Dynophore-Based Approach in Virtual Screening: A Case of Human DNA Topoisomerase IIα.

In this study, we utilized human DNA topoisomerase IIα as a model target to outline a dynophore-based approach to catalytic inhibitor design. Based on MD simulations of a known catalytic inhibitor and the native ATP ligand analog, AMP-PNP, we derived a joint dynophore model that supplements the static structure-based-pharmacophore information with a dynamic component. Subsequently, derived pharmacophore models were employed in a virtual screening campaign of a library of natural compounds. Experimental evaluation identified flavonoid compounds with promising topoisomerase IIα catalytic inhibition and binding studies confirmed interaction with the ATPase domain. We constructed a binding model through docking and extensively investigated it with molecular dynamics MD simulations, essential dynamics, and MM-GBSA free energy calculations, thus reconnecting the new results to the initial dynophore-based screening model. We not only demonstrate a new design strategy that incorporates a dynamic component of molecular recognition, but also highlight new derivates in the established flavonoid class of topoisomerase II inhibitors.

Unveiling the interaction profile of rosmarinic acid and its bioactive substructures with serum albumin.

Rosmarinic acid, a phytochemical compound, bears diverse pharmaceutical profile. It is composed by two building blocks: caffeic acid and a salvianic acid unit. The interaction profile, responsible for the delivery of rosmarinic acid and its two substructure components by serum albumin remains unexplored. To unveil this, we established a novel low-cost and efficient method to produce salvianic acid from the parent compound. To probe the interaction profile of rosmarinic acid and its two substructure constituents with the different serum albumin binding sites we utilised fluorescence spectroscopy and competitive saturation transfer difference NMR experiments. These studies were complemented with transfer NOESY NMR experiments. The thermodynamics of the binding profile of rosmarinic acid and its substructures were addressed using isothermal titration calorimetry. In silico docking studies, driven by the experimental data, have been used to deliver further atomic details on the binding mode of rosmarinic acid and its structural components.

Evaluation of the published kinase inhibitor set to identify multiple inhibitors of bacterial ATP-dependent mur ligases.

The Mur ligases form a series of consecutive enzymes that participate in the intracellular steps of bacterial peptidoglycan biosynthesis. They therefore represent interesting targets for antibacterial drug discovery. MurC, D, E and F are all ATP-dependent ligases. Accordingly, with the aim being to find multiple inhibitors of these enzymes, we screened a collection of ATP-competitive kinase inhibitors, on Escherichia coli MurC, D and F, and identified five promising scaffolds that inhibited at least two of these ligases. Compounds 1, 2, 4 and 5 are multiple inhibitors of the whole MurC to MurF cascade that act in the micromolar range (IC50, 32-368 µM). NMR-assisted binding studies and steady-state kinetics studies performed on aza-stilbene derivative 1 showed, surprisingly, that it acts as a competitive inhibitor of MurD activity towards D-glutamic acid, and additionally, that its binding to the D-glutamic acid binding site is independent of the enzyme closure promoted by ATP.

Discovery of (phenylureido)piperidinyl benzamides as prospective inhibitors of bacterial autolysin E from Staphylococcus aureus.

Autolysin E (AtlE) is a cell wall degrading enzyme that catalyzes the hydrolysis of the ß-1,4-glycosidic bond between the N-acetylglucosamine and N-acetylmuramic acid units of the bacterial peptidoglycan. Using our recently determined crystal structure of AtlE from Staphylococcus aureus and a combination of pharmacophore modeling, similarity search, and molecular docking, a series of (Phenylureido)piperidinyl benzamides were identified as potential binders and surface plasmon resonance (SPR) and saturation-transfer difference (STD) NMR experiments revealed that discovered compounds bind to AtlE in a lower micromolar range. (phenylureido)piperidinyl benzamides are the first reported non-substrate-like compounds that interact with this enzyme and enable further study of the interaction of small molecules with bacterial AtlE as potential inhibitors of this target.

A novel synthetic luteinizing hormone-releasing hormone (LHRH) analogue coupled with modified ß-cyclodextrin: insight into its intramolecular interactions.

