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HIV Reverse Transcriptase Target for AIDS Treatment

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The polymerase HIV-1 RT is the primary target of current drug design. ... C.; Sudbeck, E. A.; Venkatachalam, T. K.; Uckun, F. M. Biochemical Pharmacology. ... – PowerPoint PPT presentation

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Title: HIV Reverse Transcriptase Target for AIDS Treatment


1
HIV Reverse TranscriptaseTarget for AIDS
Treatment
  • AIDS, though an incurable disease, can be
    significantly slowed in its onset through HIV
    chemotherapy. The polymerase HIV-1 RT is the
    primary target of current drug design. This
    enzyme converts the RNA code contained in the HIV
    retrovirus into DNA that can be spliced into the
    host genome for viral replication. Two classes
    of HIV-1 RT inhibitors have emerged these are
    nucleoside analogs and non-nucleoside inhibitors
    (NNRTIs). In the following presentation, the
    general structure of HIV-1 RT will be
    investigated, followed by a demonstration of the
    means by which these inhibitors halt
    transcriptase function in vivo. Furthermore,
    computational methods of drug design will be
    explored.

2
  • General Structure of HIV-1 RT
  • The enzyme is a heterodimer of two subunits, p66
    p51. Both subunits are derived from a common
    peptide precursor, since mutations in the HIV
    genome replace aas in both subunits.1
  • Both subunits (shown only for the p66 domain)
    have 4 well defined regions Finger, Thumb, Palm
    and Connection. The p66 domain also has an
    additional RNase H domain, which cleaves the
    template RNA strand following its conversion into
    DNA.2
  • The arrangement of subdomains in the two
    subunits is dramatically different p66 is
    organized to form a cleft into which the RNA
    templateDNA primer bind.
  • The 3-OH of the primer terminus lies in
    proximity to a triad of conserved aspartic acid
    residues in the active site.
  • There is only a single active site in each HIV-1
    RT.

3
Crystal Structures Provide Different Insight
  • Multiple crystal structures of HIV-1 RT have
    been obtained under different conditions, though
    perhaps the most revealing of the enzyme mode of
    action was recently obtained by the Verdine
    group.2
  • This structure utilizes the formation of a
    disulfide bond formed between the protein and a
    DNA template strand (note that HIV-1 RT can
    polymerize DNA and RNA) to stall the enzyme
    immediately before the incorporation of a 4th
    dNTP into a growing DNA duplex.
  • Binding of the templateprimer and dNTP induce
    p66 conformation changes that shift the finger
    domain nearer to the palm region.
  • This movement brings several new residues in
    contact with the bound dNTP.
  • The dNTP site, well resolved in Verdines model
    (unable to be seen w/ a dNTP present in earlier
    structures), shows that the coordination of a
    lysine, arginine, and two magnesium ions form the
    active complex that incorporates the base into
    the growing duplex.

Finger Region
Palm Region
ThumbRegion
4
Further Investigation of the Active Site and
Conformational Changes
  • A further image of the active site is shown to
    the right in which Mg (B) can be seen to possess
    an octahedral type geometry reminiscent of T7 DNA
    polymerases.
  • To the left, Verdines structure (red) is
    superimposed on several other crystal structures
    such as the unligated (green) and NNRTI bound
    structure (blue) to demonstrate the
    conformational changes occurring in each case.
    Note that the finger domain in the Verdine
    structure is the most altered, indicative of its
    importance in the incorporation of additional
    dNTPs to the growing duplex.

