While IN inhibitors are a relatively new addition to the

While IN inhibitors are a relatively new addition to the clinician's palette of drugs to formulate HAART regimes, resistance to both INSTIs and LEDGINs has been observed. For example, even within Australia where it is estimated that 25,313 people are currently living with HIV and the infection rate has remained at a relatively stable rate of ≈1000 cases per year, resistance to IN inhibitors has been observed in a HAART naïve patient. Four point mutations conferring resistance to DTG have been characterised since the drug received FDA approval in mid-2013, and various mutations conferring resistance to LEDGINs have also been mapped. For example, the inhibitory activity of BI-224436 is reduced by 2.6- and 64-fold against the A128T and A128N mutants respectively relative to the wild-type virus. Consequently, if IN inhibitors are to remain a vital component of HAART regimes, it is essential that new generations of inhibitors are continually entering the drug development pipeline. To this end, we recently identified a series of allyltyrosine based tri-peptides which displayed specific ST inhibitory activity. In an initial attempt to enhance potency, the scaffold was subjected to a comprehensive structure-activity-relationship (SAR) campaign, and while analogues with incrementally enhanced IC50 values were developed (i.e. pp3 5, Fig. 2), significant activity enhancements were not forthcoming. Hence, as a consequence of the recent plethora of HIV IN crystal structures deposited in the protein databank, we were moved to explore the possibility of enhancing the inhibitory activity of the scaffold through a structure-based approach. IN is comprised of three independently folded domains.12, 18, 19, 20, 21, 22 The N-terminal domain (NTD; residues 1–50) binds Zn2+ via a conserved His-His-Cys-Cys motif. The catalytic core domain (CCD) (residues 50–212) adopts an RNase H superfamily fold and contains a DDE motif that binds Mg2+ or Mn2+ ions and mediates DNA cleaving and joining. The C-terminal domain (CTD; residues 223–268) features a Src homology domain 3 (SH3)-like fold that contributes to DNA binding and is connected to the catalytic core domain by a α-helical linker (residues 213–222). All three domains are required for activity, and each contributes to the correct assembly of a catalytically active IN tetramer.12, 18, 19, 20, 21, 22 However as the CCD contains the crucial determinants required for catalytic activity including the DDE catalytic triad, which mediates both 3′P and ST, we elected to focus on the active site of the CCD for the structure-based design development of the allyltyrosine analogues. The results of this work and some biological evaluations are now reported in this paper.
Results and discussion
Conclusions In relation to the allyltyrosine scaffold, from docking runs of the first generation series, a 3-point binding motif required for inhibitory activity was proposed and subsequently exploited to develop a new set of four peptide analogues which displayed comparable activity to the lead compound 5. Additionally from docking studies focusing on “in-house” compound libraries a nitrile based analogue 13 displaying an IC50 value of 0.5 μM in the combined 3′-P and ST was identified. While seven of the eight investigated derivatives were devoid of whole cell activity, the most potent tripeptide 9 displayed promising whole cell activity (EC50 10 ± 5 μM) and as the initial lead compound 5 was devoid of whole cell activity this represents a significant advancement of the scaffold. Moreover, whilst the in vitro activity of 9 is significantly higher than the majority of most current INSTIs inhibitors the whole-cell activity is comparable to a number of first generation analogues. For example the activity of 9 is equivalent to L-708906, (EC50 10.1 μM, CC50 88.3 μM), and L-731988 (EC50 1 μM, CC50 520 μM), and only one order of magnitude lower than S-136076, 77 (EC50 0.2 μM, CC50 110 μM) which was the first integrase inhibitor to reach clinical studies.