BACKGROUND: Cyclodextrins (CDs) in combination with therapeutic proteins and other bioactive compounds have been proposed as candidates that show enhanced chemical and enzymatic stability, better absorption, slower plasma clearance and improved dose-response curves or immunogenicity. As a result, an important number of therapeutic complexes between cyclodextrins and bioactive compounds capable to control several diseases have been developed. RESULTS: In this article, the synthesis and the structural study of a conjugate between a luteinizing hormone-releasing hormone (LHRH) analogue, related to the treatment of hormone dependent cancer and fertility, and modified ß-cyclodextrin residue are presented. The results show that both the phenyl group of tyrosine (Tyr) as well as the indole group of tryptophan (Trp) can be encapsulated inside the cyclodextrin cavity. Solution NMR experiments provide evidence that these interactions take place intramolecularly and not intermolecularly. CONCLUSIONS: The study of a LHRH analogue conjugated with modified ß-cyclodextrin via high field NMR and MD experiments revealed the existence of intramolecular interactions that could lead to an improved drug delivery. GENERAL SIGNIFICANCE: NMR in combination with MD simulation is of great value for a successful rational design of peptide-cyclodextrin conjugates showing stability against enzymatic proteolysis and a better pharmacological profile.

Insights into the molecular basis of action of the AT1 antagonist losartan using a combined NMR spectroscopy and computational approach.

The drug:membrane interactions for the antihypertensive AT1 antagonist losartan, the prototype of the sartans class, are studied herein using an integrated approach. The pharmacophore arrangement of the drug was revealed by rotating frame nuclear Overhauser effect spectroscopy (2D ROESY) NMR spectroscopy in three different environments, namely water, dimethyl sulfoxide (DMSO), and sodium dodecyl sulfate (SDS) micellar solutions mimicking conditions of biological transport fluids and membrane lipid bilayers. Drug association with micelles was monitored by diffusion ordered spectroscopy (2D DOSY) and drug:micelle intermolecular interactions were characterized by ROESY spectroscopy. The localisation of the drug in the micellar environment was investigated by introducing 5-doxyl and 16-doxyl stearic acids. The use of spin labels confirmed that losartan resides close to the micelle:water interface with the hydroxymethyl group and the tetrazole heterocyclic aromatic ring facing the polar surface with the potential to interact with SDS charged polar head groups in order to increase amphiphilic interactions. The spontaneous insertion, the diffusion pathway and the conformational features of losartan were monitored by Molecular Dynamics (MD) simulations in a modeled SDS micellar aggregate environment and a long exploratory MD run (580ns) in a phospholipid dipalmitoylphosphatidylcholine (DPPC) bilayer with the AT1 receptor embedded. MD simulations were in excellent agreement with experimental results and further revealed the molecular basis of losartan:membrane interactions in atomic-level detail. This applied integrated approach aims to explore the role of membranes in losartan's pathway towards the AT1 receptor.

Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization.

Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.

Stability and binding effects of silver(I) complexes at lipoxygenase-1.

An anti-inflammatory complex of Ag(I), namely [Ag(tpp)3(asp)](dmf) [tpp = triphenylphosphine, aspH = aspirin, dmf = N,N-dimethylformamide], was synthesized in an attempt to develop novel metallotherapeutic molecules. STD (1)H NMR experiments were used to examine if this complex binds to LOX-1. The (1)H NMR spectra in buffer Tris/D2O betrayed the existence of two complexes: the complex of aspirin and the complex of salicylic acid produced after deacetylation of aspirin. Nevertheless, the STD spectra showed that only the complex of salicylic acid is bound to the enzyme. Molecular docking and dynamics were used to complement our study. The complexes were stabilized inside a large LOX-1 cavity by establishing a network of hydrogen bonds and steric interactions. The complex formation with salicylic acid was more favorable. The in silico results provide a plausible explanation of the experimental results, which showed that only the complex with salicylic acid enters the binding cavity.

Discovery of new Hsp90-Cdc37 protein-protein interaction inhibitors: in silico screening and optimization of anticancer activity.

