Molecular Dynamics and Theratyping in Airway and Gut Organoids Reveal R352Q-CFTR Conductance Defect.

A significant challenge to making targeted cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q, with a potential role contributing to intrapore chloride ion permeation, in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using an ion transport assay and forskolin-induced swelling assay, respectively. CFTR potentiators (VX-770, GLPG1837, and VX-445) and correctors (VX-809, VX-445, with or without VX-661) were tested. Data from R352Q-CFTR were compared with data of 20 participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function that was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics simulations of R352Q-CFTR were carried out, which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico molecular dynamics simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.

Computational Design of High-Affinity Blockers for Sodium Channel NaV1.2 from µ-Conotoxin KIIIA.

The voltage-gated sodium channel subtype 1.2 (NaV1.2) is instrumental in the initiation of action potentials in the nervous system, making it a natural drug target for neurological diseases. Therefore, there is much pharmacological interest in finding blockers of NaV1.2 and improving their affinity and selectivity properties. An extensive family of peptide toxins from cone snails (conotoxins) block NaV channels, thus they provide natural templates for the design of drugs targeting NaV channels. Unfortunately, progress was hampered due to the absence of any NaV structures. The recent determination of cryo-EM structures for NaV channels has finally broken this impasse. Here, we use the NaV1.2 structure in complex with µ-conotoxin KIIIA (KIIIA) in computational studies with the aim of improving KIIIA's affinity and blocking capacity for NaV1.2. Only three KIIIA amino acid residues are available for mutation (S5, S6, and S13). After performing molecular modeling and simulations on NaV1.2-KIIIA complex, we have identified the S5R, S6D, and S13K mutations as the most promising for additional contacts. We estimate these contacts to boost the affinity of KIIIA for NaV1.2 from nanomole to picomole domain. Moreover, the KIIIA[S5R, S6D, S13K] analogue makes contacts with all four channel domains, thus enabling the complete blocking of the channel (KIIIA partially blocks as it has contacts with three domains). The proposed KIIIA analogue, once confirmed experimentally, may lead to novel anti-epileptic drugs.

Structural Determinants Mediating Tertiapin Block of Neuronal Kir3.2 Channels.

Tertiapin (TPN) is a 21 amino acid venom peptide from Apis mellifera that inhibits certain members of the inward rectifier potassium (Kir) channel family at a nanomolar affinity with limited specificity. Structure-based computational simulations predict that TPN behaves as a pore blocker; however, the molecular determinants mediating block of neuronal Kir3 channels have been inconclusive and unvalidated. Here, using molecular docking and molecular dynamics (MD) simulations with 'potential of mean force' (PMF) calculations, we investigated the energetically most favored interaction of TPN with several Kir3.x channel structures. The resulting binding model for Kir3.2-TPN complexes was then tested by targeted mutagenesis of the predicted contact sites, and their impact on the functional channel block was measured electrophysiologically. Together, our findings indicate that a high-affinity TPN block of Kir3.2 channels involves a pore-inserting lysine side chain requiring (1) hydrophobic interactions at a phenylalanine ring surrounding the channel pore and (2) electrostatic interactions with two adjacent Kir3.2 turret regions. Together, these interactions collectively stabilize high-affinity toxin binding to the Kir3.2 outer vestibule, which orients the ε-amino group of TPN-K21 to occupy the outermost K+ binding site of the selectivity filter. The structural determinants for the TPN block described here also revealed a favored subunit arrangement for assembled Kir3.x heteromeric channels, in addition to a multimodal binding capacity of TPN variants consistent with the functional dyad model for polybasic peptide pore blockers. These novel findings will aid efforts in re-engineering the TPN pharmacophore to develop peptide variants having unique and distinct Kir channel blocking properties.

Free-Energy Simulations Resolve the Low-Affinity Na+-High-Affinity Asp Binding Paradox in GltPh.

