Identification of a Natural Product Antagonist against the Botulinum Neurotoxin Light Chain Protease

Abstract

Botulinum neurotoxins (BoNTs) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. BoNTs are the most lethal known poisons affecting humans and has been recognized as a potential bioterrorist threat. Current treatments for botulinum poisoning are predominately prophylactic in nature relying on passive immunization with antitoxins. Inhibition of the BoNT light chain metalloprotease (LC) has emerged as a new therapeutic strategy for the treatment of botulism that may provide an effective post-exposure remedy. A high-throughput screening effort against the light chain of BoNT serotype A (LC/A) was conducted with the John Hopkins Clinical Compound Library comprised of over 1,500 existing drugs. Lomofungin, a natural product first isolated in the late 1960’s, was identified as an inhibitor of LC/A, displaying classical noncompetitive inhibition kinetics with a Ki of 6.7 ± 0.7 µM. Inhibitor combination studies reveal that lomofungin binding is nonmutually exclusive (synergistic). The inhibition profile of lomofungin has been delineated by the use of both an active site inhibitor, 2,4-dichlorocinnamic hydroxamate, and a noncompetitive inhibitor d-chicoric acid; the mechanistic implications of these observations are discussed. Lastly, cellular efficacy was investigated using a rat primary cell model which demonstrated that lomofungin can protect against SNAP-25 cleavage, the intracellular protein target of LC/A.

Structural insight into exosite binding and discovery of novel exosite inhibitors of botulinum neurotoxin serotype A through in silico screening

“…2,4-dichlorocinnamic hydroxamate (DCH) is the smallest, most potent BoNT/A inhibitor yet (K i = 0.30 μM) and the crystal structure in complex with BoNT/A has been determined (PDB code 2IMA) [23]. The D-chicoric acid and lomofungin are exosite inhibitors of BoNT/A that have been reported from studies of Janda [21,22]. The structures of compounds were built using Discovery Studio (Accelrys, San Diego, CA) and energy minimized prior to docking.…”

“…The combination of D-chicoric acid with an active-site inhibitor, 2,4-dichlorocinnamic hydroxamate, displayed nonmutually exclusive inhibition. More interestingly, another non-competitive inhibitor, lomofungin, was identified which also exhibited synergistic inhibition against BoNT/A when used in combination with 2,4-dichlorocinnamic hydroxamate and chicoric acid [22]. While no structural evidence has been generated, it has been speculated based upon kinetic data that the binding regions of the two small molecules might map to the α- and β-exosites [22].…”

Small molecule metalloprotease inhibitor with in vitro, ex vivo and in vivo efficacy against botulinum neurotoxin serotype A

“…From the time that BoNT was revealed to be a Zn 2+ metalloprotease (Schiavo et al, 1992), efforts have focused on developing small molecule inhibitors (SMIs) targeting the protease activity of the BoNT LCs (Adler et al, 1998; Anne et al, 2001; Eubanks et al, 2010; Šilhár et al, 2013a; Duplantier et al, 2016; Bremer et al, 2017). Despite considerable effort, development of suitable drug candidates with significant in vivo efficacy has not been achieved.…”

Redoxal, an inhibitor of de novo pyrimidine biosynthesis, augments APOBEC3G antiviral activity against human immunodeficiency virus type 1

“…Lomofungin, a natural product compound first isolated from the soil-dwelling Gram-positive bacteria Streptomyces lomodensis , was previously shown to inhibit RNA synthesis in yeast, fungi, and Gram-positive and Gram-negative bacteria (Cano et al, 1973; Johnson and Dietz, 1969; Klo et al, 1973). Lomofungin was subsequently identified as an inhibitor of botulinum neurotoxin light chain protease (Eubanks et al, 2010) and as a potential therapeutic for myotonic dystrophy type 1 (Hoskins et al, 2014a). Redoxal is a synthetic compound originally identified by a computer algorithm as an inhibitor of dihydroorotate dehydrogenase (DHODH), an essential enzyme in the de novo pyrimidine synthesis pathway (Fig.…”

“…Lomofungin is a natural product originally isolated from bacteria. While the specific cellular targets of lomofungin have not been identified, its inhibition of RNA synthesis is well characterized (Cano et al, 1973; Johnson and Dietz, 1969; Klo et al, 1973) and it has also been shown to inhibit the botulinum neurotoxin light chain protease (Eubanks et al, 2010) and was identified as a potential therapeutic for myotonic dystrophy type 1 (Hoskins et al, 2014b). However, its therapeutic potential is limited by cytotoxicity, demonstrated in the present study as well as prior studies.…”