binding proteins with tuned interaction energies is now feasible entirely from computation. We expect these methods to open many opportunities in biomedicine, including rapid sensor development, antidote design, and drug delivery vehicles.

SARS-CoV-2 Envelope Protein Forms Clustered Pentamers in Lipid Bilayers.

The SARS-CoV-2 envelope (E) protein is a viroporin associated with the acute respiratory symptoms of COVID-19. E forms cation-selective ion channels that assemble in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment. The channel activity of E is linked to the inflammatory response of the host cell to the virus. Like many viroporins, E is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the E stoichiometry have led to inconclusive results and suggested mixtures of oligomers whose exact nature might vary with the detergent used. Here, we employ 19F solid-state nuclear magnetic resonance and the centerband-only detection of exchange (CODEX) technique to determine the oligomeric number of E's transmembrane domain (ETM) in lipid bilayers. The CODEX equilibrium value, which corresponds to the inverse of the oligomeric number, indicates that ETM assembles into pentamers in lipid bilayers, without any detectable fraction of low-molecular-weight oligomers. Unexpectedly, at high peptide concentrations and in the presence of the lipid phosphatidylinositol, the CODEX data indicate that more than five 19F spins are within a detectable distance of about 2 nm, suggesting that the ETM pentamers cluster in the lipid bilayer. Monte Carlo simulations that take into account peptide-peptide and peptide-lipid interactions yielded pentamer clusters that reproduced the CODEX data. This supramolecular organization is likely important for E-mediated virus assembly and budding and for the channel function of the protein.

Soluble TREM2 inhibits secondary nucleation of Aß fibrillization and enhances cellular uptake of fibrillar Aß.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a single-pass transmembrane receptor of the immunoglobulin superfamily that is secreted in a soluble (sTREM2) form. Mutations in TREM2 have been linked to increased risk of Alzheimer's disease (AD). A prominent neuropathological component of AD is deposition of the amyloid-ß (Aß) into plaques, particularly Aß40 and Aß42. While the membrane-bound form of TREM2 is known to facilitate uptake of Aß fibrils and the polarization of microglial processes toward amyloid plaques, the role of its soluble ectodomain, particularly in interactions with monomeric or fibrillar Aß, has been less clear. Our results demonstrate that sTREM2 does not bind to monomeric Aß40 and Aß42, even at a high micromolar concentration, while it does bind to fibrillar Aß42 and Aß40 with equal affinities (2.6 ± 0.3 µM and 2.3 ± 0.4 µM). Kinetic analysis shows that sTREM2 inhibits the secondary nucleation step in the fibrillization of Aß, while having little effect on the primary nucleation pathway. Furthermore, binding of sTREM2 to fibrils markedly enhanced uptake of fibrils into human microglial and neuroglioma derived cell lines. The disease-associated sTREM2 mutant, R47H, displayed little to no effect on fibril nucleation and binding, but it decreased uptake and functional responses markedly. We also probed the structure of the WT sTREM2-Aß fibril complex using integrative molecular modeling based primarily on the cross-linking mass spectrometry data. The model shows that sTREM2 binds fibrils along one face of the structure, leaving a second, mutation-sensitive site free to mediate cellular binding and uptake.

Brilacidin, a COVID-19 drug candidate, demonstrates broad-spectrum antiviral activity against human coronaviruses OC43, 229E, and NL63 through targeting both the virus and the host cell.