Glutamate transporters clear up excess extracellular glutamate by cotransporting three Na+ and one H+ with the countertransport of one K+. The archaeal homologs are selective to aspartate and only cotransport three Na+. The crystal structures of GltPh from archaea have been used in computational studies to understand the transport mechanism. Although some progress has been made with regard to the ligand-binding sites, a consistent picture of transport still eludes us. A major concern is the discrepancy between the computed binding free energies, which predict high-affinity Na+-low-affinity aspartate binding, and the experimental results in which the opposite is observed. Here, we show that the binding of the first two Na+ ions involves an intermediate state near the Na1 site, where two Na+ ions coexist and couple to aspartate with similar strengths, boosting its affinity. Binding free energies for Na+ and aspartate obtained using this intermediate state are in good agreement with the experimental values. Thus, the paradox in binding affinities arises from the assumption that the ligands bind to the sites observed in the crystal structure following the order dictated by their binding free energies with no intermediate states. In fact, the presence of an intermediate state eliminates such a correlation between the binding free energies and the binding order. The intermediate state also facilitates transition of the first Na+ ion to its final binding site via a knock-on mechanism, which induces substantial conformational changes in the protein consistent with experimental observations.

A simple, parameter-free method for computing solvation free energies of ions.

Solvation free energies of ions are difficult to determine from molecular dynamics simulations due to the long-range Coulomb interactions. Various approximations and corrections are introduced to enable their calculation in small systems, which, however, raises issues of robustness. We show that solvation free energies of ions can be calculated using the spherical boundary conditions without introducing any corrections at the boundary via a buffer zone. The results are shown to converge for a droplet size of 21 Å and are independent of the parameters used for confining water or restraining the ion. The proposed method thus resolves the robustness issues in solvation free energy calculations of ions and can be used with confidence to determine force field parameters from such calculations. We apply the method to calculate the solvation free energies of the side chain analogs of charged amino acids. Tests using periodic boundary conditions show that similar results are also obtained in that case.

Computational Study of Binding of µ-Conotoxin GIIIA to Bacterial Sodium Channels NaVAb and NaVRh.

Structures of several voltage-gated sodium (NaV) channels from bacteria have been determined recently, but the same feat might not be achieved for the mammalian counterparts in the near future. Thus, at present, computational studies of the mammalian NaV channels have to be performed using homology models based on the bacterial crystal structures. A successful homology model for the mammalian NaV1.4 channel was recently constructed using the extensive mutation data for binding of µ-conotoxin GIIIA to NaV1.4, which was further validated through studies of binding of other µ-conotoxins and ion permeation. Understanding the similarities and differences between the bacterial and mammalian NaV channels is an important issue, and the NaV1.4-GIIIA system provides a good opportunity for such a comparison. To this end, we study the binding of GIIIA to the bacterial channels NaVAb and NaVRh. The complex structures are obtained using docking and molecular dynamics simulations, and the dissociation of GIIIA is studied through umbrella sampling simulations. The results are compared to those obtained from the NaV1.4-GIIIA system, and the differences in the binding modes arising from the changes in the selectivity filters are highlighted.

Na+ interactions with the neutral amino acid transporter ASCT1.

The alanine, serine, cysteine transporters (ASCTs) belong to the solute carrier family 1A (SLC1A), which also includes the excitatory amino acid transporters (EAATs) and the prokaryotic aspartate transporter GltPh. Acidic amino acid transport by the EAATs is coupled to the co-transport of three Na(+) ions and one proton, and the counter-transport of one K(+) ion. In contrast, neutral amino acid exchange by the ASCTs does not require protons or the counter-transport of K(+) ions and the number of Na(+) ions required is not well established. One property common to SLC1A family members is a substrate-activated anion conductance. We have investigated the number and location of Na(+) ions required by ASCT1 by mutating residues in ASCT1 that correspond to residues in the EAATs and GltPh that are involved in Na(+) binding. Mutations to all three proposed Na(+) sites influence the binding of substrate and/or Na(+), or the rate of substrate exchange. A G422S mutation near the Na2 site reduced Na(+) affinity, without affecting the rate of exchange. D467T and D467A mutations in the Na1 site reduce Na(+) and substrate affinity and also the rate of substrate exchange. T124A and D380A mutations in the Na3 site selectively reduce the affinity for Na(+) and the rate of substrate exchange without affecting substrate affinity. In many of the mutants that reduce the rate of substrate transport the amplitudes of the substrate-activated anion conductances are not substantially affected indicating altered ion dependence for channel activation compared with substrate exchange.

