A 3-D QSAR study of Efflux Pump Substrates

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A 3-D QSAR study of Efflux Pump Substrates Powered By Docstoc
					            Kelcey Anderson
Bioinformatics and Bioengineering Summer Institute
         Virginia Commonwealth University
                   August 4, 2009
Drug Action in the Cell




 Cannon, et al. 2009. Clin. Microbiol. Rev. 22,2:291-321
            Efflux Pumps
 Found in Gram-negative
  bacteria, including E. coli
 AcrAB-TolC
 Composed of three parts
  spanning both the inner
  and outer cell membranes
 Responsible for extruding
  a variety of mainly
  lipophilic substances
                                Symmons, et al. 2009. PNAS 106:7173-78
          Why Study Efflux Pumps?
         Also responsible for extrusion of
          antibiotics
         Multidrug resistant strains
          overexpress efflux pump proteins
         Development of new antibiotics
Penicillin G                            Minocycline
                     Acriflavine
      Research Questions
 Can we predict the antibiotics that are
  more likely to be extruded by the efflux
  pump?
 How can we use these predictions for
  the development of new drugs?
        Experimental Data
 Knock out efflux pump
                                Literature
  genes
                                Sources:
 Measure MIC values for     Mazzariol et al. 2000
  different substances       Gotoh et al. 2003
                             Nishino et al. 2003
 Ratio of normal vs.        Sulavik et al. 2001
  knocked out MIC values     Lee et al. 2000
                             McMurry et al. 1998
 Is there a correlation     Yang et al. 2003
  between the ratio of       Thanassi et al. 1997
                             Sanchez et al. 1997
  efflux and the structure   Nikaido et al. 1998
  of the compounds?
    Quantitative Structure-Activity
        Relationship (QSAR)
   Basic Principle: The structure and properties of a
    molecule determine the activity of the molecule, or in
    this case, how much it is effluxed




   The difference in efflux can be explained
    by the difference of one chlorine atom
   How do you quantify the difference of one
    chlorine atom?
                     QSAR
   Many different parameters
    calculated, such as:
     Molecular size/shape
     Dipole moment
     logP
   While this study correlates
    parameters with efflux, it
    doesn’t give direct
    information about structural
    modifications that can reduce
    efflux                          Kiralj, R. 2006. J. Mol. Graph. Mod. 25:126-45
    3-D QSAR: Comparative
Molecular Field Analysis (CoMFA)
   Comparative
    Molecular Field
    Analysis (CoMFA) –
    Three-dimensional
    noncovalent
    interactions
     Sterics
     Electrostatics
 Test the field at each
  point
 HINTCoMFA
     Hydrophobicity
         Here’s What We Did
 Drew 27 structures of penicillins and
  cephalosporins in Sybyl 8.1
 All contained common β-lactam
  substructure




                        Cephalothin

Penicillin G
              Alignment
 Molecules were
  aligned according
  to common
  substructure
 Alignment of
  molecules is critical
  for 3-D QSAR
Aligned Structures
      Tweaking the Alignment
   Rotatable bonds      Similar functional
                          groups




                           Naphthalene   Phenyloxazole
Partial Least Squares (PLS)
   Statistical technique for finding a model
    relating efflux and molecular properties
     HINTCoMFA
     Sterics
     Electrostatics
     H-bond Sterics
     H-bond Electrostatics
   Cross-validation (Q2)
     Important because more columns than rows
     may cause overfitting
 Charged vs. Neutral




