I performed post-MD MMGBSA for one of my best active compounds (IC50: 2 nM).

But the ΔG value is positive.

What can be the reason?

a. Is the MD simulation time not sufficient? (But the ligand is almost stable after 300 ns)

b. Am I choosing the wrong binding site?

c. Should I try with MMPBSA or normal mode instead?

d. Is the initial starting ligand pose not OK?

I tried with other moderately or weakly active compounds. they showed ΔG negative.