Molecular dynamics simulations elucidate the mechanism of proton transport in the glutamate transporter EAAT3.

The uptake of glutamate in nerve synapses is carried out by the excitatory amino acid transporters (EAATs), involving the cotransport of a proton and three Na(+) ions and the countertransport of a K(+) ion. In this study, we use an EAAT3 homology model to calculate the pKa of several titratable residues around the glutamate binding site to locate the proton carrier site involved in the translocation of the substrate. After identifying E374 as the main candidate for carrying the proton, we calculate the protonation state of this residue in different conformations of EAAT3 and with different ligands bound. We find that E374 is protonated in the fully bound state, but removing the Na2 ion and the substrate reduces the pKa of this residue and favors the release of the proton to solution. Removing the remaining Na(+) ions again favors the protonation of E374 in both the outward- and inward-facing states, hence the proton is not released in the empty transporter. By calculating the pKa of E374 with a K(+) ion bound in three possible sites, we show that binding of the K(+) ion is necessary for the release of the proton in the inward-facing state. This suggests a mechanism in which a K(+) ion replaces one of the ligands bound to the transporter, which may explain the faster transport rates of the EAATs compared to its archaeal homologs.

Why the Drosophila Shaker K+ channel is not a good model for ligand binding to voltage-gated Kv1 channels.

The Drosophila Shaker K(+) channel is the first cloned voltage-gated potassium channel and has, therefore, played an important role in structural and functional studies of those channels. While such a role is well justified for ion permeation, it is not clear whether this also extends to ligand binding. Despite the high degree of homology among Shaker and Kv1 channels, κ-conotoxin PVIIA (κ-PVIIA) binds to Shaker with high affinity but not to Kv1 channels. Here we address this issue by studying binding of κ-PVIIA to Shaker and Kv1 channels using molecular dynamics (MD) simulations. The structures of the channel-toxin complexes are constructed via docking and refinement with MD. The binding mode of each complex is characterized and compared to available mutagenesis data to validate the complex models. The potential of mean force for dissociation of the Shaker-κ-PVIIA complex is calculated from umbrella sampling MD simulations, and the corresponding binding free energy is determined, which provides further validation of the complex structure. Comparison of the Shaker and Kv1 complex models shows that a few mutations in the turret and extended regions are sufficient to abolish the observed sensitivity of Shaker to κ-PVIIA. This study demonstrates that Shaker is not always a good model for Kv1 channels for ligand binding. It also provides insights into the binding of the toxin to potassium channels that will be useful for improving affinity and selectivity properties of Kv1 channels.

Computational studies of marine toxins targeting ion channels.

Toxins from marine animals offer novel drug leads for treatment of diseases involving ion channels. Computational methods could be very helpful in this endeavour in several ways, e.g., (i) constructing accurate models of the channel-toxin complexes using docking and molecular dynamics (MD) simulations; (ii) determining the binding free energies of toxins from umbrella sampling MD simulations; (iii) predicting the effect of mutations from free energy MD simulations. Using these methods, one can design new analogs of toxins with improved affinity and selectivity properties. Here we present a review of the computational methods and discuss their applications to marine toxins targeting potassium and sodium channels. Detailed examples from the potassium channel toxins-ShK from sea anemone and κ-conotoxin PVIIA-are provided to demonstrate capabilities of the computational methods to give accurate descriptions of the channel-toxin complexes and the energetics of their binding. An example is also given from sodium channel toxins (µ-conotoxin GIIIA) to illustrate the differences between the toxin binding modes in potassium and sodium channels.

Q1291H-CFTR molecular dynamics simulations and ex vivo theratyping in nasal epithelial models and clinical response to elexacaftor/tezacaftor/ivacaftor in a Q1291H/F508del patient.

