Background
Materials and Methods
Computational and analytical methods
Amino-acid | EIS hydropathy (log scale) | side-chain volume Å3 | side-chain pKa | formal side-chain charge (pH 7.0) |
---|---|---|---|---|
A ala | 0.25 | 156.55 | 0 | |
C cys | 0.04 | 219.43 | 8.33 | 0 |
D asp | -0.72 | 232.12 | 3.86 | -1 |
E glu | -0.62 | 284.70 | 4.25 | -1 |
F phe | 0.61 | 382.16 | 0 | |
G gly | 0.16 | 85.18 | 0 | |
H his | -0.40 | 334.84 | 6.0 | +1 |
I ile | 0.73 | 313.43 | 0 | |
K lys | -1.10 | 366.25 | 10.28 | +1 |
L leu | 0.53 | 315.53 | 0 | |
M met | 0.26 | 327.41 | 0 | |
N asn | -0.64 | 256.12 | 0 | |
P pro | -0.07 | ND | 0 | |
Q gln | -0.69 | 308.35 | 0 | |
R arg | -1.80 | 421.32 | 12.48 | +1 |
S ser | -0.26 | 183.07 | 0 | |
T thr | -0.18 | 238.55 | 0 | |
V val | 0.54 | 267.78 | 0 | |
W trp | 0.37 | 462.28 | 0 | |
Y tyr | 0.02 | 407.07 | 10.07 | 0 |
PFCRT : clone GC03 | |||||
---|---|---|---|---|---|
EIS | SCV | CH | |||
residue | amino acid | ||||
72 | C | 0.04 | 219.43 | 0 | |
73 | M | 0.26 | 327.41 | 0 | |
75 | N | -0.64 | 256.12 | 0 | |
76 | K | -1.1 | 366.25 | 1 | |
SUM | -1.44 | 1169.21 | 1 | ||
MEAN | -0.275 | 292.303 | 0.25 | ||
72 | C | 0.04 | 219.43 | 0 | |
74 | M | 0.26 | 327.41 | 0 | |
75 | N | -0.64 | 256.12 | 0 | |
76 | K | -1.1 | 366.25 | 1 | |
97 | H | -0.4 | 334.84 | 1 | |
220 | A | 0.25 | 156.55 | 0 | |
271 | Q | -0.69 | 308.35 | 0 | |
326 | N | -0.64 | 256.12 | 0 | |
356 | I | 0.73 | 313.43 | 0 | |
371 | R | -1.8 | 421.32 | 1 | |
SUM | -3.99 | 2959.82 | 3 | ||
MEAN | -0.399 | 295.982 | 0.3 |
clone | c1GC03 | c2GC03 | c3dd2 | c4dd2 | c5k76I | c67g8 |
---|---|---|---|---|---|---|
mutable residues 72-76 | CMNK | CMNK | CIET | CIET | CIEI | SMNT |
mutable residues 72-371 | CMNK HAQNIR | CMNK HAQNIR | CIET HSESTI | CIET HSESTI | CIEI HSESII | SMNT HSQDLR |
mean EIS 72-76
| -0.275 | -0.275 | -0.0075 | -0.0075 | 0.22 | -0.205 |
mean EIS 72-371
| -0.399 | -0.399 | -0.102 | -0.102 | 0.08 | -0.416 |
mean SCV 72-76
| 292.3025 | 292.3025 | 264.0275 | 264.0275 | 282.7475 | 251.2875 |
mean SCV 72-371
| 295.982 | 295.982 | 260.1521 | 260.1521 | 275.7521 | 275.2463 |
mean CH 72-76
| 0.25 | 0.25 | -0.25 | -0.25 | -0.25 | 0 |
mean CH 72-371
| 0.3 | 0.3 | -0.1 | -0.1 | -0.1 | 0.1 |
CQ IC50 nM
| 27.4 | 22.9 | 143.8 | 147.4 | 101.7 | 126.9 |
DCQ IC50 nM
| ND | 37.7 | 875.2 | 1182.2 | 635.3 | 649.4 |
AQ IC50 nM
| 20.2 | 18.2 | 31.6 | 25.8 | 28.9 | 35.8 |
DAQ IC50 nM
| ND | 45.2 | 70.2 | 67.9 | 54.8 | 86.2 |
C-V IC50 nM
| 27.9 | 21.8 | 76.2 | 71.3 | 49.4 | 106.2 |
Statistics
drug | logD5.2 | Vol(Å3) (base) | surface area (Å2) (base) | protons (positive charges) |
---|---|---|---|---|
CQ | -3.44 | 1010 | 606 | 2 |
DCQ | -4.61 | 910 | 555 | 2 |
AQ | -1.06 | 996 | 568 | 2 |
DAQ | -2.54 | 930 | 554 | 2 |
VP | 0.59 | 1340 | 734 | 1 |
RESIDUE | mutants | Δ sc charge res-sens | Δ EIS res-sens | Δ SCV Å3 res/sens |
---|---|---|---|---|
CYS 72 | SER | 0 | -0.30 | 0.834 |
MET 74 | ILE | 0 | 0.47 | 0.957 |
ASN 75 | GLU | -1 | 0.02 | 1.111 |
LYS 76 | THR ILE* ASN* | -1 -1 -1 | 0.92 1.83 0.46 | 0.652 0.855 0.699 |
