Molecular basis of USP7 inhibition by selective small-molecule inhibitors.

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.

Heteropoda toxin 2 interaction with Kv4.3 and Kv4.1 reveals differences in gating modification.

Kv4 (Shal) potassium channels are responsible for the transient outward K(+) currents in mammalian hearts and central nervous systems. Heteropoda toxin 2 (HpTx2) is an inhibitor cysteine knot peptide toxin specific for Kv4 channels that inhibits gating of Kv4.3 in the voltage-dependent manner typical for this type of toxin. HpTx2 interacts with four independent binding sites containing two conserved hydrophobic amino acids in the S3b transmembrane segments of Kv4.3 and the closely related Kv4.1. Despite these similarities, HpTx2 interaction with Kv4.1 is considerably less voltage-dependent, has smaller shifts in the voltage-dependences of conductance and steady-state inactivation, and a 3-fold higher K(d) value. Swapping four nonconserved amino acids in S3b between the two channels exchanges the phenotypic response to HpTx2. To understand these differences in gating modification, we constructed Markov models of Kv4.3 and Kv4.1 activation gating in the presence of HpTx2. Both models feature a series of voltage-dependent steps leading to a final voltage-independent transition to the open state and closely replicate the experimental data. Interaction with HpTx2 increases the energy barrier for channel opening by slowing activation and accelerating deactivation. The greater degree of voltage-dependence in Kv4.3 occurs because it is the voltage-dependent transitions that are most affected by HpTx2; in contrast, it is the voltage-independent step in Kv4.1 that is most affected by the presence of toxin. These data demonstrate the basis for subtype-specificity of HpTx2 and point the way to a general model of gating modifier toxin interaction with voltage-gated ion channels.

Heteropoda toxin 2 is a gating modifier toxin specific for voltage-gated K+ channels of the Kv4 family.

Kv4 voltage-gated K(+) channels are responsible for transient K(+) currents in the central nervous system and in the heart. HpTx2 is a peptide toxin that selectively inhibits these currents; making it a useful probe for understanding Kv4 channel structure and drug binding. Therefore, we developed a method to produce large amounts of recombinant HpTx2. Recombinant toxin inhibits all three Kv4 isoforms to the same degree; however, the voltage-dependence of inhibition is less apparent for Kv4.1 than for Kv4.3. Similarly, recombinant HpTx2(GS) effects gating characteristics of both channels, but Kv4.1 to a much lesser degree. The toxin lacks affinity for Kv1.4, Kv2.1, and Kv3.4. To locate the binding site, the amino acids linking the third and forth membrane spanning segments of Kv4.3 were replaced with analogous amino acids of Kv1.4. The chimeric K(+) channel was completely insensitive to block by rHpTx2, suggesting that its binding site is near the channel's voltage sensor. These data show that rHpTx2(GS) is a gating modifier toxin that binds to a site remote from the pore.