The interaction between heat shock protein 90 (Hsp90) and Hsp90 co-chaperone cell-division cycle 37 (Cdc37) is crucial for the folding and maturation of several oncogenic proteins, particularly protein kinases. This makes the inhibition of this protein-protein interaction (PPI) an interesting target for developing new anticancer compounds. However, due to the large interaction surface, developing PPI inhibitors is challenging. In this work, we describe the discovery of new Hsp90-Cdc37 PPI inhibitors using a ligand-based virtual screening approach. Initial hit compounds showed Hsp90 binding, resulting in anticancer activity in the MCF-7 breast cancer cell line. To optimize their antiproliferative effect, 35 analogs were prepared. Binding affinity for Hsp90 was determined for the most promising compounds, 8c (K d = 70.8 µM) and 13g (K d = 73.3 µM), both of which interfered with the binding of Cdc37 to Hsp90. This resulted in anticancer activity against Ewing sarcoma (SK-N-MC), breast cancer (MCF-7), and leukemia (THP-1) cell lines in vitro. Furthermore, compounds 8c and 13g demonstrated the ability to induce apoptosis in the Ewing sarcoma cell line and caused a decrease in the levels of several known Hsp90 client proteins in MCF-7 cells, all without inducing the heat shock response.

Simulation- and AI-directed optimization of 4,6-substituted 1,3,5-triazin-2(1H)-ones as inhibitors of human DNA topoisomerase IIα.

The 4,6-substituted-1,3,5-triazin-2(1H)-ones are promising inhibitors of human DNA topoisomerase IIα. To further develop this chemical class targeting the enzyme´s ATP binding site, the triazin-2(1H)-one substitution position 6 was optimized. Inspired by binding of preclinical substituted 9H-purine derivative, bicyclic substituents were incorporated at position 6 and the utility of this modification was validated by a combination of molecular simulations, dynamic pharmacophores, and free energy calculations. Considering also predictions of Deepfrag, a software developed for structure-based lead optimization based on deep learning, compounds with both bicyclic and monocyclic substitutions were synthesized and investigated for their inhibitory activity. The SAR data showed that the bicyclic substituted compounds exhibited good inhibition of topo IIα, comparable to their mono-substituted counterparts. Further evaluation on a panel of human protein kinases showed selectivity for the inhibition of topo IIα. Mechanistic studies indicated that the compounds acted predominantly as catalytic inhibitors, with some exhibiting topo IIα poison effects at higher concentrations. Integration of STD NMR experiments and molecular simulations, provided insights into the binding model and highlighted the importance of the Asn120 interaction and hydrophobic interactions with substituents at positions 4 and 6. In addition, NCI-60 screening demonstrated cytotoxicity of the compounds with bicyclic substituents and identified sensitive human cancer cell lines, underlining the translational relevance of our findings for further preclinical development of this class of compounds. The study highlights the synergy between simulation and AI-based approaches in efficiently guiding molecular design for drug optimization, which has implications for further preclinical development of this class of compounds.

Nature-inspired substituted 3-(imidazol-2-yl) morpholines targeting human topoisomerase IIα: Dynophore-derived discovery.

The molecular nanomachine, human DNA topoisomerase IIα, plays a crucial role in replication, transcription, and recombination by catalyzing topological changes in the DNA, rendering it an optimal target for cancer chemotherapy. Current clinical topoisomerase II poisons often cause secondary tumors as side effects due to the accumulation of double-strand breaks in the DNA, spurring the development of catalytic inhibitors. Here, we used a dynamic pharmacophore approach to develop catalytic inhibitors targeting the ATP binding site of human DNA topoisomerase IIα. Our screening of a library of nature-inspired compounds led to the discovery of a class of 3-(imidazol-2-yl) morpholines as potent catalytic inhibitors that bind to the ATPase domain. Further experimental and computational studies identified hit compound 17, which exhibited selectivity against the human DNA topoisomerase IIα versus human protein kinases, cytotoxicity against several human cancer cells, and did not induce DNA double-strand breaks, making it distinct from clinical topoisomerase II poisons. This study integrates an innovative natural product-inspired chemistry and successful implementation of a molecular design strategy that incorporates a dynamic component of ligand-target molecular recognition, with comprehensive experimental characterization leading to hit compounds with potential impact on the development of more efficient chemotherapies.

