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Int J Parasitol Drugs Drug Resist
2016 Feb 12;61:74-84. doi: 10.1016/j.ijpddr.2016.02.003.
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Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors.
Qvit N
,
Schechtman D
,
Pena DA
,
Berti DA
,
Soares CO
,
Miao Q
,
Liang LA
,
Baron LA
,
Teh-Poot C
,
Martínez-Vega P
,
Ramirez-Sierra MJ
,
Churchill E
,
Cunningham AD
,
Malkovskiy AV
,
Federspiel NA
,
Gozzo FC
,
Torrecilhas AC
,
Manso Alves MJ
,
Jardim A
,
Momar N
,
Dumonteil E
,
Mochly-Rosen D
.
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Parasitic diseases cause ∼ 500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania''s receptor for activated C-kinase) and TRACK (Trypanosoma receptor for activated C-kinase). We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase), may be interaction sites for signaling proteins that are critical for the parasites'' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs.
Fig. 1. Rational design of peptides with anti-parasitic activity. Sequence alignment between human RACK (P63244) and L. donovani LACK (Q76LS6). (A) The alignment identified eight short regions labeled L1-L8 of low similarity between LACK and RACK. The alignment between the proteins was done using the FASTA server, where: (:) represents identical amino acids, and (.) represents similar amino acids. (B) The position of identified L regions in A are highlighted in the structure of the Trypanosoma LACK homolog, TRACK (PDB: 4V8M) and colored as in A. We used the TRACK structure because the structure of LACK is not available and there is high similarity between LACK and TRACK (over 75% similarity). The sequence of the L4 region in LACK is also provided on the structure.
Fig. 5. Structure activity relationship (SAR) studies: Alanine scan of the cyclic peptide in culture. Peptides were added once to L. donovani promastigotes or to epimastigote forms of T. cruzi and their leishmanicidal and trypanocidal activity was evaluated after 48 or 24 h, respectively. (A) Alanine scan of p4d. Anti-parasitic activity was assessed after treatment with p4d and alanine-scan analogs (given once, at 100 μM). Substitution of the amino acids from the N-terminal (R, N, G, or Q) only slightly reduced the anti-parasitic activity of the peptide. Though, substitution of any one of the amino acids in the C-terminal half (C, Q, R or K) caused a reduction or a complete loss of anti-parasitic activity. (B) Sequence alignment of L04 region between L. donovani LACK (Q76LS6) and T. cruzi TRACK (Q4DTN2). L4 region is 50% identical and 88% similar between LACK and TRACK, where: (:) represents identical amino acids, and (.) represents similar amino acids. Data are representative of three independent experiments and presented as mean ± SEM. Statistical analysis was performed using two-tailed unpaired Student's t-test (#p < 0.05, *p < 0.001 vs. TAT). The observer was blinded to the experimental conditions.
Fig. 6. The effect of p4d on T. cruzi infections in vivo. T. cruzi parasitemia level, mortality, and parasite burden in infected mice (see Materials and Methods for more experimental details). Mice treated with the cyclic peptide, p4d, showed a 65% reduction in parasitemia (A), increase in survival by three-fold (B) and significantly less parasite burden of amastigotes in the cardiac muscle at the peak days (days 39–42) (C). The observer was blinded to the experimental conditions. Data presented as mean ± SEM. Statistical analysis was performed using two-tailed unpaired Student's t-test (§p < 0.01, #p < 0.05, *p < 0.001 vs. control).
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