Brilacidin, a mimetic of host defense peptides (HDPs), is currently in Phase 2 clinical trial as an antibiotic drug candidate. A recent study reported that brilacidin has antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by inactivating the virus. In this study, we discovered an additional mechanism of action of brilacidin by targeting heparan sulfate proteoglycans (HSPGs) on the host cell surface. Brilacidin, but not acetyl brilacidin, inhibits the entry of SARS-CoV-2 pseudovirus into multiple cell lines, and heparin, an HSPG mimetic, abolishes the inhibitory activity of brilacidin on SARS-CoV-2 pseudovirus cell entry. In addition, we found that brilacidin has broad-spectrum antiviral activity against multiple human coronaviruses (HCoVs) including HCoV-229E, HCoV-OC43, and HCoV-NL63. Mechanistic studies revealed that brilacidin has a dual antiviral mechanism of action including virucidal activity and binding to coronavirus attachment factor HSPGs on the host cell surface. Brilacidin partially loses its antiviral activity when heparin was included in the cell cultures, supporting the host-targeting mechanism. Drug combination therapy showed that brilacidin has a strong synergistic effect with remdesivir against HCoV-OC43 in cell culture. Taken together, this study provides appealing findings for the translational potential of brilacidin as a broad-spectrum antiviral for coronaviruses including SARS-CoV-2.

Integrin α2ß1 regulates collagen I tethering to modulate hyperresponsiveness in reactive airway disease models.

Severe asthma remains challenging to manage and has limited treatment options. We have previously shown that targeting smooth muscle integrin α5ß1 interaction with fibronectin can mitigate the effects of airway hyperresponsiveness by impairing force transmission. In this study, we show that another member of the integrin superfamily, integrin α2ß1, is present in airway smooth muscle and capable of regulating force transmission via cellular tethering to the matrix protein collagen I and, to a lesser degree, laminin-111. The addition of an inhibitor of integrin α2ß1 impaired IL-13-enhanced contraction in mouse tracheal rings and human bronchial rings and abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. We confirmed that this effect was not due to alterations in classic intracellular myosin light chain phosphorylation regulating muscle shortening. Although IL-13 did not affect surface expression of α2ß1, it did increase α2ß1-mediated adhesion and the level of expression of an activation-specific epitope on the ß1 subunit. We developed a method to simultaneously quantify airway narrowing and muscle shortening using 2-photon microscopy and demonstrated that inhibition of α2ß1 mitigated IL-13-enhanced airway narrowing without altering muscle shortening by impairing the tethering of muscle to the surrounding matrix. Our data identified cell matrix tethering as an attractive therapeutic target to mitigate the severity of airway contraction in asthma.

Visualization of Platelet Integrins via Two-Photon Microscopy Using Anti-transmembrane Domain Peptides Containing a Blue Fluorescent Amino Acid.

The fluorescent reporters commonly used to visualize proteins can perturb both protein structure and function. Recently, we found that 4-cyanotryptophan (4CN-Trp), a blue fluorescent amino acid, is suitable for one-photon imaging applications. Here, we demonstrate its utility in two-photon fluorescence microscopy by using it to image integrins on cell surfaces. Specifically, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4-cyanoindole (4CNI) at their N-termini to image integrins on cell surfaces. CHAMP (computed helical anti-membrane protein) peptides spontaneously insert into membrane bilayers to target integrin transmembrane domains and cause integrin activation. We found that 4CNI labeling did not perturb the ability of CHAMP peptides to insert into membranes, bind to integrins, or cause integrin activation. We then used two-photon fluorescence microscopy to image 4CNI-containing integrins on the surface of platelets. Compared to a 4CNI-labeled scrambled peptide that uniformly decorated cell surfaces, 4CNI-labeled CHAMP peptides were present in discrete blue foci. To confirm that these foci represented CN peptide-containing integrins, we co-stained platelets with integrin-specific fluorescent monoclonal antibodies and found that CN peptide and antibody fluorescence coincided. Because 4CNI can readily be biosynthetically incorporated into proteins with little if any effect on protein structure and function, it provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments. In addition, these results clearly demonstrate that the two-photon excitation cross section of 4CN-Trp is sufficiently large to make it a useful two-photon fluorescence reporter for biological applications.

Dual antagonists of α5ß1/αvß1 integrin for airway hyperresponsiveness.

