X-ray Crystal Structures of Escherichia coli RNA Polymerase with Switch Region Binding Inhibitors Enable Rational Design of Squaramides with an Improved Fraction Unbound to Human Plasma Protein

Abstract

Squaramides constitute a novel class of RNA polymerase inhibitors of which genetic evidence and computational modeling previously have suggested an inhibitory mechanism mediated by binding to the RNA polymerase switch region. An iterative chemistry program increased the fraction unbound to human plasma protein from below minimum detection levels, i.e. <1%, to 4~6%, while retaining biochemical potency. Since in vitro antimicrobial activity against an efflux-negative strain of Haemophilus influenzae was 4~8-fold higher, the combined improvement was at least 20~60-fold. Co-crystal structures of Escherichia coli RNA polymerase with two key squaramides showed displacement of the switch 2, predicted to interfere with the conformational change of clamp domain and/or with binding of non-template DNA, a mechanism akin to that of natural product myxopyronin. Furthermore, the structures confirmed the chemical features required for biochemical potency. The terminal isoxazole and benzyl rings bind into distinct relatively narrow, hydrophobic pockets and both are required for biochemical potency. In contrast, the linker composed of squarate and piperidine accesses different conformations in their respective co-crystal structures with RNA polymerase, reflecting its main role of proper orientation of the aforementioned terminal rings. These observations further explain the tolerance of hydrophilic substitutions in the linker region that was exploited to improve the fraction unbound to human plasma protein while retaining biochemical potency.

An Introduction to the Structure and Function of the Catalytic Core Enzyme of Escherichia coli RNA Polymerase

“…The near-atomic resolution X-ray crystal structure of E. coli RNAP was determined first as holoenzyme containing σ 70 (Murakami, 2013). X-ray crystal and cryo-electron microscopy (cryo-EM) structures of E. coli RNAP are now available in several forms such as holoenzymes containing alternative σ factors (Liu et al, 2016; Yang et al, 2015), promoter DNA complexes (Glyde et al, 2017; Zuo and Steitz, 2015), elongation complex (Kang et al, 2017), in complex with transcription factors (Bae et al, 2013; Liu et al, 2017; Molodtsov et al, 2018), and with inhibitors/antibiotics (Bae et al, 2015; Chen et al, 2017; Degen et al, 2014; Molodtsov et al, 2015; Molodtsov et al, 2013), providing details of the structure and function of E. coli RNAP transcription and regulation.…”

Bacterial Transcription as a Target for Antibacterial Drug Development

“…2, circles 3, 4, and 5, respectively). Cocrystal structures of two squaramides with E. coli RNAP holoenzyme have recently been published, which showed that their major binding site was broadly similar to that of the myxopyronins within the switch 1 and 2 regions, with less substantial interaction with switch regions 3 and 4 (140). The cocrystal structures also demonstrated that the binding of squaramide compounds to RNAP pushed switch 2 into the DNA binding main channel of RNAP, which would prevent the correct positioning of the melted template DNA.…”

Bacterial RNA Polymerase-DNA Interaction—The Driving Force of Gene Expression and the Target for Drug Action

“…Compounds like myxopyronins, corallopyronin (Mukhopadhyay et al, 2008), ripostatin (Mukhopadhyay et al, 2008; Belogurov et al, 2009), and squaramides (Buurman et al, 2012; Molodtsov et al, 2015) bind to RNAP β′ switch-2 region that controls the hinged, swinging motion of β′ clamp, which in turn is responsible for the opening and closing of the primary channel (Srivastava et al, 2011). Binding of these compounds prevents the β′ clamp from opening, stabilizes the β′ clamp/switch regions in a partly closed/fully closed conformation, and prevents template DNA from reaching the active site.…”

“…Binding of these compounds prevents the β′ clamp from opening, stabilizes the β′ clamp/switch regions in a partly closed/fully closed conformation, and prevents template DNA from reaching the active site. In particular, squaramide, in its co-crystal structure with RNAP, is shown to displace β′ switch-2 into the DNA binding main channel of RNAP (Molodtsov et al, 2015), which would interfere with proper placement of the melted template DNA (Bae et al, 2015b). Fidaxomicin and lipiarmycin (Tupin et al, 2010; Artsimovitch et al, 2012; Morichaud et al, 2016) are structurally closely related natural compounds that also bind to the β′ switch-2 region and prevent t-strand DNA from accessing the RNAP active-site cleft.…”

“…Beginning in 2013, high-resolution structures of E. coil ( Eco ) RNAP holoenzyme and its complexes with nucleic acid and inhibitors began to emerge from several groups. Altogether more than 24 high resolution structures of RNAP/RNAP complexes to date have been deposited to database (Zhang et al, 2012, 2014; Bae et al, 2013, 2015a,b), (Bae et al, 2015c; Molodtsov et al, 2013, 2015; Murakami, 2013; Sarkar et al, 2013), (Zuo et al, 2013; Basu et al, 2014; Degen et al, 2014; Feng et al, 2015, 2016; Liu et al, 2015, 2016; Yang et al, 2015b; Zuo and Steitz, 2015). These include: the structures of Taq core; Eco core with transcription factor, RapA; Taq, Tth , and Eco holoenzymes and open promoter complexes; Tth ternary elongation complexes with and without transcription factors (Gfh1, GreA); Taq and Tth RNAP complexes with small-molecule inhibitors and antibiotics.…”