5
Investigation of Drug Binding Sites in HIV-1 RT
  • As mentioned previously, the two types of HIV
    chemotherapy drugs, nucleoside analogs and
    NNRTIs, bind to two distinct sites in the HIV-1
    RT complex.
  • Nucleoside analogs bind to the dNTP site (here
    labeled dTTP), as shown above, and become
    directly incorporated into the growing duplex.
  • Due to the lack of a 3-OH group, the strand
    cannot be further elongated, hence the complex is
    halted. Though effective, the nucleoside
    analogues are non-specific and can cause severe
    damage to host cells.1
  • NNRTIs bind allosterically to a distinct NNIBP
    site nearly 10A away from the catalytically
    active site. It is framed by two tryptophan
    residues that swivel to incorporate the NNRTI
    drugs. The NNRTIs are thought to prevent the
    conformational changes demonstrated in Verdines
    structure to be necessary for elongation of the
    DNA strand.3

6
Examples of Drugs Inhibiting HIV-1 RT
  • The following drugs strongly inhibit the action
    of HIV-1 RT, by either binding to the nucleoside
    binding site or the NNIBP site.
  • Note that the nucleoside analogs all lack a
    3-OH, a requisite for their ability to halt
    polymerization.
  • Note also that the NNRTIs all possess structures
    with large phenyl substituents. This common
    structural feature has been attributed to their
    binding through pi-stacking interactions with the
    swiveling tryptophan residues in the NNIBP site
    of HIV-1 RT.1

Nucleoside Analogs
NNRTIs
7
NNRTI Binding Site Use of Rational Drug Design
to Find New Leads
  • Given that the nucleoside analogs are
    non-specific and generally toxic, developing new
    NNRTIs is of the foremost interest.
  • Over the last 20 years, multiple crystal
    structures of HIV-1 RT complexed with various
    NNRTIs have supplied a wide range of models, all
    of which have significant differences in the
    demonstrated NNIBP. This fact makes rational
    drug design impossible to implement.
  • Recently, however, several groups have
    investigated the idea of using composite binding
    pockets in which several crystal structures have
    been hybridized to give an averaged consensus
    binding site that can be used to target new
    analogs.
  • This idea is rational, given that evidence
    indicates that the NNIBP is quite flexible. This
    is demonstrated by its ability of to bind drugs
    that appear to be too large by initial analysis,
    but fit comfortably in the product crystal
    structures.
  • The results of this composite binding site are
    shown above. The grid lines represent the Van
    der Waals surface, while the internal molecules
    show the superimposed structures of several
    NNRTIs from their crystal structures.3

8
Use of Rational Drug Design to Find New Leads,
Continued
  • With this composite binding site, Mao et al.
    were able to identify several new drugs with
    IC-50 values higher than those found
    conventionally, with equal to or lower than
    typical cytotoxicity.3
  • Demonstration of their binding to the composite
    binding site is also displayed to the left.

9
Use of Rational Drug Design to Find New Leads,
Continued
  • As further evidence to the validity of the
    techniques used, Mao et al. superimposed the
    composite site into the active site residues of
    NNIBP (right). Those areas where a great deal of
    overlap occur indicate NNIBP site residues that
    are more flexible than others.
  • In addition, demonstration of the derivative,
    HI-244 is demonstrated in both the composite site
    and among the binding site residues (left) (Wing
    1 and 2 represent domains of the NNIBP).

10
Conclusions
  • Targeting of HIV-1 RT by chemical agents has
    provided a way by which to slow the onset of
    AIDS. Though not a cure, these drugs provide
    individuals afflicted with the disease prolonged
    life spans, or in rare cases, near eradication of
    the virus. Use of nucleoside analogs together
    with NNRTIs provides a synergistic, two-front
    attack on the enzyme. This is possible given
    that these drugs act at distinct sites and by
    different mechanisms. Though direct use of HIV-1
    RT crystal structures is not amenable to rational
    drug design, use of composite binding sites has
    been demonstrated to provide leads into potential
    therapeutic targets.
  • References
  • Tantillo, C. Ding, J. Jacob-Molina, A. Nanni,
    R. G. Boyer, P. L. Hughes, S. H. Pauwels, R.
    Andries, K. Janssen, P. A. J. Arnold, E. J.
    Mol. Biol. 1994, 243, 369-387.
  • Huang, H. Chopra, R. Verdine, G. Harrison, S.
    C. Science. 1998, 282, 1669-1675.
  • Mao, C. Sudbeck, E. A. Venkatachalam, T. K.
    Uckun, F. M. Biochemical Pharmacology. 2000, 60,
    1251-1265.
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