Inhibition of integrin α5ß1 emerges as a novel therapeutic option to block transmission of contractile forces during asthma attack. We designed and synthesized novel inhibitors of integrin α5ß1 by backbone replacement of known αvß1 integrin inhibitors. These integrin α5ß1 inhibitors also retain the nanomolar potency against αvß1 integrin, which shows promise for developing dual integrin α5ß1/αvß1 inhibitor. Introduction of hydrophobic adamantane group significantly boosted the potency as well as selectivity over integrin αvß3. We also demonstrated one of the inhibitors (11) reduced airway hyperresponsiveness in ex vivo mouse tracheal ring assay. Results from this study will help guide further development of integrin α5ß1 inhibitors as potential novel asthma therapeutics.

Swellable and porous bilayer tablet for gastroretentive drug delivery: Preparation and in vitro-in vivo evaluation.

The purpose of this study was to develop a novel gastroretentive drug delivery system with immediate buoyancy and high wet strength. The proposed bilayer tablet was composed of a drug layer and a highly porous and swellable gastroretentive (GR) layer. The highly porous GR layer was prepared by sublimating the volatile materials after compaction with swellable polymers. This pore-forming process decreased the density of the GR layer and enabled the tablet to float immediately on the dissolution media. The GR layer formulation was optimized by comparing the swelling, erosion, and mechanical properties of candidate swellable polymers. The release rates were conveniently controlled by changing the polymer content in the drug layer, while the swelling and floating properties were provided by the GR layer. The application of percolation theory revealed that the polymer content above the estimated threshold was required for a reliable drug release profile. In vivo study in fed beagle dogs confirmed the enhanced gastric retention time of the tablets compared to that of conventional single layer tablets. Taken together, our data suggest that the proposed system can be a promising platform technology with superior GR properties and a convenient formulation process.

Synthesis and application of the blue fluorescent amino acid l-4-cyanotryptophan to assess peptide-membrane interactions.

Recently, l-4-cyanotryptophan has been shown to be an efficient blue fluorescence emitter, with the potential to enable novel applications in biological spectroscopy and microscopy. However, lack of facile synthetic routes to this unnatural amino acid limits its wide use. Herein, we describe an expedient approach to synthesize Fmoc protected l-4-cyanotryptophan with high optical purity (>99%). Additionally, we test the utility of this blue fluorophore in imaging cell-membrane-bound peptides and in determining peptide-membrane binding constants.

Glutamine Side Chain 13C═18O as a Nonperturbative IR Probe of Amyloid Fibril Hydration and Assembly.

Infrared (IR) spectroscopy has provided considerable insight into the structures, dynamics, and formation mechanisms of amyloid fibrils. IR probes, such as main chain 13C═18O, have been widely employed to obtain site-specific structural information, yet only secondary structures and strand-to-strand arrangements can be probed. Very few nonperturbative IR probes are available to report on the side-chain conformation and environments, which are critical to determining sheet-to-sheet arrangements in steric zippers within amyloids. Polar residues, such as glutamine, contribute significantly to the stability of amyloids and thus are frequently found in core regions of amyloid peptides/proteins. Furthermore, polyglutamine (polyQ) repeats form toxic aggregates in several neurodegenerative diseases. Here we report the synthesis and application of a new nonperturbative IR probe-glutamine side chain 13C═18O. We use side chain 13C═18O labeling and isotope dilution to detect the presence of intermolecularly hydrogen-bonded arrays of glutamine side chains (Gln ladders) in amyloid-forming peptides. Moreover, the line width of the 13C═18O peak is highly sensitive to its local hydration environment. The IR data from side chain labeling allows us to unambiguously determine the sheet-to-sheet arrangement in a short amyloid-forming peptide, GNNQQNY, providing insight that was otherwise inaccessible through main chain labeling. With several different fibril samples, we also show the versatility of this IR probe in studying the structures and aggregation kinetics of amyloids. Finally, we demonstrate the capability of modeling amyloid structures with IR data using the integrative modeling platform (IMP) and the potential of integrating IR with other biophysical methods for more accurate structural modeling. Together, we believe that side chain 13C═18O will complement main chain isotope labeling in future IR studies of amyloids and integrative modeling using IR data will significantly expand the power of IR spectroscopy to elucidate amyloid assemblies.

