ANTI-RETROVIRAL
DRUG DESIGN BASED ON STRUCTURE ANALYSIS OF PROTEASES
1Jindřich
Hašek , 1Jan
Dohnálek , 1Jarmila
Dušková, 2Jan
Konvalinka, 2Martin
Hradilek, 2Milan
Souček, 3Juraj
Sedláček, 3Jiří
Brynda, 4Eva
Buchtelová
1Institute
of Macromolecular Chemistry Acad.Sci.CR, Heyrovského nám.2, 162
06 Praha 6
2Institute
of Organic Chemistry and Biochemistry of Acad.Sci.CR, Flemingovo
nám.2., 166 37 Praha 6
3Institute
of Molecular Genetics Acad.Sci.CR, Flemingovo nám.2, 166 37
Praha 6
4Sincrotone
Elettra, Padriciano 99, 34012 Trieste, Italy
Rational drug design based
on experimental determination of enzyme-inhibitor bonding using
x-ray diffraction is used in parallel with random screening of
large numbers of potential inhibitors using combinatorial
chemistry. In spite of more than 70 determined structures of HIV
protease-inhibitor complexes of different types no satisfactory
rules for the best inhibitor design were found. In addition, it
seems that virtually all residues forming binding site of the HIV
protease (8,23,81,82,84,150), (50,108,123,181,182,184),
(28,29,30,32,47,84), (128,129,130,132,147,184) except of the
catalytic aspartates 25 and 125 can be naturally mutated forming
thus a new generations of viruses resistant against inhibitors
originally applied [1].
Our project deals with
experimental determination of HIV proteases complexed with
ligands of different affinity resulting from screening.
Monitoring the changes in interactions in sequences of inhibitors
with protease we can follow some rules according to which new
screening collections should be proposed. The series of 20
wide-spectral (with good mutational resistance) inhibitors
denoted according to the scheme shown in the following table as SE,
SQ, SI, RE, RQ, RI, OE OI, SRE, SRQ, SRI, SSE, SSQ, SSI, RSE,
RSQ, RSI, RRE, RRQ, RRI is designed to explain [2,3]:
- the influence of
hydroxyl group originally supposed to replace the
activated water molecule in the cleft site,
- the influence of the
(S/R) configuration of CH(OH) group, and
- the influence of the
charge, polarization and hydrophobicity of the residue in
the P2' site.
Table 1. Ki
values [picomol.l-1] and
shorthand notation for native HIV-1 protease complexed with 22
hydroxyethylamine, ethylamine and hydroxyethylene inhibitors of
the general type Boc-Phe-[X]-Phe-P2'-Phe-NH2
and C9H6NCO-Asn-Phe-[X]-NC9H14CO-NHC(CH3)3.
Bold face numbers
denote an area of the reasonably efficient inhibitors. Compound
code (bold
letters) consists of two parts: code
for P3-P2-P1-[X]-P1’ and code for P2’-P3’. For example, the
complex HIV-1 protease with Boc-Phe-[(R) CH(OH) CH2
NH]-Phe-Glu-Phe-NH2 is denoted as RE.
Table 1 summarizes Ki
values [in picomol.l-1] of native HIV-1 protease with
22 different inhibitors of the general type P3-P2-P1-[X]-P1'-P2'-P3’
belonging to the classes of hydroxyethylamine, ethylamine and
hydroxyethylene inhibitors [4]. The table shows data for
complexes native HIV-1 protease with 20 inhibitors synthetised in
IOCB [2] and saquinavir - Ro-31-8959 ( C9H6NCO-Asn-P1-[X]-C9NH14CO-NH2
C(CH3)3 ),
here denoted as RoB. The groups related to S3, S2, S1,
S1’,S2’ ,S3’ are:
P3 none in
all cases except for saquinavir where two condensed rings C9H6N
form the end of the chain.
P2 is Boc
in all cases, except saquinavir where Asn is the P2 position.
P1 The function
group at the P1 site is Phe in all cases.
[X]
Hydroxyethylene (R or S), hydroxyethylamine (SR, SS,
RS, RR) and ethylamine (Ea) spacers are used as
isoster spacers.
P1' Hydrophobic
groups Phe or DIQ (NC9H14) are
available in both possible configurations R/S.
P2' The
influence of four different residues (Glu, Gln, t-But, Ile)
in the P2' position make difference in Ki from one to
three orders in magnitude. The data for saquinavir (Ro-31-8959
) are given in the eigth column.
P3´ is Phe-NH2
in all cases except saquinavir, where the chain is terminated at
P2’ by t-But.
The data for Saquinavir are
given in the eigth line. Dash means that the experimental value
of Ki is not available. The symbol* denotes structures that have
been measured in the IMC, the symbola
denotes structures under
investigation, the symbol + denotes structures determined by a group of
prof.Hingenfeld (to be published) and the symbol # refers to
molecular structures determined by Krohn et al [5].
Crystal structure of the
complex SQ clearly shows that the NH of P1’(Phe) forms
hydrogen bridges with the catalytic aspartates D25, D125 and thus
the hydroxy group does not play the role designed by
hydroxyethylamine-inhibitor designers. It explains better
inhibition properties of EoE complex (where CH(OH) group
is replaced by CH2) in comparison with SE and RE
complexes. It seems that the interaction of hydroxy group
need not be decisive for a design of the best inhibitor (notice
that EoE belongs to the few best known inhibitors Kbest=
50 pM ).
New structure
determinations show that some rules for good inhibitors derived
by different authors from structure and function correlation on
limited number of structures are of limited validity. It seems
that:
- The best inhibitor
need not replace the activated water molecule by hydroxyl
group.
- The configuration at
the hydroxyl group need not be S (resembling L-amino
acids) as it has already been confirmed with many
inhibitors designed later.
- The inhibitor need not
possess two-fold symmetry.
- Varying biochemical
properties in P2’ need not lead to the monotoneous
variation of the binding affinity because of possible
conformational changes in other parts if inhibitor or
protease.
The work is supported
by A4050811 and 203/97/PO31.
- Babime R.E., Bender
S.L.: Molecular Recognition of Protein-Ligand Complexes:
Applications to Drug Design. Chem.Rev.97,
1359-1472 (1997).
- Konvalinka
J., Litera J., Weber J., Vondrášek J., Hradilek M.,
Souček M., Pichová I., Majer P., Štrop P., Sedláček J., Heuser A.-M.,
Kottler H., Kräusslich H.-G.:
Configurations of Diastereomeric Hydroxyethylene Isosteres Strongly Affect
Biological Activities of Series of Specific Inhibitors of
Human-Immunodeficiency -Virus Proteinase. Eur.J.Biochem.,250,559-566(1997).
- Sedláček
J., Fábry M., Hořejší M., Brynda J., Luftig R.B.,
Majer P.: A Rapid Screening Method
for Biological Activity of Human Immunodeficiency Virus
Proteinase Inhibitors by Using a Recombinant DNA-Derived Bacterial System. Anal.Biochem.
215, 306-309 (1993).
- Dohnálek
J., Hašek J., Brynda J., Fábry M., Sedláček J.,
Hradilek M., Konvalinka J., Souček M., Adams M.J.,
- Naylor
C.E.: Complex of HIV-1 Protease with (S)-Boc-Phe-y[CH(OH)CH2NH]-Phe-Glu-Phe-NH2. Protein Sci.,
5, 167 (1996).
- Krohn A., Redshaw S.,
Ritchie J.C., Graves B.J., Hatada M.H.: J.Med.Chem.,
3340-3342 (1991).