HIS 97 | GLN | -1 | -0.29 | 0.921 |
ALA 220 | SER | 0 | -0.51 | 1.170 |
GLN 271 | GLU | -1 | 0.07 | 0.923 |
ASN 326 | SER ASP | 0 -1 | 0.38 -0.08 | 0.714 0.907 |
ILE 356 | THR LEU | 0 0 | -0.91 -0.20 | 0.761 1.007 |
ARG 371 | ILE THR | -1 -1 | 2.53 1.62 | 0.744 0.566 |
MEAN (± S.E.) | -0.60 (0.13) | 0.40 (0.25) | 0.86 (0.04) |
Results
Initial analysis of the effects of residue changes on physicochemical characteristics of the protein. (Table 5)
Correlation of physicochemical characteristics with drug activity in transfectants
Chloroquine-resistant transfectants (Table 6: Figures 1,2,3,4,5,6,7)
drug | ratio R+/R | EIS 72-76 | EIS 72-371 | SCV 72-76 | SCV 72-371 |
---|---|---|---|---|---|
CQ | 1.5 | 0.29 | 0.11 | 0.37 | 0.77 |
DCQ | 1.9 | 0 | 0.02 | 0.02 | 0.76 |
AQ | 1.4 | 0.41 | 0.56 | 0.35 | 0.23 |
DAQ | 1.6 | 0.98* | 0.98* | 0.96* | 0 |
CQ-VE | 2.2 | 0.97* | 0.99* | 0.94* | 0.01 |
Desethylamodiaquine (DAQ)
The verapamil (VE) effect
Chloroquine (CQ)-sensitive and resistant transfectants (see Table 7: Figures 8,9,10,11,12,13)
drug | ratio R/S | EIS 72-76 | EIS 72-371 | SCV 72-76 | SCV 72-371 | CH 72-76 | CH 72-371 |
---|---|---|---|---|---|---|---|
CQ | 6.4 | 0.35 | 0.30 | 0.77* | 0.95* | 0.80* | 0.80* |
DCQ | 31.4 | 0.28 | 0.26 | 0.45 | 0.93* | 0.75* | 0.75* |
AQ | 2.0 | 0.17 | 0.08 | 0.76* | 0.47 | 0.41 | 0.41 |
DAQ | 1.9 | 0.01 | 0.01 | 0.98* | 0.34 | 0.10 | 0.10 |
CQ-VE | 4.9 | 0.04 | 0.01 | 0.98* | 0.53 | 0.32 | 0.32 |
Discussion and Conclusions
Effects of residue changes on physicochemical characteristics of the protein
Conclusion
Correlation of physicochemical characteristics with drug activity in transfectants
CQ-resistant transfected lines
Response to CQ and DCQ
Response to AQ
Response to DAQ
Antiparasitic effect of CQ in combination with a resistance-reversing concentration of VE
CQ-sensitive and – resistant transfected lines
EIS | C5K76I Laboratory | C34Dd2 SE Asia | C67G8Brazil | Jav/IAJColombia | Ecu1110 Ecuador | Cambodia | Cambodia | Cambodia |
---|---|---|---|---|---|---|---|---|
HAPLO.
| CIEI | CIET | SMNT | CMET | CMNT | CMNT | CIDT | CTNT |
72
| 0.04 | 0.04 | -0.26 | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 |
74
| 0.73 | 0.73 | 0.26 | 0.26 | 0.26 | 0.26 | 0.73 | -0.18 |
75
| -0.62 | -0.62 | -0.64 | -0.62 | -0.64 | -0.64 | -0.72 | -0.64 |
76
| 0.73 | -0.18 | -0.18 | -0.18 | -0.18 | -0.18 | -0.18 | -0.18 |
SUM
| 0.88 | -0.03 | -0.82 | -0.5 | -0.52 | -0.52 | -0.13 | -0.96 |
MEAN
| 0.22 | -0.0075 | -0.205 | -0.125 | -0.13 | -0.13 | -0.0325 | -0.24 |
predict IC50 DAQ (nM)
| 53.6 | 70.3 | 84.8 | 79.0 | 79.3 | 79.3 | 72.2 | 87.4 |
ACTUAL
| 54.8 | 69.05 | 86.2 | |||||
PGH-1 factor
| Y86---N86 | Y86---N86 | S. America | S. America | S. America | Y86---N86 | Y86---N86 | Y86---N86 |
1.4------1.0 | 1.4------1.0 | ----------2.7 | ----------2.7 | ----------2.7 | 1.4------1.0 | 1.4------1.0 | 1.4------1.0 | |
predict field sensitivity (IC50) nM
| ||||||||
IC50 DAQ (nM)
| 76-------54 | 98-------70 | ---------230 | ---------213 | ---------213 | 111------79 | 101-----72 | 122-----87 |