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Question 13.5

This original paper describes exactly what you see: Hydrophilic peptides derived from the

transframe region of Gag-Pol inhibit the HIV-1 protease (Louis JM, Dyda F, Nashed NT,

Kimmel AR, Davies DR (1998). Hydrophilic peptides derived from the transframe region

of Gag-Pol inhibit the HIV-1 protease. Biochemistry. 37(8):2105–10. https://doi.org/https://

doi.org/10.1021/bi972059x).

The HIV-1 transframe region (TFR) is between the structural and functional domains

of the Gag-Pol polyprotein, flanked by the nucleocapsid and the protease domains at its N

and C termini, respectively. Transframe octapeptide (TFP) Phe-Leu-Arg-Glu-Asp-Leu-

Ala-Phe, the N terminus of TFR, and its analogues are competitive inhibitors of the action

of the mature HIV-1 protease. The smallest, most potent analogues are tripeptides: Glu-­

Asp-­Leu and Glu-Asp-Phe with Ki values of approximately 50 and approximately 20

microM, respectively. Substitution of the acidic amino acids in the TFP by neutral amino

acids and d or retro-d configurations of Glu-Asp-Leu results in a > 40-fold increase in Ki.

Protease inhibition by Glu-Asp-Leu is dependent on a protonated form of a group with a

pKa of 3.8; unlike other inhibitors of HIV-1 protease which are highly hydrophobic, Glu-­

Asp-­Leu is extremely soluble in water, and its binding affinity decreases with increasing

NaCl concentration. However, Glu-Asp-Leu is a poor inhibitor (Ki approximately 7.5 mM)

of the mammalian aspartic acid protease pepsin. X-ray crystallographic studies at pH 4.2

show that the interactions of Glu at P2 and Leu at P1 of Glu-Asp-Leu with residues of the

active site of HIV-1 protease are similar to those of other product-enzyme complexes. It

was not feasible to understand the interaction of intact TFP with HIV-1 protease under

conditions of crystal growth due to its hydrolysis giving rise to two products. The sequence-­

specific, selective inhibition of the HIV-1 protease by the viral TFP suggests a role for TFP

in regulating protease function during HIV-1 replication.

Chellappan S, Kiran Kumar Reddy GS, Ali A et al. (2007) Design of mutation-resistant

HIV protease inhibitors with the substrate envelope hypothesis. Chem Biol Drug Des.

2007 May; 69(5): 298–313.

There is a clinical need for HIV protease inhibitors that can evade resistance muta­

tions. One possible approach to designing such inhibitors relies upon the crystallographic

observation that the substrates of HIV protease occupy a rather constant region within the

binding site. In particular, it has been hypothesized that inhibitors which lie within this

region will tend to resist clinically relevant mutations. The present study offers the first

prospective evaluation of this hypothesis, via computational design of inhibitors predicted

to conform to the substrate envelope, followed by synthesis and evaluation against wild-­

type and mutant proteases, as well as structural studies of complexes of the designed

inhibitors with HIV protease. The results support the utility of the substrate envelope

hypothesis as a guide to the design of robust protease inhibitors.

CARB-AD37 docked into HIV protease from crystal structure. Inhibitors were tested

against wild-type HIVP and a panel of three proteases with clinically relevant mutation

sets: M1 (L10I/G48V/ I54V/L63P/V82A), M2 (D30N/L63P/N88D), and M3 (L10I/L63P/

A71V/ G73S/I84V/L90M).

20  Solutions to the Exercises