key: cord-102151-26lumewy
authors: Cox, Robert M.; Sourimant, Julien; Govindarajan, Mugunthan; Natchus, Michael G.; Plemper, Richard K.
title: Therapeutic Targeting of Measles Virus Polymerase with ERDRP-0519 Suppresses All RNA Synthesis Activity
date: 2020-09-24
journal: bioRxiv
DOI: 10.1101/2020.09.23.311043
sha: 
doc_id: 102151
cord_uid: 26lumewy

Morbilliviruses, such as measles virus (MeV) and canine distemper virus (CDV), are highly infectious members of the paramyxovirus family. MeV is responsible for major morbidity and mortality in non-vaccinated populations. ERDRP-0519, a pan-morbillivirus small molecule inhibitor for the treatment of measles, targets the morbillivirus RNA-dependent RNA-polymerase (RdRP) complex and displayed unparalleled oral efficacy against lethal infection of ferrets with CDV, an established surrogate model for human measles. Resistance profiling identified the L subunit of the RdRP, which harbors all enzymatic activity of the polymerase complex, as the molecular target of inhibition. Here, we examined binding characteristics, physical docking site, and the molecular mechanism of action of ERDRP-0519 through label-free biolayer interferometry, photoaffinity cross-linking, and in vitro RdRP assays using purified MeV RdRP complexes and synthetic templates. Results demonstrate that unlike all other mononegavirus small molecule inhibitors identified to date, ERDRP-0519 inhibits all phosphodiester bond formation in both de novo initiation of RNA synthesis at the promoter and RNA elongation by a committed polymerase complex. Photocrosslinking and resistance profiling-informed ligand docking revealed that this unprecedented mechanism of action of ERDRP-0519 is due to simultaneous engagement of the L protein polyribonucleotidyl transferase (PRNTase)-like domain and the flexible intrusion loop by the compound, pharmacologically locking the polymerase in pre-initiation conformation. This study informs selection of ERDRP-0519 as clinical candidate for measles therapy and identifies a previously unrecognized druggable site in mononegavirus L polymerase proteins that can silence all synthesis of viral RNA. Importance The mononegavirus order contains major established and recently emerged human pathogens. Despite the threat to human health, antiviral therapeutics directed against this order remain understudied. The mononegavirus polymerase complex represents a promising drug target due to its central importance for both virus replication and viral mitigation of the innate host antiviral response. In this study, we have mechanistically characterized a clinical candidate small-molecule MeV polymerase inhibitor. The compound blocked all phosphodiester bond formation activity, a unique mechanism of action unlike all other known mononegavirus polymerase inhibitors. Photocrosslinking-based target site mapping demonstrated that this class-defining prototype inhibitor stabilizes a pre-initiation conformation of the viral polymerase complex that sterically cannot accommodate template RNA. Function-equivalent druggable sites exist in all mononegavirus polymerases. In addition to its direct anti-MeV impact, the insight gained in this study can therefore serve as a blueprint for indication spectrum expansion through structure-informed scaffold engineering or targeted drug discovery.

The mononegavirus order contains major established and recently emerged human pathogens.

Despite the threat to human health, antiviral therapeutics directed against this order remain 22 understudied. The mononegavirus polymerase complex represents a promising drug target due to its 23 central importance for both virus replication and viral mitigation of the innate host antiviral response. In 24 this study, we have mechanistically characterized a clinical candidate small-molecule MeV polymerase 25 inhibitor. The compound blocked all phosphodiester bond formation activity, a unique mechanism of 26 action unlike all other known mononegavirus polymerase inhibitors. Photocrosslinking-based target site 27 mapping demonstrated that this class-defining prototype inhibitor stabilizes a pre-initiation conformation 28 of the viral polymerase complex that sterically cannot accommodate template RNA. Function-equivalent 29 druggable sites exist in all mononegavirus polymerases. In addition to its direct anti-MeV impact, the 30 insight gained in this study can therefore serve as a blueprint for indication spectrum expansion through

Morbilliviruses belong to the paramyxovirus family of highly contagious respiratory RNA viruses

Several cryo-electron microscopy-based reconstructions of mononegavirus polymerase proteins 100 have been reported, including structural models of PIV-5 and RSV L polymerases that are closely 101 related to MeV L (31, 32). We have previously identified signature resistance hot-spots for ERDRP-102 0519 in MeV and CDV L through viral adaptation (10, 12) . Escape sites locate to the polymerase and 

A catalytically defective L mutant harboring an N774A substitution in the polymerase active site 153 confirmed that RNA synthesis was MeV P-L specific and not due to co-purified cellular contaminants.

Polymerase complexes containing an ERDRP-0519 resistance mutations H589Y or T776A showed 155 greatly reduced susceptibility to compound-mediated suppression of de novo polymerase initiation, 156 reflected by inhibitory concentrations of 3.64 µM and 3.22 µM, respectively, which represents a 24-and backpriming and/or primer extension (27, 28, 34) . Backpriming refers to the spontaneous formation of a resulting paired 3'-ends beyond the actual length of the template (figure 4A). To assess whether the 179 same limitation applies to ERDRP-0519, we applied the MeV polymerase complex to a previously 180 described 25-mer RNA template derived from an authentic RSV promoter sequence that is capable of 181 efficient backpriming (35). The RSV template was efficiently recognized as a suitable substrate for de 

In conclusion, these results consistently demonstrate that ERDRP-0519 directly suppresses all 192 phosphodiester bond formation, setting the compound mechanistically apart from all other allosteric 193 mononegavirus inhibitor classes characterized to date.