4-Cyanoindole-2'-deoxyribonucleoside as a Dual Fluorescence and Infrared Probe of DNA Structure and Dynamics.

Unnatural nucleosides possessing unique spectroscopic properties that mimic natural nucleobases in both size and chemical structure are ideally suited for spectroscopic measurements of DNA/RNA structure and dynamics in a site-specific manner. However, such unnatural nucleosides are scarce, which prompts us to explore the utility of a recently found unnatural nucleoside, 4-cyanoindole-2'-deoxyribonucleoside (4CNI-NS), as a site-specific spectroscopic probe of DNA. A recent study revealed that 4CNI-NS is a universal nucleobase that maintains the high fluorescence quantum yield of 4-cyanoindole and that among the four natural nucleobases, only guanine can significantly quench its fluorescence. Herein, we further show that the C≡N stretching frequency of 4CNI-NS is sensitive to the local environment, making it a useful site-specific infrared probe of oligonucleotides. In addition, we demonstrate that the fluorescence-quencher pair formed by 4CNI-NS and guanine can be used to quantitatively assess the binding affinity of a single-stranded DNA to the protein system of interest via fluorescence spectroscopy, among other applications. We believe that this fluorescence binding assay is especially useful as its potentiality allows high-throughput screening of DNA⁻protein interactions.

Inhibitor binding mode and allosteric regulation of Na+-glucose symporters.

Sodium-dependent glucose transporters (SGLTs) exploit sodium gradients to transport sugars across the plasma membrane. Due to their role in renal sugar reabsorption, SGLTs are targets for the treatment of type 2 diabetes. Current therapeutics are phlorizin derivatives that contain a sugar moiety bound to an aromatic aglycon tail. Here, we develop structural models of human SGLT1/2 in complex with inhibitors by combining computational and functional studies. Inhibitors bind with the sugar moiety in the sugar pocket and the aglycon tail in the extracellular vestibule. The binding poses corroborate mutagenesis studies and suggest a partial closure of the outer gate upon binding. The models also reveal a putative Na+ binding site in hSGLT1 whose disruption reduces the transport stoichiometry to the value observed in hSGLT2 and increases inhibition by aglycon tails. Our work demonstrates that subtype selectivity arises from Na+-regulated outer gate closure and a variable region in extracellular loop EL5.

Entry from the Lipid Bilayer: A Possible Pathway for Inhibition of a Peptide G Protein-Coupled Receptor by a Lipophilic Small Molecule.

The pathways that G protein-coupled receptor (GPCR) ligands follow as they bind to or dissociate from their receptors are largely unknown. Protease-activated receptor-1 (PAR1) is a GPCR activated by intramolecular binding of a tethered agonist peptide that is exposed by thrombin cleavage. By contrast, the PAR1 antagonist vorapaxar is a lipophilic drug that binds in a pocket almost entirely occluded from the extracellular solvent. The binding and dissociation pathway of vorapaxar is unknown. Starting with the crystal structure of vorapaxar bound to PAR1, we performed temperature-accelerated molecular dynamics simulations of ligand dissociation. In the majority of simulations, vorapaxar exited the receptor laterally into the lipid bilayer through openings in the transmembrane helix (TM) bundle. Prior to full dissociation, vorapaxar paused in metastable intermediates stabilized by interactions with the receptor and lipid headgroups. Derivatives of vorapaxar with alkyl chains predicted to extend between TM6 and TM7 into the lipid bilayer inhibited PAR1 with apparent on rates similar to that of the parent compound in cell signaling assays. These data are consistent with vorapaxar binding to PAR1 via a pathway that passes between TM6 and TM7 from the lipid bilayer, in agreement with the most consistent pathway observed by molecular dynamics. While there is some evidence of entry of the ligand into rhodopsin and lipid-activated GPCRs from the cell membrane, our study provides the first such evidence for a peptide-activated GPCR and suggests that metastable intermediates along drug binding and dissociation pathways can be stabilized by specific interactions between lipids and the ligand.