Background: Cystic fibrosis (CF) is caused by a wide spectrum of mutations in the CF transmembrane conductance regulator (CFTR) gene, with some leading to non-classical clinical presentations. We present an integrated in vivo, in silico and in vitro investigation of an individual with CF carrying the rare Q1291H-CFTR allele and the common F508del allele. At age 56 years, the participant had obstructive lung disease and bronchiectasis, qualifying for Elexacaftor/Tezacaftor/Ivacaftor (ETI) CFTR modulator treatment due to their F508del allele. Q1291H CFTR incurs a splicing defect, producing both a normally spliced but mutant mRNA isoform and a misspliced isoform with a premature termination codon, causing nonsense mediated decay. The effectiveness of ETI in restoring Q1291H-CFTR is largely unknown. Methods: We collected clinical endpoint measurements, including forced expiratory volume in 1 s percent predicted (FEV1pp) and body mass index (BMI), and examined medical history. In silico simulations of the Q1291H-CFTR were compared to Q1291R, G551D, and wild-type (WT)-CFTR. We quantified relative Q1291H CFTR mRNA isoform abundance in patient-derived nasal epithelial cells. Differentiated pseudostratified airway epithelial cell models at air liquid interface were created and ETI treatment impact on CFTR was assessed by electrophysiology assays and Western blot. Results: The participant ceased ETI treatment after 3 months due to adverse events and no improvement in FEV1pp or BMI. In silico simulations of Q1291H-CFTR identified impairment of ATP binding similar to known gating mutants Q1291R and G551D-CFTR. Q1291H and F508del mRNA transcripts composed 32.91% and 67.09% of total mRNA respectively, indicating 50.94% of Q1291H mRNA was misspliced and degraded. Mature Q1291H-CFTR protein expression was reduced (3.18% ± 0.60% of WT/WT) and remained unchanged with ETI. Baseline CFTR activity was minimal (3.45 ± 0.25 µA/cm2) and not enhanced with ETI (5.73 ± 0.48 µA/cm2), aligning with the individual's clinical evaluation as a non-responder to ETI. Conclusion: The combination of in silico simulations and in vitro theratyping in patient-derived cell models can effectively assess CFTR modulator efficacy for individuals with non-classical CF manifestations or rare CFTR mutations, guiding personalized treatment strategies and optimizing clinical outcomes.

Investigation of polarization effects in the gramicidin A channel from ab initio molecular dynamics simulations.

Polarization is an important component of molecular interactions and is expected to play a particularly significant role in inhomogeneous environments such as pores and interfaces. Here we investigate the effects of polarization in the gramicidin A ion channel by performing quantum mechanics/molecular mechanics molecular dynamics (MD) simulations and comparing the results with those obtained from classical MD simulations with non-polarizable force fields. We consider the dipole moments of backbone carbonyl groups and channel water molecules as well as a number of structural quantities of interest. The ab initio results show that the dipole moments of the carbonyl groups and water molecules are highly sensitive to the hydrogen bonds (H-bonds) they participate in. In the absence of a K(+) ion, water molecules in the channel are quite mobile, making the H-bond network highly dynamic. A central K(+) ion acts as an anchor for the channel waters, stabilizing the H-bond network and thereby increasing their average dipole moments. In contrast, the K(+) ion has little effect on the dipole moments of the neighboring carbonyl groups. The weakness of the ion-peptide interactions helps to explain the near diffusion-rate conductance of K(+) ions through the channel. We also address the sampling issue in relatively short ab initio MD simulations. Results obtained from a continuous 20 ps ab initio MD simulation are compared with those generated by sampling ten windows from a much longer classical MD simulation and running each window for 2 ps with ab initio MD. Both methods yield similar results for a number of quantities of interest, indicating that fluctuations are fast enough to justify the short ab initio MD simulations.

S945L-CFTR molecular dynamics, functional characterization and tezacaftor/ivacaftor efficacy in vivo and in vitro in matched pediatric patient-derived cell models.

