Oxadiazole-Based Cell Permeable Macrocyclic Transition State Inhibitors of Norovirus 3CL Protease

Abstract

Human noroviruses are the primary causative agents of acute gastroenteritis and a pressing public health burden worldwide. There are currently no vaccines or small molecule therapeutics available for the treatment or prophylaxis of norovirus infections. Norovirus 3CL protease plays a vital role in viral replication by generating structural and nonstructural proteins via the cleavage of the viral polyprotein. Thus, molecules that inhibit the viral protease may have potential therapeutic value. We describe herein the structure-based design, synthesis, and in vitro and cell-based evaluation of the first class of oxadiazole-based, permeable macrocyclic inhibitors of norovirus 3CL protease.

Putative structural rearrangements associated with the interaction of macrocyclic inhibitors with norovirus 3CL protease

“…Surprisingly, with the exception of inhibitor 5 , the IC 50 values are comparable, even in the case of inhibitor 3 that lacks the H‐bonds between Thr134 and His157 and the P 1 Gln side chain, suggesting that the positioning of inhibitors 1 to 5 in the active site is sub‐optimal and that, in addition to ring size, other factors come into play, such as, for example, the nature and orientation of the heterocyclic (1,4‐triazole) and phenyl ring in the linker and “cap”, respectively, as well as conformational effects. Similar observations were made with some recently reported 1,3,4‐oxadiazole‐based macrocyclic inhibitors …”

“…We have previously reported the structure‐guided design, synthesis and evaluation of multiple series of inhibitors of NV 3CLpro, including demonstration of efficacy in a mouse model of the disease using a dipeptidyl inhibitor . We have furthermore described the structure‐guided design of oxadiazole and triazole‐based macrocyclic transition state aldehyde inhibitors of NV 3CLpro (Figure ), as well as pertinent biochemical, structural, and high‐field NMR studies . In an attempt to gain insight and understanding into the nature of the interaction of macrocyclic inhibitors with NV 3CLpro, as well as delineate the structural elements of the inhibitors responsible for the observed potency and cellular permeability, we have determined additional high resolution X‐ray structures of NV 3CLpro with triazole‐based macrocyclic transition state aldehyde inhibitors.…”

“…12,13,18 We have furthermore described the structure-guided design of oxadiazole and triazole-based macrocyclic transition state aldehyde inhibitors of NV 3CLpro ( Figure 2), as well as pertinent biochemical, structural, and high-field NMR studies. 27,28 In an attempt to gain insight and understanding into the nature of the interaction of macrocyclic inhibitors with NV 3CLpro, as well as delineate the structural elements of the inhibitors responsible for the observed potency and cellular permeability, we have determined additional high resolution X-ray structures of NV 3CLpro with triazole-based macrocyclic transition state aldehyde inhibitors. It was envisaged that the results of our studies would place the design of macrocyclic inhibitors of NV 3CLpro on a more secure structural footing and suggest opportunities for optimizing potency, permeability, and pharmacokinetics.…”

Design, synthesis, and evaluation of a novel series of macrocyclic inhibitors of norovirus 3CL protease

“…The aldehyde and aldehyde bisulfite adducts had comparable potency, however, replacement of the warhead with an α-ketoamide (Table 1, compounds 13 and 25 versus α-ketoamide 31 ) diminished activity. These observations are congruent with the results of previous studies with peptidyl and macrocyclic inhibitors of NV 3CLpro [48–49]. Replacement of the P 2 Leu (R 2 ) residue with cyclohexylalanine (Cha) had a minor effect on potency.…”

Structure-based exploration and exploitation of the S4 subsite of norovirus 3CL protease in the design of potent and permeable inhibitors

“…Prominent viral targets that are particularly well-suited as targets for anti-noroviral drug development include the viral-encoded 3CL protease (3CLpro) and RNA dependent RNA polymerase (RdRp) because of the essential roles they play in viral replication [18]. Following translation of the viral genome, the viral polyprotein is cleaved by 3CLpro, a cysteine protease with a prototypical catalytic triad (Cys 139, His30, Glu54) and a strong preference for a P 1 Gln (or Glu) residue [21], to generate structural and nonstructural proteins [22–24] Norovirus 3CLpro has been the focus of intense investigations and an array of inhibitors of 3CLpro that display anti-norovirus activity have been reported [11–14,19], including peptidyl [25–26] and macrocyclic [27–28] transition state inhibitors and transition state mimics [29]. Importantly, in vivo proof of concept in a mouse model using a dipeptidyl transition state inhibitor of norovirus 3CLpro has also been demonstrated [25].…”