Author response: Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action

Abstract

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition.

Elucidation of Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein 1 Using a Chemical “Toolkit”

“…Dynapyrazole, Ciliobrevin and their Analogs In vitro and in vivo studies of ciliobrevin A and D, the two ATP-competitive ligands, have shown that they non-selectively bind to the ATP binding sites of the hexameric head of both cytoplasmic dynein 1 and dynein 2 [7,10]. Dynapyrazole A and B resulted from a chemical structure modification to produce ciliobrevin analogs with higher potency [26], for overcoming geometric isomerization complexity caused by the C8-C11 double bond in ciliobrevin. (Figure 4) Unlike the ciliobrevin analogs, which abrogate both MT-stimulated and basal ATPase activity, dynapyrazole analogs inhibit MT-stimulated ATPase activity with high potency without affecting basal ATPase activity [26].…”

“…Dynapyrazole A and B resulted from a chemical structure modification to produce ciliobrevin analogs with higher potency [26], for overcoming geometric isomerization complexity caused by the C8-C11 double bond in ciliobrevin. (Figure 4) Unlike the ciliobrevin analogs, which abrogate both MT-stimulated and basal ATPase activity, dynapyrazole analogs inhibit MT-stimulated ATPase activity with high potency without affecting basal ATPase activity [26]. This feature resembles She1, a microtubule-associated protein (MAP) that effectively reduces MT-stimulated ATPase activity without significantly decreasing its basal activity [35].…”

“…isomer since the one-dimensional NMR spectrum and the result of a 2D Nuclear Overhauser Effect Spectroscopy (NOESY) of ciliobrevin D, has shown an intramolecular H-bond between the hydrogen atom on the N7 and the O22 that stabilizes ciliobrevin D in solution [26]. The N7 is directly attached to the C8, and the O22 relates to the C11 via the double bond to the C13, which is covalently attached to the C11.…”

“…Three-dimensional structures of the ligands were built up individually and minimized stepwise using the steepest descent algorithm according to the Tripos force field, with 0.0001 kJ/mol energy gradient and 10,000,000 iterations. The library contained previously synthesized and in vitro studied 46 analogs of ciliobrevin [10], dynapyrazole A and B [26], and the protonated forms of the lead compounds ciliobrevin A and D, dynapyrazole A and B modeled in silico. It also included the nucleotides ATP, ADP, and AMPPNP, a non-hydrolyzable analog of ATP.…”

“…The potency of the ciliobrevin was thought to be affected by isomerization, where there is only a fraction of the isomer abrogating dynein [4]. The benzoylacrylonitrile group of the molecule favors the E isomer since the one-dimensional NMR spectrum and the result of a 2D Nuclear Overhauser Effect Spectroscopy (NOESY) of ciliobrevin D, has shown an intramolecular H-bond between the hydrogen atom on the N7 and the O22 that stabilizes ciliobrevin D in solution [26]. The N7 is directly attached to the C8, and the O22 relates to the C11 via the double bond to the C13, which is covalently attached to the C11.…”