TY - JOUR
T1 - Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing
AU - Whitehead, Timothy A.
AU - Chevalier, Aaron
AU - Song, Yifan
AU - Dreyfus, Cyrille
AU - Fleishman, Sarel J.
AU - De Mattos, Cecilia
AU - Myers, Chris A.
AU - Kamisetty, Hetunandan
AU - Blair, Patrick
AU - Wilson, Ian A.
AU - Baker, David
N1 - We thank D. Fowler and S. Fields for helpful discussions and use of their in-house software to process sequencing data, C. Lee, J. Shendure and M. Dunham for experimental expertise in DNA prep and sequencing, C. Sitz and C. Santiago for technical help and the Joint Center for Structural Genomics for crystallization using the JCSG/IAVI/TSRI Rigaku Crystalmation system. This work was funded by Defense Advanced Research Projects Agency (DARPA) and the Defense Threat Reduction Agency (DTRA), and US National Institutes of Health, National Institute of Allergy and Infectious Diseases and National Institute of General Medical Sciences. The GM/CA CAT 23-ID-B beamline has been funded in whole or in part with federal funds from National Cancer Institute (Y1-CO-1020) and NIGMS (Y1-GM-1104). Use of the Advanced Photon Source (APS) was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. DE-AC02-06CH11357. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIGMS or the NIH. Contributions T.A.W. and A.C. conceived the idea, performed yeast display selections, analyzed deep sequencing data, performed hemagglutinin binding experiments, and performed computational modeling. Y.S. developed the electrostatics model and ran computational modeling code. C.D. expressed and purified hemagglutinin proteins, determined and analyzed the crystal structures with the guidance of I.A.W., and performed hemagglutinin binding experiments. S.J.F. assisted with structural analysis and developed the computational modeling code. C.D.M. performed the viral neutralization experiments under the guidance of C.A.M. and P.B. H.K. carried out covariance analysis on deep sequencing data. D.B. conceived the idea, analyzed deep sequencing data, and developed the electrostatics model. All authors discussed the results and wrote the manuscript.
PY - 2012/6
Y1 - 2012/6
N2 - We show that comprehensive sequence-function maps obtained by deep sequencing can be used to reprogram interaction specificity and to leapfrog over bottlenecks in affinity maturation by combining many individually small contributions not detectable in conventional approaches. We use this approach to optimize two computationally designed inhibitors against H1N1 influenza hemagglutinin and, in both cases, obtain variants with subnanomolar binding affinity. The most potent of these, a 51-residue protein, is broadly cross-reactive against all influenza group 1 hemagglutinins, including human H2, and neutralizes H1N1 viruses with a potency that rivals that of several human monoclonal antibodies, demonstrating that computational design followed by comprehensive energy landscape mapping can generate proteins with potential therapeutic utility.
AB - We show that comprehensive sequence-function maps obtained by deep sequencing can be used to reprogram interaction specificity and to leapfrog over bottlenecks in affinity maturation by combining many individually small contributions not detectable in conventional approaches. We use this approach to optimize two computationally designed inhibitors against H1N1 influenza hemagglutinin and, in both cases, obtain variants with subnanomolar binding affinity. The most potent of these, a 51-residue protein, is broadly cross-reactive against all influenza group 1 hemagglutinins, including human H2, and neutralizes H1N1 viruses with a potency that rivals that of several human monoclonal antibodies, demonstrating that computational design followed by comprehensive energy landscape mapping can generate proteins with potential therapeutic utility.
UR - http://www.scopus.com/inward/record.url?scp=84862025262&partnerID=8YFLogxK
U2 - https://doi.org/10.1038/nbt.2214
DO - https://doi.org/10.1038/nbt.2214
M3 - مقالة
C2 - 22634563
SN - 1087-0156
VL - 30
SP - 543
EP - 548
JO - Nature biotechnology
JF - Nature biotechnology
IS - 6
ER -