Cryptosporidiosis is a diarrheal disease caused by the parasite resulting in over 100,000 deaths annually. Here, we present a structure-activity relationship study of the benzoic acid position (R) of pyrazolo[3,4-]pyrimidine lead SLU-2815 (), an inhibitor of parasite phosphodiesterase PDE1, resulting in the discovery of benzoxaborole SLU-10906 () as a benzoic acid bioisostere. Benzoxaborole is 10-fold more potent than against the parasite in a cell-based infection model (EC = 0.19 μM) and non-cytotoxic. Furthermore, has a fast rate of parasite-killing and is orally efficacious in a mouse infection model (50 mg/kg BID), although relapse was observed 7 days post-drug treatment. The partial selectivity profile versus human phosphodiesterases is preserved with the benzoxaborole motif and represents an important feature to improve in future optimization. Benzoxaborole represents an important advance toward the optimization of the pyrazolo[3,4-]pyrimidine series and the identification of a drug to treat cryptosporidiosis.

Facioscapulohumeral muscular dystrophy (FSHD) is a common and progressive muscle wasting disease that is characterized by muscle weakness often first noticed in the face, the shoulder girdle and upper arms before progressing to the lower limb muscles. FSHD is caused by the misexpression of the Double Homeobox 4 (DUX4) transcription factor in skeletal muscle. While epigenetic derepression of D4Z4 macrosatellite repeats underlies DUX4 misexpression, our understanding of the complex transcriptional activation of DUX4 is incomplete.

Facioscapulohumeral muscular dystrophy (FSHD) is a degenerative muscle disease caused by loss of epigenetic silencing and ectopic reactivation of the embryonic double homeobox protein 4 gene (DUX4) in skeletal muscle. The p38 MAP kinase inhibitor losmapimod is currently being tested in FSHD clinical trials due to the finding that p38 inhibition suppresses DUX4 expression in preclinical models. However, the role of p38 in regulating DUX4 at different myogenic stages has not been investigated. We used genetic and pharmacologic tools in FSHD patient-derived myoblasts/myocytes to explore the temporal role of p38 in differentiation-induced DUX4 expression. Deletion of MAPK14/11 or inhibition of p38α/β caused a significant reduction in early differentiation-dependent increases in DUX4 and DUX4 target gene expression. However, in MAPK14/11 knockout cells, there remains a differentiation-associated increase in DUX4 and DUX4 target gene expression later in differentiation. Furthermore, pharmacologic inhibition of p38α/β only partially decreased DUX4 and DUX4 target gene expression in late differentiating myotubes. In xenograft studies, p38α/β inhibition by losmapimod failed to suppress DUX4 target gene expression in late FSHD xenografts. Our results show that while p38 is critical for DUX4 expression during early myogenesis, later in myogenesis a significant level of DUX4 expression is independent of p38α/β activity.

Our previous work identified compound (SLU-2633) as a potent lead compound toward the identification of a novel treatment for cryptosporidiosis, caused by the parasite (EC = 0.17 μM). While this compound is potent and orally efficacious, the mechanism of action and biological target(s) of this series are currently unknown. In this study, we synthesized 70 compounds to develop phenotypic structure-activity relationships around the aryl “tail” group. In this process, we found that 2-substituted compounds are inactive, confirmed that electron withdrawing groups are preferred over electron donating groups, and that fluorine plays a remarkable role in the potency of these compounds. The most potent compound resulting from this work is SLU-10482 (, EC = 0.07 μΜ), which was found to be orally efficacious with an ED < 5 mg/kg BID in a -infection mouse model, superior to SLU-2633.