Table 2.

Robustness of minimax-optimal inhibitors

Minimax-optimal inhibitors	Fitness of best protease
	0	1	2	3	4	5
	(1)	(119)	(6288)	(1.9 × 105)	(3.9 × 106)	(5.1 × 107)
GWQFAQAG	0.0071	2.9291	3.1456	5.9740	8.4034	8.5782
GLQFAQAG	0.0105	0.0548	0.2312	0.5605	0.8769	1.5725
GFTFAQAG	0.0229	0.0747	0.1467	0.3394	0.5791	1.0133
GFVYAQTG	0.0205	0.0984	0.3060	0.3060	0.9070	1.0752
GFVYWLGT*	0.2769	0.2769	0.4477	0.4477	0.4829	0.7753
GFVFYQAG	0.0369	0.1485	0.3833	0.6660	0.6660	0.6660

The minimax inhibitor optimized against each set of mutant proteases, given in Table 1, was subsequently subjected to the other five sets of proteases. Each column corresponds to a set of proteases with a different number of simultaneous mutations, from wild type to pentuple mutants (left to right). Figures in parentheses are the number of different mutant proteases in each set. Values in bold are the viral fitnesses obtained during the initial search for each inhibitor (identical to those values in Table 1); values in plain type are fitnesses when the inhibitor then was subjected to the other five sets of mutants. 

^*The inhibitor selected against the set of quadruple mutants, GFVYWLGT, shows less robust behavior than the inhibitors selected against the other sets and also shows a sharp dip in both inhibitor and substrate binding free energy compared with the other inhibitors (see Fig. 3). This is because of the structural mode used to evade inhibitors: The best quadruple mutant reduces the size of the P1 and P1′ sites whereas the best proteases selected from the other sets increase P2 and P2′ and decrease P3 and P3′ (data not shown). Examples of both modes can be found within 20% of the minimax-optimal inhibitor in the sets of triple, quadruple, and pentuple mutants.