Cystic Fibrosis (CF) results from over 400 different disease-causing mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. These CFTR mutations lead to numerous defects in CFTR protein function. A novel class of targeted therapies (CFTR modulators) have been developed that can restore defects in CFTR folding and gating. This study aimed to characterize the functional and structural defects of S945L-CFTR and interrogate the efficacy of modulators with two modes of action: gating potentiator [ivacaftor (IVA)] and folding corrector [tezacaftor (TEZ)]. The response to these modulators in vitro in airway differentiated cell models created from a participant with S945L/G542X-CFTR was correlated with in vivo clinical outcomes of that participant at least 12 months pre and post modulator therapy. In this participants' airway cell models, CFTR-mediated chloride transport was assessed via ion transport electrophysiology. Monotherapy with IVA or TEZ increased CFTR activity, albeit not reaching statistical significance. Combination therapy with TEZ/IVA significantly (p = 0.02) increased CFTR activity 1.62-fold above baseline. Assessment of CFTR expression and maturation via western blot validated the presence of mature, fully glycosylated CFTR, which increased 4.1-fold in TEZ/IVA-treated cells. The in vitro S945L-CFTR response to modulator correlated with an improvement in in vivo lung function (ppFEV1) from 77.19 in the 12 months pre TEZ/IVA to 80.79 in the 12 months post TEZ/IVA. The slope of decline in ppFEV1 significantly (p = 0.02) changed in the 24 months post TEZ/IVA, becoming positive. Furthermore, there was a significant improvement in clinical parameters and a fall in sweat chloride from 68 to 28 mmol/L. The mechanism of dysfunction of S945L-CFTR was elucidated by in silico molecular dynamics (MD) simulations. S945L-CFTR caused misfolding of transmembrane helix 8 and disruption of the R domain, a CFTR domain critical to channel gating. This study showed in vitro and in silico that S945L causes both folding and gating defects in CFTR and demonstrated in vitro and in vivo that TEZ/IVA is an efficacious modulator combination to address these defects. As such, we support the utility of patient-derived cell models and MD simulations in predicting and understanding the effect of modulators on CFTR function on an individualized basis.

Molecular dynamics and functional characterization of I37R-CFTR lasso mutation provide insights into channel gating activity.

Characterization of I37R, a mutation located in the lasso motif of the CFTR chloride channel, was conducted by theratyping several CFTR modulators from both potentiator and corrector classes. Intestinal current measurements in rectal biopsies, forskolin-induced swelling (FIS) in intestinal organoids, and short circuit current measurements in organoid-derived monolayers from an individual with I37R/F508del CFTR genotype demonstrated that the I37R-CFTR results in a residual function defect amenable to treatment with potentiators and type III, but not type I, correctors. Molecular dynamics of I37R using an extended model of the phosphorylated, ATP-bound human CFTR identified an altered lasso motif conformation which results in an unfavorable strengthening of the interactions between the lasso motif, the regulatory (R) domain, and the transmembrane domain 2 (TMD2). Structural and functional characterization of the I37R-CFTR mutation increases understanding of CFTR channel regulation and provides a potential pathway to expand drug access to CF patients with ultra-rare genotypes.

Comparative study of the energetics of ion permeation in Kv1.2 and KcsA potassium channels.

Biological ion channels rely on a multi-ion transport mechanism for fast yet selective permeation of ions. The crystal structure of the KcsA potassium channel provided the first microscopic picture of this process. A similar mechanism is assumed to operate in all potassium channels, but the validity of this assumption has not been well investigated. Here, we examine the energetics of ion permeation in Shaker Kv1.2 and KcsA channels, which exemplify the six-transmembrane voltage-gated and two-transmembrane inward-rectifier channels. We study the feasibility of binding a third ion to the filter and the concerted motion of ions in the channel by constructing the potential of mean force for K(+) ions in various configurations. For both channels, we find that a pair of K(+) ions can move almost freely within the filter, but a relatively large free-energy barrier hinders the K(+) ion from stepping outside the filter. We discuss the effect of the CMAP dihedral energy correction that was recently incorporated into the CHARMM force field on ion permeation dynamics.

Accurate determination of the binding free energy for KcsA-charybdotoxin complex from the potential of mean force calculations with restraints.

Free energy calculations for protein-ligand dissociation have been tested and validated for small ligands (50 atoms or less), but there has been a paucity of studies for larger, peptide-size ligands due to computational limitations. Previously we have studied the energetics of dissociation in a potassium channel-charybdotoxin complex by using umbrella sampling molecular-dynamics simulations, and established the need for carefully chosen coordinates and restraints to maintain the physiological ligand conformation. Here we address the ligand integrity problem further by constructing additional potential of mean forces for dissociation of charybdotoxin using restraints. We show that the large discrepancies in binding free energy arising from simulation artifacts can be avoided by using appropriate restraints on the ligand, which enables determination of the binding free energy within the chemical accuracy. We make several suggestions for optimal choices of harmonic potential parameters and restraints to be used in binding studies of large ligands.

