NECA derivatives exploit the paralog-specific properties of the site 3 side pocket of Grp94, the endoplasmic reticulum Hsp90

Abstract

The hsp90 chaperones govern the function of essential client proteins critical for normal cell function as well as cancer initiation and progression. Hsp90 activity is driven by ATP, which binds to the N-terminal domain and induces large conformational changes that are required for client maturation. Inhibitors targeting the ATP-binding pocket of the N-terminal domain have anticancer effects, but most bind with similar affinity to cytosolic Hsp90␣ and Hsp90␤, endoplasmic reticulum Grp94, and mitochondrial Trap1, the four cellular hsp90 paralogs. Paralog-specific inhibitors may lead to drugs with fewer side effects. The ATP-binding pockets of the four paralogs are flanked by three side pockets, termed sites 1, 2, and 3, which differ between the paralogs in their accessibility to inhibitors. Previous insights into the principles governing access to sites 1 and 2 have resulted in development of paralog-selective inhibitors targeting these sites, but the rules for selective targeting of site 3 are less clear. Earlier studies identified 5Nethylcarboxamido adenosine (NECA) as a Grp94-selective ligand. Here we use NECA and its derivatives to probe the properties of site 3. We found that derivatives that lengthen the 5 moiety of NECA improve selectivity for Grp94 over Hsp90␣. Crystal structures reveal that the derivatives extend further into site 3 of Grp94 compared with their parent compound and that selectivity is due to paralog-specific differences in ligand pose and ligand-induced conformational strain in the protein. These studies provide a structural basis for Grp94-selective inhibition using site 3.

Bisubstrate-Type Chemical Probes Identify GRP94 as a Potential Target of Cytosine-Containing Adenosine Analogs

“…Our compound 4 bears an adenosine moiety (FIG. 7), shared by GRP94-selective inhibitor NECA, 40 that binds to the ATP binding site. Additionally, NECA was also shown to interact with Site 3 of GRP94.…”

Inhibitors of the Plasmodium falciparum Hsp90 towards Selective Antimalarial Drug Design: The Past, Present and Future

“…Lastly, it has been shown that the small differences in amino acid sequences resulted in some structural variations between Hsp90 proteins from different organisms and in different cellular compartments [ 111 ]. The elucidation of several Hsp90 crystal structures has provided more avenues to identify drug targets based on unique conformations of the Hsp90 proteins [ 112 ]. These structural insights gained from the high-resolution crystal structures have led to the screening and identification of novel Hsp90 inhibitors.…”

“…This leads to a loss of membrane potential and the release of cytochrome c, resulting in selective cancer cell apoptosis [ 150 , 152 ]. Another purine-scaffold Hsp90 paralog selective inhibitor of Grp94, N-ethyl-carboximido adenosine (NECA), was identified ( Figure 7 ) [ 112 , 113 ]. The selectivity of NECA and its derivatives N-propylcarboxamido adenosine (NPCA), N-hydroxyethylcarboxamido adenosine (NEoCA), and N-aminoethycarboxamido adenosine (NEaCA) [ 112 ] for Grp94 are based on the N-ethyl carboximido moiety that extends into the third hydrophobic pocket that surrounds the ATP binding pocket of Grp94.…”

“…Another purine-scaffold Hsp90 paralog selective inhibitor of Grp94, N-ethyl-carboximido adenosine (NECA), was identified ( Figure 7 ) [ 112 , 113 ]. The selectivity of NECA and its derivatives N-propylcarboxamido adenosine (NPCA), N-hydroxyethylcarboxamido adenosine (NEoCA), and N-aminoethycarboxamido adenosine (NEaCA) [ 112 ] for Grp94 are based on the N-ethyl carboximido moiety that extends into the third hydrophobic pocket that surrounds the ATP binding pocket of Grp94. Furthermore, three purine derivatives, 8-((2,4-Dimethylphenyl)thio)-3-(pent-4-yn-1-yl)-3H-purin-6-amine (PU-H54), 6-amino-8-(C3,5-dichlorophenyl)thio)-N-(1-methylethyl)-9H-purine-9-propanamine (PU-WS13) and 8-((2,4-Dichlorophenyl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (PU-H39) were identified as potential paralog selective inhibitors ( Figure 6 ) [ 99 , 149 ].…”

“…On the other hand, with high sequence similarities between Hsp90s localized in equivalent cell compartments from different species, the development of paralog-specific Hsp90 inhibitors is an attractive avenue worth exploring. As such, several Hsp90 paralog selective inhibitors have been developed [ 112 , 162 , 163 , 187 , 188 ]. These paralog selective inhibitors, if adopted to the parasitic PfHsp90_A apicoplast resident chaperone, may offer interesting possibilities, since this target is only present in apicomplexans and not in humans.…”