Neutral           Charged
       PLS Results - Neutral
                     H-bond    H-bond                    Standard
HINT Sterics Electro Sterics   Electro     R2      Q2      Error Comp
1.000                                    0.697   0.425     1.405   2
      1.000                              0.945   0.568     0.611   3
              1.000                      0.837   0.460     1.076   4
                      0.521    0.479     0.913   0.556     0.768   3
0.511 0.489                              0.808   0.502     1.119   2
0.656         0.344                      0.759   0.459     1.281   3
0.297                 0.368    0.335     0.820   0.504     1.084   2
      0.628 0.372                        0.937   0.624     0.668   4
      0.265           0.364    0.371     0.945   0.591     0.612   3
              0.203 0.429      0.368     0.894   0.563     0.847   3
0.387 0.367 0.246                        0.867   0.561     0.952   3
0.230 0.211           0.287    0.272     0.854   0.541     0.976   2
0.231         0.171 0.298      0.300     0.905   0.570     0.805   3
      0.232 0.169 0.306        0.294     0.931   0.608     0.683   3
0.179 0.181 0.153 0.234        0.253     0.930   0.598     0.690   3
      PLS Results - Charged
                     H-bond    H-bond                    Standard
HINT Sterics Electro Sterics   Electro     R2      Q2      Error Comp
1.000                                    0.854   0.589     0.995   3
      1.000                              0.944   0.525     0.619   3
              1.000                      0.783   0.348     1.243   4
                      0.598    0.402     0.877   0.548     0.915   3
0.556 0.444                              0.863   0.618     0.946   2
0.745         0.255                      0.870   0.598     0.939   3
0.319                 0.350    0.331     0.825   0.557     1.067   2
      0.664 0.336                        0.971   0.594     0.451   4
      0.273           0.378    0.349     0.842   0.531     1.014   2
              0.161   0.522    0.318     0.870   0.550     0.941   3
0.449 0.388 0.163                        0.954   0.660     0.559   3
0.252 0.200           0.274    0.275     0.864   0.588     0.943   2
0.295         0.112   0.363    0.230     0.905   0.592     0.805   3
      0.250 0.119     0.365    0.266     0.925   0.581     0.716   3
0.233 0.198 0.090     0.275    0.204     0.948   0.619     0.595   3
         Further Validation
 3 randomly selected training sets of 18
  molecules
 3 randomly selected test sets of 9
  molecules
 Predict efflux of test set from training set
 Calculate R2 of predicted versus actual
  efflux
                                        Results - Charged
                       HINTCoMFA and                                               HINTCoMFA,                                            HINTCoMFA, Sterics,
                                                                                                                                         Electrostatics, H-bond
                           Sterics                                                  Sterics and                                           Sterics, and H-bond
                     14                                                            Electrostatics                                            Electrostatics
                     12          R² = 0.2011
                                                                              15
                     10                                                                 R² = 0.1386
                                                                                                                                        30                 R² = 0.1267
  Predicted Efflux




                                                                                                                                        20



                                                           Predicted Efflux
                      8




                                                                                                                      redicted Efflux
                                                                              10
                      6                                                                                                                 10
                      4                                                        5                                                         0
                      2                                                                                                                       0             5             10
                                                                                                                                        -10
                      0                                                        0
                     -2 0   2       4        6       8                             0    2      4       6      8                         -20
                     -4                                                       -5                                                        -30
                            Experimental Efflux                                        Experimental Efflux                                         Experimental Efflux


                            Experimental     Predicted                                  Experimental    Predicted                                 Experimental
  Compound                     Efflux          Efflux      Compound                        Efflux         Efflux     Compound                        Efflux    Predicted Efflux
    azlocillin                    4            4.0762        azlocillin                       4          4.29914      ampicillin                        1          25.7836
  cefaloridine                  0.5           5.76296      cefaloridine                     0.5          4.8379     cefamandole                       1.5          -6.8309
   cefpirome                      1           4.59915       cefpirome                         1          3.60789      cefepime                          0         -7.38936
  ceftazidime                     0           4.67878      ceftazidime                        0          3.74119      cefoxitin                       1.5         -3.83287
cephalosporinC                    0           12.1518    cephalosporinC                       0          12.0176     ceftazidime                        0         -18.8207
  faropenem                       2           -1.4075      faropenem                          2         -1.67211     ceftriaxone                        0         -10.4137
    nafcillin                   7.5           3.51439        nafcillin                      7.5          3.57557       nafcillin                      7.5          3.82874
   penicillinN                    0           8.99761       penicillinN                       0          8.94097       oxacillin                      8.5          8.61405
  piperacillin                    3           3.20176      piperacillin                       3          3.30456     sulbenicillin                      1           7.4452
                                                              Results
                      Neutral Molecules using Sterics, Electrostatics,
                        H-bond Sterics, and H-bond Electrostatics
                      10                                                          8                                                             5
                                 R² = 0.8406                                                                                                             R² = 0.4717
   Predicted Efflux




                                                               Predicted Efflux




                                                                                                                            Predicted Efflux
                       8                                                          6          R² = 0.7237                                        4
                       6                                                                                                                        3
                                                                                  4
                       4                                                                                                                        2
                       2                                                          2
                                                                                                                                                1
                       0                                                          0                                                             0
                           0            5              10                             0            5               10
                                                                                                                                               -1 0    2      4        6     8
                                Experimental Efflux                                        Experimental Efflux                                        Experimental Efflux