Exploration and optimisation of structure-activity relationships of new triazole-based C-terminal Hsp90 inhibitors towards in vivo anticancer potency.

The development of new anticancer agents is one of the most urgent topics in drug discovery. Inhibition of molecular chaperone Hsp90 stands out as an approach that affects various oncogenic proteins in different types of cancer. These proteins rely on Hsp90 to obtain their functional structure, and thus Hsp90 is indirectly involved in the pathophysiology of cancer. However, the most studied ATP-competitive inhibition of Hsp90 at the N-terminal domain has proven to be largely unsuccessful clinically. Therefore, research has shifted towards Hsp90 C-terminal domain (CTD) inhibitors, which are also the focus of this study. Our recent discovery of compound C has provided us with a starting point for exploring the structure-activity relationship and optimising this new class of triazole-based Hsp90 inhibitors. This investigation has ultimately led to a library of 33 analogues of C that have suitable physicochemical properties and several inhibit the growth of different cancer types in the low micromolar range. Inhibition of Hsp90 was confirmed by biophysical and cellular assays and the binding epitopes of selected inhibitors were studied by STD NMR. Furthermore, the most promising Hsp90 CTD inhibitor 5x was shown to induce apoptosis in breast cancer (MCF-7) and Ewing sarcoma (SK-N-MC) cells while inducing cause cell cycle arrest in MCF-7 cells. In MCF-7 cells, it caused a decrease in the levels of ERα and IGF1R, known Hsp90 client proteins. Finally, 5x was tested in zebrafish larvae xenografted with SK-N-MC tumour cells, where it limited tumour growth with no obvious adverse effects on normal zebrafish development.

Hiding in plain sight: Optimizing topoisomerase IIα inhibitors into Hsp90ß selective binders.

Due to their impact on several oncogenic client proteins, the Hsp90 family of chaperones has been widely studied for the development of potential anticancer agents. Although several Hsp90 inhibitors have entered clinical trials, most were unsuccessful because they induced a heat shock response (HSR). This issue can be circumvented by using isoform-selective inhibitors, but the high similarity in the ATP-binding sites between the isoforms presents a challenge. Given that Hsp90 shares a conserved Bergerat fold with bacterial DNA gyrase B and human topoisomerase IIα, we repurposed our ATP-competitive inhibitors of these two proteins for Hsp90 inhibition. We virtually screened a library of in-house inhibitors and identified eleven hits for evaluation of Hsp90 binding. Among these, compound 11 displayed low micromolar affinity for Hsp90 and demonstrated a 12-fold selectivity for Hsp90ß over its closest isoform, Hsp90α. Out of 29 prepared analogs, 16 showed a preference for Hsp90ß over Hsp90α. Furthermore, eleven of these compounds inhibited the growth of several cancer cell lines in vitro. Notably, compound 24e reduced intracellular levels of Hsp90 client proteins in MCF-7 cells, leading to cell cycle arrest in the G0/G1 phase without inducing HSR. This inhibitor exhibited at least a 27-fold preference for Hsp90ß and was selective against topoisomerase IIα, a panel of 22 representative protein kinases, and proved to be non-toxic in a zebrafish larvae toxicology model. Finally, molecular modeling, corroborated by STD NMR studies, and the binding of 24e to the S52A mutant of Hsp90α confirmed that the serine to alanine switch drives the selectivity between the two cytoplasmic isoforms.

Thermal, dynamic and structural properties of drug AT1 antagonist olmesartan in lipid bilayers.

