Inhibitor Design

2 hybridized α-carbon relative to the hydroxamate, Several strategies exist to achieve selectivity for HDAC6 over other HDAC paralogues with hydroxamate inhibitors. These include the use of (1) large polycyclic capping groups, (3, 20, 21) (2) a substituted sphybridized α-carbon relative to the hydroxamate, (22) and (3) sterically demanding substitution close to the chelating group. (7, 8) On the basis of these strategies, combined with our experience in PET radiotracer design for HDACs, (23-26) we developed a small library of potential CNS-penetrant HDAC6 inhibitors. Of these, Bavarostat ( Figure 1 A) emerged as unique. In Figure 1 B,C, the structure of Bavarostat is shown docked into the catalytic domain 2 (CD2) of human HDAC6, providing a model to the contribution of structural elements to its selectivity for HDAC6 over all other HDAC paralogues ( Figure 1 D) and exemplified by comparison to HDAC2.

Figure 1 Figure 1. A: Structure of Bavarostat (1). B: Alignment of hHDAC6’s catalytic domain 2 (CD2) (white and cyan protein, PDB 5EDU) and the 15 zHDAC6 CD2 crystal structures available in the PDB database (wheat colored proteins), showing similar L1 loop and L1 helix conformations. HDAC6-selective inhibitor Bavarostat was docked into the hHDAC6 complex described in the Experimental Section. Bavarostat is predicted to form a hydrogen bond (yellow dashed line) with Ser568 on the hHDAC6 L1 loop segment represented in cyan. C: Alignment of hHDAC6 CD2 (white and cyan) and the five hHDAC2 catalytic site crystal structures (pink proteins) in PDB. Large movement of both the L1 loop segment and the L1 helix is represented by arrows (arrows depict movement of analogous amino acids from hHDAC6 to hHDAC2: left arrow depicts hHDAC6’s Ile571 going to corresponding hHDAC2’s Asp100′, and right arrow depicts hHDAC6’s Arg557 going to corresponding hHDAC2’s Ser86′). Prime indicates hHDAC2. The hypothesized steric and electrostatic clash between Bavarostat and the hHDAC2 L1 loop is represented in red. D: IC 50 values for Bavarostat inhibition of HDAC1–11 were determined in a fluorescence assay (BPS Bioscience, San Diego, CA) measuring acetylation of a synthetic substrate. E: Bavarostat docked into a hHDAC6 CD2 complex incorporating a conserved water. Key amino acids chelating the catalytic zinc or lining the 10 Å channel are labeled. F/G: Significant topography differences between the surface of the catalytic domains of hHDAC6 (F) and hHDAC2 (G) are highlighted by the pink contouring of the L1 loop segment of hHDAC2.

2-hybridized hydroxamate α-carbon atom) in Bavarostat prevents an effective κ2-binding mode of the hydroxamate warhead. Since a high-resolution crystal structure of zHDAC6 bound to a phenyl-linked HDAC6-selective inhibitor, HPOB in PDB Bavarostat was docked into an hHDAC6 complex to rationalize its selectivity (see Experimental Section for further details). Briefly, the hydroxamic acid of Bavarostat was modeled to chelate the catalytic zinc and form a hydrogen bond to an ordered water in a similar fashion as seen in the zHDAC6 structure (PDB 5EF7 ) recently published by Hai et al. (27) Indeed, the rigidity introduced by the linker phenyl (sp-hybridized hydroxamate α-carbon atom) in Bavarostat prevents an effective κ-binding mode of the hydroxamate warhead. Since a high-resolution crystal structure of zHDAC6 bound to a phenyl-linked HDAC6-selective inhibitor, HPOB in PDB 5EF7 , showed a unique mode of binding to the catalytic zinc ion via a conserved water molecule, (27) we applied it to Bavarostat. The presence of this water molecule greatly improved the fit of Bavarostat to the protein, suggesting that the rigid phenyl linker moiety is partially conveying HDAC6 selectivity. Additional discriminating factors, such as sterically demanding moieties, were employed to convey paralogue selectivity and potency for HDAC6. First, the fluorophenyl of Bavarostat occupying the 10 Å channel leading to the protein surface forms a favorable noncovalent interaction with Phe620 via a parallel-displaced π-stacking configuration ( Figure 1 E). The fluorine substituent, installed with imaging applications in mind in a position not expected to interfere with HDAC binding, (26) contributes to favorable van der Waals interactions by vectoring in a small divot in the 10 Å channel ( Figure SI3 ).

Second, the proton of the tertiary amine off of the linker phenyl ring directly hydrogen bonds to Ser568 on the L1 loop ( Figures 1 B and 1 E). Finally, the adamantyl group in Bavarostat is predicted to fill a hydrophobic surface groove adjacent to the L1 loop in hHDAC6 complex’s CD2 ( Figure 1 F). To understand if the conformation of the L1 loop and L1 helix found in the hHDAC6 CD2 structure (PDB 5EDU ) was an anomaly due to the crystallization technique used or the result of the bound ligand that was crystallized with it, all zHDAC6 CD2 structures available were aligned to hHDAC6 CD2 (27) Figure 1 B). The L1 loop and L1 helix in CD2 of hHDAC6 and CD2 of zHDAC6 structures consistently folded in the same conformation, suggesting that the L1 loop and L1 helix of hHDAC6 are in a stable low energy conformation. The selectivity profile of Bavarostat ( Figure 1 D) shows that it is highly selective for hHDAC6 over hHDAC2. To rationalize how Bavarostat may have gained such selectivity, the catalytic site of hHDAC2 was used as a comparison and a representative for class I HDACs. We aligned the CD2 structure of hHDAC6 (PDB 5EDU ) to all available CD1 hHDAC2 structures to see if the L1 loop and L1 helix were in a similar conformation since the adamantyl of Bavarostat sits on a hydrophobic groove next to the L1 loop. The L1 loop and L1 helix of hHDAC2 were found to be in a consistent conformation across all available structures, suggesting that this folded state is also stable. This conformation, however, is significantly different relative to CD2 hHDAC6 as exemplified by the large movement of both the L1 loop segment and the L1 helix depicted by arrows in Figures 1 B and 1 C). The L1 loop/L1 helix conformation of CD1 of hHDAC2 would sterically clash with the bound Bavarostat compound ( Figure 1 C), preventing its positioning into the analogous hydrophobic groove ( Figure 1 G).