4-Hydroxy-1α,25-Dihydroxyvitamin D₃: Synthesis and Structure–Function Study

Round 1

Reviewer 1 Report

The article by Rochel et al. is interesting for scientists involved in the vitamin D research, but the potential audience can be even broader. There is continuing interest in the vitamin D metabolism and synthesis of different calcitriol analogs with the modified biological activity. The reviewed manuscript is devoted to two less explored A-ring natural metabolites of vitamin D₃ resulting from hydroxylation at C-4. The synthesis of both epimeric compounds, 1α,4α,25-(OH)₃D₃ and 1α,4β,25-(OH)₃D₃ (Authors use different but acceptable abbreviations), has been achieved using Trost’s approach (palladium-catalyzed coupling of epimeric A-ring enyne synthons with a C/D-ring fragment). This synthesis was well planned and successfully executed.

The obtained target calcitriol analogs, hydroxylated at C-4, were then tested by different biological assays. An important finding was the observation that these metabolites bind effectively to the zebrafish (z)VDR LBD and they are potent VDR agonist ligands with a potency approaching that of 1α,25-(OH)₂D₃. Also, they regulate the expression of renal VDR target genes, and increase serum calcium levels in vivo in mice. All these data indicate close similarity of the biological potencies of both C-4 modified analogs to their parent compound (calcitriol).  

Additionally, the Authors have solved the crystal structures of their complexes with the zVDR LBD and they have drawn general conclusions regarding orientation of the ligands in the binding pocket, identifying the most important interactions for the ligand binding and discussing conformations of the receptor-bound ligands. The article is clear and well written.

Taking these facts into consideration this reviewer recommends the article for publication in the Biomolecules if the Authors revise the manuscript according to the comments shown in the attached file.

Comments for author File: Comments.pdf

Author Response

We thank the reviewer for his very positive feedback and acknowledge for the corrections. We made all suggested corrections.

Reviewer 2 Report

The manuscript "4-Hydroxy-1α,25-Dihydroxyvitamin D3: Synthesis and Structure-Function Study" is very comprehensive and interesting. It significantly delves into the synthesis, structure, and receptor interactions of the 4(OH) ligands. The work is clear in the materials and methods section as well as in presenting the results. The only part that should be further elaborated is explaining why 1,4alpha,25D3 and 1,4beta,25D3 could have clinical applications. In my opinion, both in the introductory part and in the discussion, this aspect is underdeveloped.

Author Response

We thank the reviewer for his very positive feedback and acknowledge him for the valuable comments.

We have included a discussion on the biological properties of natural metabolites and application of the new analogs.