It is proposed that AT1 antagonists (ARBs) exert their biological action by inserting into the lipid membrane and then diffuse to the active site of AT1 receptor. Thus, lipid bilayers are expected to be actively involved and play a critical role in drug action. For this reason, the thermal, dynamic and structural effects of olmesartan alone and together with cholesterol were studied using differential scanning calorimetry (DSC), 13C magic-angle spinning (MAS) nuclear magnetic resonance (NMR), cross-polarization (CP) MAS NMR, and Raman spectroscopy as well as small- and wide angle X-ray scattering (SAXS and WAXS) on dipalmitoyl-phosphatidylcholine (DPPC) multilamellar vesicles. 13C CP/MAS spectra provided direct evidence for the incorporation of olmesartan and cholesterol in lipid bilayers. Raman and X-ray data revealed how both molecules modify the bilayer's properties. Olmesartan locates itself at the head-group region and upper segment of the lipid bilayers as 13C CP/MAS spectra show that its presence causes significant chemical shift changes mainly in the A ring of the steroidal part of cholesterol. The influence of olmesartan on DPPC/cholesterol bilayers is less pronounced. Although, olmesartan and cholesterol are residing at the same region of the lipid bilayers, due to their different sizes, display distinct impacts on the bilayer's properties. Cholesterol broadens significantly the main transition, abolishes the pre-transition, and decreases the membrane fluidity above the main transition. Olmesartan is the only so far studied ARB that increases the gauche:trans ratio in the liquid crystalline phase. These significant differences of olmesartan may in part explain its distinct pharmacological profile.

Conformational analysis of two novel cytotoxic C2-substituted pyrrolo[2,3-f]quinolines in aqueous media, organic solvents, membrane bilayers and at the putative active site.

We have performed: (i) conformational analysis of two novel cytotoxic C2-substituted pyrrolo[2,3-f]quinolines 5e and 5g in deuterated dimethylsulfoxide (DMSO-d(6)) utilizing NOE results from NMR spectroscopy; (ii) molecular dynamics (MD) calculations in water, DMSO and dimyristoyl phosphatidylcholine bilayers and (iii) molecular docking and MD calculations on DNA nucleotide sequences. The obtained results for the two similar in structure molecules showed differences in: (i) their conformational properties in silico and in media that reasonably simulate the biological environment; (ii) the way they are incorporated into the lipid bilayers and therefore their diffusion ability and (iii) molecular docking capacity as it is depicted from their different binding scores.

Determination of conformational preferences of dipeptides using vibrational spectroscopy.

The NMR coupling constants ((3)J(H(N), H(alpha))) of dipeptides indicate that the backbone conformational preferences vary strikingly among dipeptides. These preferences are similar to those of residues in small peptides, denatured proteins, and the coil regions of native proteins. Detailed characterization of the conformational preferences of dipeptides is therefore of fundamental importance for understanding protein structure and folding. Here, we studied the conformational preferences of 13 dipeptides using infrared and Raman spectroscopy. The main advantage of vibrational spectroscopy over NMR spectroscopy is in its much shorter time scale, which enables the determination of the conformational preferences of short-lived states. Accuracy of structure determination using vibrational spectroscopy depends critically on identification of the vibrational parameters that are sensitive to changes in conformation. We show that the frequencies of the amide I band and the A12 ratio of the amide I components of dipeptides correlate with the (3)J(H(N), H(alpha)). These two infrared vibrational parameters are thus analogous to (3)J(H(N), H(alpha)), indicators for the preference for the dihedral angle phi. We also show that the intensities of the components of the amide III bands in infrared spectra and the intensities of the skeletal vibrations in Raman spectra are indicators of populations of the P(II), beta, and alpha(R) conformations. The results show that alanine dipeptide adopts predominantly a PII conformation. The population of the beta conformation increases in valine dipeptides. The populations of the alpha(R) conformation are generally small. These data are in accord with the electrostatic screening model of conformational preferences.

A combined NMR and molecular dynamics simulation study to determine the conformational properties of agonists and antagonists against experimental autoimmune encephalomyelitis.

Myelin basic protein (MBP) is one of the best characterized autoantigens causing multiple sclerosis (MS), via a procedure that involves a stable formation of the trimolecular complex of a T-cell Receptor (TCR), an MBP epitope, and the receptor HLA-DR2b. Experimental autoimmune encephalomyelitis (EAE) is considered as an instructive model for MS in humans, and plenty of X-ray data is available for a number of EAE inducing peptide-receptor complexes. To date, though, there are no data available for complexes involving peptides reversing EAE, namely antagonists. Conformational properties of the EAE inducing epitope MBP(87-99) were analyzed in DMSO using the NOE connectivities and vicinal H(N)-H(alpha) coupling constants, and compared with the antagonist altered peptide ligands. A robust method, which is based on a combination of molecular dynamics and energy minimization, is proposed for identifying the putative bioactive conformations. Generated conformations are compared with the known X-ray structure of MBP(83-96) (human sequence numbering) in the HLA-DR2b complex. The structural motif for the agonist-antagonist activity is discussed.