Photoaffinity labeling maps the ERDRP-0519 target site to the central L cavity

To map the physical target site of ERDRP-0519, we synthesized a photo-activatable analog of the 196 compound, ERDRP-0519 az , through installation of an aryl azide moiety at C-2 position of the ERDRP-197 0519 piperidine ring via a short tether (figure 5A). Analog design was guided by our extensive insight 198 into the structure-activity relationship (SAR) of the ERDRP-0519 chemotype that we had acquired in 199 previous work (11). Bioactivity testing of ERDRP-0519 az against MeV revealed dose-dependent 200 suppression of virus replication and an EC 50 of 12.1 µM (figure 5B). No cytotoxicity was detectable at 201 the highest concentration tested (100 µM), indicating specific virus inhibition by the photo-activatable probe. Compared to ERDRP-0519, however, antiviral potency of ERDRP-0519 az was reduced 203 approximately 50-fold, which may reflect reduced plasma membrane permeability of the azide analog in 204 cell-based assays or partially impaired target access of the modified compound. We compensated for 205 this reduction in activity by incubating the P-L complexes in the presence of 40 µM compound prior to 206 photo-activation.

Photo-coupling of ERDRP-0519 az to purified L followed by LC-MS/MS analysis after trypsin 208 digestion of the ligand-L complexes identified three discrete peptides that are located in the capping 

We therefore concentrated further analysis on peptides 1 and 2, which are near the intersection 217 between the polymerase capping, connector and MTase domains, and in close proximity to highly 218 conserved polymerase motifs such as the proposed PRNTase domain (HR moiety of motif D and 219 G1156 of motif A) as well as both the postulated paramyxovirus L priming and "intrusion" loops (32) 220 (figure 5E).

Overlaying the photo-crosslinking results and resistance maps in the MeV L structural model 223 revealed a circular arrangement of all potential ERDRP-0519 anchor points along the interior lining of 224 the central polymerase cavity (figure 6A). Docking of ERDRP-0519 into the L structure was guided by 225 the following constraints: positioning of the ligand is compatible with the formation of covalent bonds 226 with residues in photo-crosslinking defined peptides 1 and 2; and the docked compound is in equal ERDRP-0519, two chemical analogs, the original screening hit 16677 (13) and the first-generation lead the intersection of MeV L capping and RdRP domains, between motifs A and D of the predicted and 2.7-10.4Å distance to peptide 2, establishing hydrogen bond interactions between residue Y1155 in 

Individual models were ranked based on goodness of fit (R 2 ) and predictive capacity (q 2 ) against the 

Consistent with this conclusion, none of these three inhibitor classes affects extension of the RNA 303 template after backpriming (27, 28, 34) , which is considered to mimic RNA elongation by a committed

Characterization of ERDRP-0519 demonstrated that preventing the switch to elongation mode is 306 not the only mechanism available to allosteric small molecule inhibitors to block mononegavirus 307 polymerases, since the compound interrupted both initiation at the promoter and RNA elongation after 308 backpriming with equal potency. Underscoring a unique mechanism of action of ERDRP-0519, 

Although three peptides were identified by photocrosslinking, we primarily focused on peptides 1 320 and 2. Peptide 3 is located in a highly variable and unstructured region that is completely solvent 

Hydrogen bonding between the sulfonyl group of ERDRP-0519 and this residue is very likely 342 catastrophic for the formation of productive initiation complexes.

Confirmed resistance sites to ERDRP-0519 line the internal wall of the central polymerase cavity, 344 but individual hot-spots do not cluster in the native structure and none is predicted to be in direct 345 contact with the docked ligand. Remarkably, however, resistance sites were located in highly sequence 346 conserved L domains such as on the priming and intrusion loops (i.e. H589Y, R1233Q, and V1239A) 347 and/or in immediate proximity of functional motifs (i.e. S768A and T776A framing the GDNQ catalytic 348 center). We conclude that escape from ERDRP-0519 is mediated by secondary structural effects rather 349 than due to primary resistance, which is unusual for non-nucleoside analog polymerase inhibitors (51- Purified MeV L 1708 was incubated with 40 µM ERDRP-0519 az for 15 minutes prior to activating the 510 crosslinker. The MeV L1708 -ERDRP-0519 az mixture was incubated on ice and exposed to UV light 511 (365 nm) for 30 min. The sample was then exposed to additional UV light (254 nm) for 15 minutes.

Protein was then collected using FLAG resin. Bound resin was then incubated with Laemmli buffer at 513 56°C for 15 minutes. SDS-PAGE electrophoreses was then performed using Laemmli buffer on 4-15% 514 acrylamide gels. Bands of interest were excised and analyzed by mass spectrometry. ERDRP-0519 az 515 crosslinked peptides were identified by the Proteomics & Metabolomics Facility at the Wistar Institute 516 as previously described (28). In order to identify ERDRP-0519 az crosslinked peptides, mass addition of and created 287 initial pharmacophore models based on the training set. After electrostatic, steric, and 532 space filling model building was performed, a partial-least squares analysis was performed and each 533 model was validated, scored, and ranked based on LOO correlation (q 2 ) and goodness of fit (R 2 ). The predictive potential of the model was then tested using a conformation database of the test set. 

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