                               Experimental     Predicted                                 Experimental                                                Experimental    Predicted
Compound                          Efflux          Efflux     Compound                        Efflux    Predicted Efflux     Compound                     Efflux          Efflux
 amoxicillin                         0           1.50566      ampicillin                        1         0.771516            azlocillin                    4           2.89042
  cefalotin                          1           1.4389     cefamandole                       1.5         0.377938          cefaloridine                  0.5         -0.378696
  cefazolin                          0            1.558       cefepime                          0         1.16332            cefpirome                      1          0.462454
cefotaxime                         1.5           1.3924       cefoxitin                       1.5         2.08809           ceftazidime                     0          0.419119
cefuroxime                           3           2.49287     ceftazidime                        0         0.60735         cephalosporinC                    0           1.15945
 cloxacillin                         8           8.41614     ceftriaxone                        0         1.35764           faropenem                       2           0.93727
 imipenem                            0           2.55825       nafcillin                      7.5         3.39569             nafcillin                   7.5           3.90485
 penicillinG                       2.5           2.89332       oxacillin                      8.5         7.22959            penicillinN                    0           3.14532
sulbenicillin                        1           2.93117     sulbenicillin                      1         2.87694           piperacillin                    3           2.92451
                                                              Results
                      Neutral Molecules using Sterics, Electrostatics,
                        H-bond Sterics, and H-bond Electrostatics
                      10                                                          8                                                             5
                                 R² = 0.8406                                                                                                             R² = 0.4717
   Predicted Efflux




                                                               Predicted Efflux




                                                                                                                            Predicted Efflux
                       8                                                          6          R² = 0.7237                                        4
                       6                                                                                                                        3
                                                                                  4
                       4                                                                                                                        2
                       2                                                          2
                                                                                                                                                1
                       0                                                          0                                                             0
                           0            5              10                             0            5               10
                                                                                                                                               -1 0    2      4        6     8
                                Experimental Efflux                                        Experimental Efflux                                        Experimental Efflux



                               Experimental     Predicted                                 Experimental                                                Experimental    Predicted
Compound                          Efflux          Efflux     Compound                        Efflux    Predicted Efflux     Compound                     Efflux          Efflux
 amoxicillin                         0           1.50566      ampicillin                        1         0.771516            azlocillin                    4           2.89042
  cefalotin                          1           1.4389     cefamandole                       1.5         0.377938          cefaloridine                  0.5         -0.378696
  cefazolin                          0            1.558       cefepime                          0         1.16332            cefpirome                      1          0.462454
cefotaxime                         1.5           1.3924       cefoxitin                       1.5         2.08809           ceftazidime                     0          0.419119
cefuroxime                           3           2.49287     ceftazidime                        0         0.60735         cephalosporinC                    0           1.15945
 cloxacillin                         8           8.41614     ceftriaxone                        0         1.35764           faropenem                       2           0.93727
 imipenem                            0           2.55825       nafcillin                      7.5         3.39569             nafcillin                   7.5           3.90485
 penicillinG                       2.5           2.89332       oxacillin                      8.5         7.22959            penicillinN                    0           3.14532
sulbenicillin                        1           2.93117     sulbenicillin                      1         2.87694           piperacillin                    3           2.92451
                 Visualization
Neutral Molecules using Sterics, Electrostatics, H-bond
          Sterics, and H-bond Electrostatics
                                        Lower Values of Efflux:
                                         Less bulk near green
                                         More bulk near yellow
                                         Less positive near blue
                                         Less negative near red




         Shown with Oxacillin
                  Docking
 Basic Assumption of CoMFA: Molecules
  with a similar structure will bind in a
  similar way
 Binding is not dependent on structure,
  but rather sterics, electrostatics, and
  hydrophobicity
 How do we know if the alignment
  reflects the way they bind in the pocket?
     Docking can provide a clue
    Diverse Efflux Substrates
 How to align diverse structures for
  QSAR?
 Use docked positions
             Summary
 Aligned structures
 Used CoMFA and HINTCoMFA to create
  fields surrounding the molecules
 Analyzed the correlation between efflux
  and structure using PLS
             Conclusions
 Antibiotics with a predicted efflux of ≤2
  are not effluxed or are poorly effluxed
 Antibiotics most likely bind to the pocket
  in their neutral forms
         Future Research
 Use a docking program to attain a
  different alignment for QSAR
 Use different data for efflux

				
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