Nitrate Esters of Heteroaromatic Compounds as Candida albicans CYP51 Enzyme Inhibitors.

Four heteroaromatic compounds bearing nitrate esters were selected using a virtual-screening procedure as putative sterol 14α-demethylase (CYP51) Candida albicans inhibitors. Compounds were examined for their inhibition on C. albicans growth and biofilm formation as well as for their toxicity. NMR spectroscopy studies, in silico docking, and molecular dynamics simulations were used to investigate further the selectivity of these compounds to fungal CYP51. All compounds exhibited good antimicrobial properties, indicated with low minimal inhibitory concentrations and ability to inhibit formation of fungal biofilm. Moreover, all of the compounds had the ability to inhibit growth of C. albicans cells. N-(2-Nitrooxyethyl)-1Η-indole-2-carboxamide was the only compound with selectivity on C. albicans CYP51 that did not exhibit cytotoxic effect on cells isolated from liver and should be further investigated for selective application in new leads for the treatment of candidiasis.

Electrostatic screening and backbone preferences of amino acid residues in urea-denatured ubiquitin.

Local structures in denatured proteins may be important in guiding a polypeptide chain during the folding and misfolding processes. Existence of local structures in chemically denatured proteins is a highly controversial issue. NMR parameters [coupling constants (3) J(H(alpha),H(N)) and chemical shifts] of chemically denatured proteins in general deviate little from their values in small peptides. These peptides were presumed to be completely unstructured; therefore, it was considered that chemically denatured proteins are random coils. But recent experimental studies show that small peptides adopt relatively stable structures in aqueous solutions. Small deviations of the NMR parameters from their values in small peptides may thus actually indicate the existence of local structures in chemically denatured proteins. Using NMR data and theoretical predictions we show here that fluctuating beta-strands exist in urea-denatured ubiquitin (8 M urea at pH 2). Residues in such beta-strands populate more frequently the left side of the broad beta region of -psi space. Urea-denatured ubiquitin contains no detectable beta-sheet secondary structures; nevertheless, the fluctuating beta-strands in urea-denatured ubiquitin coincide to the beta-strands in the native state. Formation of beta-strands is in accord with the electrostatic screening model of unfolded proteins. The free energy of a residue in an unfolded protein is in this model determined by the local backbone electrostatics and its screening by backbone solvation. These energy terms introduce strong electrostatic coupling between neighboring residues, which causes cooperative formation of beta-strands in denatured proteins. We propose that fluctuating beta-strands in denatured proteins may serve as initiation sites to form fibrils.

Comparison of proposed putative active conformations of myelin basic protein epitope 87-99 linear altered peptide ligands by spectroscopic and modelling studies: the role of positions 91 and 96 in T-cell receptor activation.

This work proposes a structural motif for the inhibition of experimental autoimmune encephalomyelitis (EAE) by the linear altered peptide ligands (APLs) [Ala91,96] MBP87-99 and [Arg91,Ala96] MBP87-99 of myelin basic protein. Molecular dynamics was applied to reveal distinct populations of EAE antagonist [Ala91,96] MBP87-99 in solution, in agreement with NOE data. The combination of the theoretical and experimental results led to the identification of a putative active conformation. This approach is of value as no crystallographic data is available for the APL-receptor complex. TCR contact residue Phe89 has an altered topology in the putative bioactive conformations of both APLs with respect to the native peptide, as found via crystallography; it is no longer prominent and solvent exposed. It is proposed that the antagonistic activity of the APLs is due to their binding to MHC, preventing the binding of self-myelin epitopes, with the absence of an immunologic response as the loss of some interactions with the TCR hinders activation of T-cells.