Disarming an Electrophilic Warhead: Retaining Potency in Tyrosine Kinase Inhibitor (TKI)‐Resistant CML Lines While Circumventing Pharmacokinetic Liabilities

Abstract

Pharmacologic blockade of STAT3 activation in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML) cell lines characterized by kinase-independent resistance re-sensitized CML cells to TKI therapy, suggesting that STAT3 inhibitors in combination with TKIs are an effective combinatorial therapeutic for the treatment of CML. Benzoic acid-based STAT3 inhibitors, SH-4-54 and SH-5-07, developed in our lab, demonstrated promising activity against these resistant CML cell lines. However, pharmacokinetic studies in murine models (CD-1 mice) revealed that both SH-4-54 and SH-5-07 are susceptible to glutathione conjugation at the para position of the pentafluorophenyl group via nucleophilic aromatic substitution (SNAr). To determine whether the electrophilicity of the pentafluorophenyl sulfonamide could be tempered, an in-depth structure activity relationship (SAR) study of the SH-4-54 scaffold was conducted. The studies revealed that AM-1-124, possessing a 2,3,5,6-tetrafluorophenylsulfonamide, retained STAT3 protein affinity (Ki = 15 μM), as well as selectivity over STAT1 (Ki >250 μM). Moreover, in both hepatocytes and in in vivo pharmacokinetic studies (CD-1 mice), AM-1-124 was found to be dramatically more stable than SH-4-54 (t1/2 = 1.42 h cf. 10 minutes, respectively). AM-1-124 represents a promising STAT3-targeting inhibitor with demonstrated bioavailability, suitable for evaluation in preclinical cancer models.

STAT3 differential scanning fluorimetry and differential scanning light scattering assays: Addressing a missing link in the characterization of STAT3 inhibitor interactions

“…All four of these agents are proposed STAT3 SH2 domain binders. 5,6,[22][23][24] Furthermore, STATTIC, S3I-201 and BP1-102 have been identified as probable covalent modifiers of STAT3 protein, [13][14][15] adding more interest to their evaluation in the DSF assay. Binding of BP1-102 to STAT3 127-688 was also confirmed by FP assay (Supplementary Figure 5).…”

“…inhibitors have the propensity to act as electrophilic alkylating agents. This has recently been highlighted using mass spectrometry 13,14 and fluorescence tagging 15 techniques with some of the most widely used STAT3 inhibitors. The majority of published STAT3 inhibitors are reported as selective Src Homology 2 (SH2) domain antagonists, however, the assays that are used to support SH2 domain binding may be also sensitive to compounds that can alkylate STAT3.…”

“…Recent efforts have demonstrated that commonly used STAT3 inhibitors BP1-102 14 which had been equilibrated with gel Filtration buffer (20 mM HEPES, 500 mM NaCl, 10% glycerol, 0.5 mM TCEP, pH 7.5, 2 mM TECP) and eluted using the same buffer. After SDS-PAGE analysis, fractions containing the desired protein were pooled and concentrated using Vivaspin concentrators (Sartorius).…”

The crystal structure of 4-bromo-N-cyclopropyl-2,5-difluorobenzenesulfonamide, C9H8BrF2NO2S

“…Phenyl sulfonamide and its derivatives can be used as an important intermediate in the synthesis of non-nucleoside thumb pocket 2 HCV NS5B polymerase inhibitors. In addition, it has been reported that phenyl sulfonamides can also be used to synthesize the tyrosine kinase inhibitor, which shows anti-cancer effect [5].…”

Identification of novel regulators of STAT3 activity

“…As a protein-protein interaction, this is a challenging drug target but at least four small-molecule inhibitors have entered early-phase clinical testing [TTI-101 (NCT03195699), WP1066 (NCT01904123), OPB-51602 (NCT02058017, NCT01423903), OPB-31121 (NCT00955812)]. However, most (if not all) reported SH2 binding STAT3 inhibitors are electrophilic resulting in reactivity with cysteine thiols and they therefore have low selectivity towards the SH2 domain in more complex biological settings and often cause off-target toxicities [14][15][16][17]. An alternative approach is to inhibit the upstream signals leading to STAT3 activation, including more readily druggable targets such as kinases and cell surface receptors for which there are several approved and investigational agents [5].…”