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The maturation of the RNA cap involving guanosine N-7 methylation, catalyzsed by the HsRNMT (RNA guanine-7 methyltransferase (HsRNMT)-RAM (RNA guanine-N7 methyltransferase activating subunit (RAM) complex, is currently under investigation as a novel strategy to combat PIK3CA -mutant breast cancer. However, the development of effective drugs is hindered by a limited understanding of the enzyme's mechanism and a lack of small molecule inhibitors. Following the elucidation of the HsRNMT-RAM molecular mechanism, we report the biophysical characterizsation of two small molecule hits. Biophysics, biochemistry and structural biology confirm that both compounds bind competitively with cap and bind effectively to HsRNMT-RAM in the presence of the co-product SAH, with a binding affinity (KD) of approximately 1 μM. This stabilisation of the enzyme--product complex results in uncompetitive inhibition. Finally, we describe the properties of the cap pocket and provided suggestions for further development of the tool compounds., (© 2025 The Author(s).)

Cryptosporidiosis is a diarrheal disease caused by infection with Cryptosporidium spp. parasites and is a leading cause of death in malnourished children worldwide. The only approved treatment, nitazoxanide, has limited efficacy in this at-risk patient population. Additional safe therapeutics are urgently required to tackle this unmet medical need. However, the development of anti-cryptosporidial drugs is hindered by a lack of understanding of the optimal compound properties required to treat this gastrointestinal infection. To address this knowledge gap, a diverse set of potent lysyl-tRNA synthetase inhibitors was profiled to identify optimal physicochemical and pharmacokinetic properties required for efficacy in a chronic mouse model of infection. The results from this comprehensive study illustrated the importance of balancing solubility and permeability to achieve efficacy in vivo. Our results establish in vitro criteria for solubility and permeability that are predictive of compound efficacy in vivo to guide the optimization of anti-cryptosporidial drugs. Two compounds from chemically distinct series (DDD489 and DDD508) were identified as demonstrating superior efficacy and prioritized for further evaluation. Both compounds achieved marked parasite reduction in immunocompromised mouse models and a disease-relevant calf model of infection. On the basis of these promising data, these compounds have been selected for progression to preclinical safety studies, expanding the portfolio of potential treatments for this neglected infectious disease.