Allosteric Drugs: The Interaction of Antitumor Compound MKT-077 with Human Hsp70 Chaperones

Abstract

The Hsp70 chaperones (Heat shock protein 70 kDa) are key to cellular protein homeostatis. However, they also have the ability to inhibit tumor apoptosis, and contribute to aberrant accumulation of hyperphosphorylated tau in neuronal cells affected by tauopathies, including Alzheimer’s disease. Hence, Hsp70 are increasingly been identified as targets for therapeutic intervention in these widely abundant diseases. Hsp70 proteins are allosteric machines and offer besides classical active site targets, also opportunities to target the mechanism of allostery. In this work, it is demonstrated that the action of the potent anti-cancer compound MKT-077, is through differential interaction with the Hsp70 allosteric states. MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4- oxothiazolidin-2-ylidenemethyl] pyridinium chloride) is therefore an “allosteric drug”. Using NMR spectroscopy, the compound’s binding site on human HSPA8 (Hsc70) is identified. The binding pose is obtained from NMR-restrained docking calculations, subsequently scored by molecular dynamics-based energy and solvation computations. Suggestions for improvement of the compound’s properties are made on the basis of the binding location and pose.

Synthesis and Initial Evaluation of YM-08, a Blood-Brain Barrier Permeable Derivative of the Heat Shock Protein 70 (Hsp70) Inhibitor MKT-077, Which Reduces Tau Levels

“…The results showed that YM-08 (30 μM) decreased the levels of pS396/404 and total tau by ∼40% and ∼60%, respectively ( Figure 4C). This cellular activity is not as dramatic as that of MKT-077, which reduced tau levels by >80% at 30 μM ( Figure 4D); 23 however, reducing tau by only ∼50% is predicted to provide benefits in some AD models. 4 Importantly, we also confirmed that, like MKT-077 and other Hsp70 inhibitors, 12,41,42 YM-08 did not induce a stress response, based on the unchanged levels of stress inducible Hsp72 (HSPA1) in the treated cell lysates ( Figure 4C).…”

“…This pocket is framed by a number of residues, including S208, S221, T222, D225, H227, and L228 ( Figure 3C) that are known to be sensitive to addition of MKT-077. 23 When the side chains of these residues were allowed to freely rotate, they adjusted to define the YM08-binding pocket (see Figure 3A). In this orientation, the benzothiazole of YM-08 was predicted to access a deep cleft composed of hydrophobic and cationic residues (T12, K70, R71, R75, V81, Y148, T203, G223, and T225).…”

“…In previous work, MKT-077 stabilized the interaction between prokaryotic Hsp70 (DnaK) and denatured luciferase. 23 Briefly, this assay involves immobilizing unfolded luciferase in microtiter plates and measuring binding to DnaK. 34 Using this approach, we tested the MKT-077 analogues and found that MKT-077, YM-01, and YM-08 (50 μM) all significantly enhanced binding by ∼30% ( Figure 4B).…”

“…In control experiments, the biotinylated MKT-077 bound to human Hsp72 (4.9 ± 0.8 μM) and DnaK (16.7 ± 3.1 μM) with similar affinities to human Hsc70 (6.4 ± 0.23 μM), reinforcing the similarity among the Hsp70 family members in the reported MKT-077 binding site. 23 Biolayer Interferometry (Octet Red). Biotinylated human Hsc70 NBD or full length Hsp72 was first immobilized on super streptavidin biosensors (ForteBio, 18-5057) as follows: biosensors were soaked with MG buffer (100 mM Tris base, 20 mM KCl, 6 mM MgCl 2 , 0.01% Triton X-100, pH 7.4) for 10 min before moving them to wells containing 200 μL of either 100 μg/mL biotinylated proteins or 100 μg/mL biocytin for 60 min.…”

The active Hsc70/tau complex can be exploited to enhance tau turnover without damaging microtubule dynamics

“…One of the reasons for this slower development of Hsp70 inhibitors has been that there are 10 isoforms in mammalian cells and selective inhibitors are currently not available (9)(10)(11)(12)(13). Moreover, Hsp70 proteins are very promiscuous, affecting many proteins in the cell, while Hsp90 proteins are slightly more discriminating in regard to their clientele.…”