Protein-binding properties of a designed steroidal lactam compound

• A designed steroidal amide compound (HAAO) was synthesized by introducing amide bonds into a steroid nucleus.
• HAAO binds to Sudlow’s site I in HSA through hydrogen bonds and van der Waals power.
• The interaction was a spontaneous and exothermic process with modest a degree of reversibility.
• The secondary structure of HSA and the microenvironment of TRP214 altered.
• The introduced amide bond in the steroid nucleus (NHCO) plays an important role in stabling the structure of macromolecules.

Introducing amide bonds into a steroid nucleus or its side chain may reduce the acute toxicity and enhance the pharmaceutical activity. In this work, a designed steroidal amide compound, named 3β-hydroxy-17-aza-d-homo-5-androsten-17-one (HAAO), was synthesized and identified. The interactions between HAAO and human serum albumin (HSA) were studied by multiple spectroscopic methods and molecular modeling procedures. It was found that HAAO locates in Sudlow’s site I in subdomain IIA of HSA molecules, relying on hydrogen bonds and van der Waals power to form HAAO–HSA complexes at ground state. The number of binding sites, binding constants, enthalpy change (ΔHθ), Gibbs free energy change (ΔGθ) and entropy change (ΔSθ) were calculated at different temperatures based on fluorescence quenching theory and classical thermodynamic equation. The percentages content of the HSA’s secondary structures in presence of HAAO were detected by circular dichroism (CD) spectra and compared with those in no presence of HAAO. In addition, the experimental results of both binding site and conformational change were further confirmed by molecular modeling investigation, in which more details of the binding were visually unfolded. The information provided by the study may be useful for designing novel chemotherapeutic drugs and be helpful both in the early stages of drug discovery and in clinical practice.