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Re-evaluating strategies of cancer treatment based on a novel class of H4-dependent PARP-1 inhibitors

1) Novel H4-dependent and classical NAD-like inhibitors act on PARP-1 through different molecular mechanisms.


The clinical potential of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors has been increasingly recognized over the past two decades. However, recent studies re-discovered most of the drug candidates inhibiting PARPs as generally lacking target specificity, with off-target activities occurring both at the intra- family and inter-family levels. Classical PARP-1 inhibitors were designed to compete with NAD at the enzyme active site (Fig. 1). However, because they are largely based on benzamide or purine structures, such compounds also inhibit other enzymes that use NAD, including other members of the PARP family, mono-ADP-ribosyl-transferases and sirtuins. Therefore, inhibition of PARP-1 by competing with NAD tends to affect a number of other metabolic processes producing multiple off-target effects. We have developed a novel class of PARP-1 inhibitors by targeting the histone H4 route of PARP-1 activation, a mechanism that is unique to PARP-1 (Fig. 1). H4-dependent PARP-1 inhibitors have no obvious structural homologues among components of eukaryotic enzymatic pathways, thus minimizing the off-target effects and ensuring greater specificity.


Mechanism of action of novel H4-dependent PARP-1 inhibitors is completely different from that of the “classical” NAD-like PARP-1 inhibitors. The classical PARP-1 inhibitors stabilize binding of PARP-1 to the activator histone H4, while H4-
dependent PARP-1 inhibitors disruptPARP-1 binding to histone H4 (Fig. 2A). Therefore, NAD competitors arrest PARP-1- activator complex in transient conformation, while new H4-dependent PARP-1 inhibitors disrupt this complex and exclude PARP-1 from functional complexes. Thus, H4-dependent PARP-1 inhibitors repress transcription of PARP-1- dependent genes more effectively than NAD-like PARP-1inhibitors. Importantly, H4-dependent PARP-1 inhibitors are active against PARP-1 isoform lacking catalytic domain, the target of NAD-like PARP-1 inhibitors (Fig. 2A and B). These results validate that H4- dependent and NAD-like PARP-1 inhibitors act through two distinct mechanisms. Notably H4-dependent PARP-1 inhibitors are highly specific for PARP-1 inhibition (Fig. 3).


Mechanism of action of novel H4-dependent PARP-1 inhibitors is completely different from that of the “classical” NAD-like PARP-1 inhibitors. The classical PARP-1 inhibitors stabilize binding of PARP-1 to the activator histone H4, while H4-
dependent PARP-1 inhibitors disrupt PARP-1 binding to histone H4 (Fig. 2A). Therefore, NAD competitors arrest PARP-1- activator complex in transient conformation, while new H4-dependent PARP-1 inhibitors disrupt this complex and exclude PARP-1 from functional complexes. Thus, H4-dependent PARP-1 inhibitors repress transcription of PARP-1- dependent genes more effectively than NAD-like PARP-1inhibitors. Importantly, H4-dependent PARP-1 inhibitors are active against PARP-1 isoform lacking catalytic domain, the target of NAD-like PARP-1 inhibitors (Fig. 2A and B). These results validate that H4- dependent and NAD-like PARP-1 inhibitors act through two distinct mechanisms. Notably,H4
-dependent PARP-1 inhibitors are highly specific for PARP-1 inhibition (Fig. 3).


Figure 3.5F02 (5mkM) suppresses PARP-1, but not Tankyrase-1 (USCN) or PARP-2


2)H4-dependent PARP-1 inhibitor 5F02 inhibits the growth of sunitinib- and olaparib-resistantxenograft tumors in an animal model of human renal cell carcinoma.
Using sunitinib-resistant renal cell carcinoma (RCC) as a model, we found that our lead compound 5F02 demonstrated significant antitumor activity against xenograft RCC tumors. As demonstrated in Figure 4, animals treated with 5F02 showed a significant inhibition of PNX0010 RCC xenograft tumor growth relative to control animals, animals treated with sunitinib and animals treated with olaparib. Notably, 5F02 demonstrated good oral activity (Fig.4). Similar results were
obtained using mice bearing castration-resistant PC-3 prostate cancer xenografts treated with 5F02 and olaparib.

Figure 4. 5F02 suppresses growth of RCC xenograft tumors. Xenograft tumors were established in 6-week-old male C.B17/Icr-scid mice using PNX0010 RCC cells generated from a clinical specimen of kidney cancer resistant to sunitinib
treatment. Animals were treated with H4- dependent PARP-1inhibitor 5F02 (4 mg/kg intravenously (i.v.) or 6 mg/kg orally(p.o.)), classical PARP-1 inhibitor olaparib (20 mg/kg, i.v.),sunitinib (40 mg/kg, p.o.) or vehicle 5 days a week. Values
shown represent means (n=5) + SEM. Data were fitted to exponential growth models using nonlinear curve fitting module of Statistica 7.0 software.


Summary:

  • We have developed a novel class of PARP-1 inhibitors by targeting the histone H4 route of PARP-1 activation, a mechanism that is unique to PARP-1. This mechanism of action is completely different from that of the “classical” NAD
  • -like PARP-1 inhibitors designed to compete with NAD at the enzyme active site.
  • H4-dependent PARP-1 inhibitors have no obvious structural homologues among components of eukaryotic enzymatic pathways.
  • H4-dependent PARP-1 inhibitors are highly specific for PARP-1 inhibition. In opposite,NAD-like PARP- 1 inhibitors inhibit other enzymes that use NAD, including other members of the PARP family.
  • H4-dependent PARP-1 inhibitors demonstrate superior antitumor activity compared with the classical NAD-like PARP-1 inhibitor olaparib in a xenograft model of human RCC. H4-dependent PARP-1 inhibitors also demonstrate prominent activity against sunitinib-resistant RCC xenografts. Similar results were obtained using mice bearing castration-resistant prostate cancer xenograft tumors.

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