Abstract: International Journal of Molecular Sciences Article Lactoferrin Inhibition of the Complex Formation between ACE2 Receptor and SARS CoV-2 Recognition Binding Domain Roberta Piacentini 1,2 , Laura Centi 1 , Mattia Miotto 2,3 , Edoardo Milanetti 2,3 , Lorenzo Di Rienzo 2 , Martina Pitea 2,4 , Paolo Piazza 5 , Giancarlo Ruocco 2,3 , Alberto Boffi 1 and Giacomo Parisi 2, * 1 2 3 4 5 * Citation: Piacentini, R.; Centi, L.; Miotto, M.; Milanetti, E.; Di Rienzo, L.; Pitea, M.; Piazza, P.; Ruocco, G.; Boffi, A.; Parisi, G. Lactoferrin Department of Biochemistry, Sapienza University, Piazzale Aldo Moro 5, 00185 Rome, Italy; roberta.piacentini@uniroma1.it (R.P.); centi.laura95@gmail.com (L.C.); alberto.boffi@uniroma1.it (A.B.) Center of Life Nano and Neuro Science, Institute of Italian Technology, Viale Regina Elena 291, 00181 Rome, Italy; miottomattia1@gmail.com (M.M.); edoardo.milanetti@uniroma1.it (E.M.); lorenzo.dirienzo@iit.it (L.D.R.); martina.pitea@iit.it (M.P.); giancarlo.ruocco@iit.it (G.R.) Department of Physics, Sapienza University, Piazzale Aldo Moro 5, 00185 Rome, Italy D-Tails s.r.l., Via di Torre Rossa 66, 00165 Rome, Italy EDIF Instruments s.r.l., Via Ardeatina 132, 00147 Rome, Italy; paolopiazza@edifinstruments.com Correspondence: giacomo.parisi@iit.it Abstract: The present investigation focuses on the analysis of the interactions among human lactoferrin (LF), SARS-CoV-2 receptor-binding domain (RBD) and human angiotensin-converting enzyme 2 (ACE2) receptor in order to assess possible mutual interactions that could provide a molecular basis of the reported preventative effect of lactoferrin against CoV-2 infection. In particular, kinetic and thermodynamic parameters for the pairwise interactions among the three proteins were measured via two independent techniques, biolayer interferometry and latex nanoparticle-enhanced turbidimetry. The results obtained clearly indicate that LF is able to bind the ACE2 receptor ectodomain with significantly high affinity, whereas no binding to the RBD was observed up to the maximum “physiological” lactoferrin concentration range. Lactoferrin, above 1 µM concentration, thus appears to directly interfere with RBD–ACE2 binding, bringing about a measurable, up to 300-fold increase of the KD value relative to RBD–ACE2 complex formation. Inhibition of the Complex Formation between ACE2 Receptor and SARS CoV-2 Recognition Binding Domain. Keywords: SARS-CoV-2 receptor-binding domain (RBD); angiotensin-converting enzyme 2 (ACE2); lactoferrin; biolayer interferometry; nanoparticle enhanced turbidimetry; kinetic analysis Int. J. Mol. Sci. 2022, 23, 5436. https://doi.org/10.3390/ijms23105436 Academic Editor: Samuel De Visser Received: 20 April 2022 Accepted: 11 May 2022 Published: 13 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).