PREreview of Design of linear and cyclic peptide binders of different lengths from protein sequence information

In this preprint, the authors describe the second generation of their EvoBind, which iteratively introduces mutations in a peptide to increase its fitness to host protein. In EvoBind2 some updates were introduced and the metrics for measuring fitness are discussed as well.

The experimental validation of the designed peptides using SPR was a very interesting aspect of the manuscript. The authors describe very good success rates (close to 50%), with the stronger binder showing higher affinity than their positive control.

Major issues

• I missed the comparison with other de novo design tools in terms of execution times. EvoBind (first generation, I will call it EvoBind1 to avoid confusion with EvoBind2) had quite long execution times due the extensive sampling of the sequence space. Also, the memory requirements were somewhat quite high. I couldn’t myself get it to run on Google Cloud machines, for example. If we compare this requirements with RFDifusion, for example, we have quite fast inference times for the later with cheap memory requirements. A few words about how EvoBind2 compares to EvoBind1 and, possibly with RFDiffusion or other comparable tools, would add a lot here.

Minor issues

• The discussion about the ‘Affinity and in silico metrics’ is very interesting. I believe I would not expect a very high correlation between affinity and pLDDT. However, how does the affinity compares to AFM interface PAE? And how does it compare with ipTM? I guess these are the metrics that were introduced specifically to account to the reliability of the prediction for an interaction, so I would expect them to perform better than pLDDT. It would be great if the authors could bring this discussion their manuscript.

• Finally, It would be great to think about the future in peptide design. How can we think in a clever way for other types of cyclization beyond the positional encoder offset (disulfide bond, for example) in AF2/EvoBind2? Is it possible to envision a way to introduce non-canonical amino acids?

Competing interests

The author declares that they have no competing interests.