Free energy simulations of ligand binding to the aspartate transporter Glt(Ph).

Glutamate/Aspartate transporters cotransport three Na(+) and one H(+) ions with the substrate and countertransport one K(+) ion. The binding sites for the substrate and two Na(+) ions have been observed in the crystal structure of the archeal homolog Glt(Ph), while the binding site for the third Na(+) ion has been proposed from computational studies and confirmed by experiments. Here we perform detailed free energy simulations of Glt(Ph), giving a comprehensive characterization of the substrate and ion binding sites, and calculating their binding free energies in various configurations. Our results show unequivocally that the substrate binds after the binding of two Na(+) ions. They also shed light into Asp/Glu selectivity of Glt(Ph), which is not observed in eukaryotic glutamate transporters.

Ab initio calculation of the potential of mean force for dissociation of aqueous Ca-Cl.

The potential of mean force for the dissociation of a Ca-Cl ion pair in water is calculated from ab initio molecular dynamics simulations. The constraint-force method is employed to enhance sampling over the entire range of the reaction coordinate (Ca-Cl distance) from 2.2 to 6.5 Å. Particular attention is paid to equilibration of the system as it is found that the potential of mean force is highly sensitive to the hydration number of the Ca(2+) ion. The structure and polarization of hydration waters are examined in detail at three ion-ion separations of interest: the contact-ion position, the solvent-separated-ion position, and the transition state between them. The ab initio results are compared to the classical ones obtained using the CHARMM force field and the parameters of Dang and Smith. There are substantial differences between the polarization of hydration waters of Ca(2+) and Cl(-) ions at all distances, which indicates that an accurate description of Ca-Cl dissociation with nonpolarizable force fields may not be feasible. The ab initio results presented here for the Ca-Cl ion pair complements our earlier results for Na-Cl, and together they provide useful benchmarks for polarizable force fields under construction.

Dissociation of NaCl in water from ab initio molecular dynamics simulations.

We perform ab initio molecular dynamics simulations to study the dissociation of NaCl in water. The potential of mean force (PMF) between the two ions is determined using the constrained-force method. The simulation windows corresponding to the contact and solvent-separated minima, and the transition state in between, are further analyzed to determine the changes in the properties of hydration waters such as coordination number, dipole moment, and orientation. The ab initio results are compared with those obtained from classical molecular dynamics simulations of aqueous NaCl using several common force fields. The ab initio PMF is found to have a shallower contact minimum and a smaller transition barrier compared with the classical ones. Also the binding free energy calculated from the ab initio PMF almost vanishes whereas it is negative for all the classical PMFs. Water dipole moments are observed to exhibit little change during dissociation, indicating that description of NaCl with a nonpolarizable force field may be feasible. However, overcoordination of the ion pair at all distances remains as a serious shortcoming of the current classical models. The ab initio results presented here provide useful guidance for alternative parametrizations of the nonpolarizable force fields as well as the polarizable ones currently under construction.

Modulation of Lymphocyte Potassium Channel KV1.3 by Membrane-Penetrating, Joint-Targeting Immunomodulatory Plant Defensin.

We describe a cysteine-rich, membrane-penetrating, joint-targeting, and remarkably stable peptide, EgK5, that modulates voltage-gated KV1.3 potassium channels in T lymphocytes by a distinctive mechanism. EgK5 enters plasma membranes and binds to KV1.3, causing current run-down by a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. EgK5 exhibits selectivity for KV1.3 over other channels, receptors, transporters, and enzymes. EgK5 suppresses antigen-triggered proliferation of effector memory T cells, a subset enriched among pathogenic autoreactive T cells in autoimmune disease. PET-CT imaging with 18F-labeled EgK5 shows accumulation of the peptide in large and small joints of rodents. In keeping with its arthrotropism, EgK5 treats disease in a rat model of rheumatoid arthritis. It was also effective in treating disease in a rat model of atopic dermatitis. No signs of toxicity are observed at 10-100 times the in vivo dose. EgK5 shows promise for clinical development as a therapeutic for autoimmune diseases.