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Directed evolution of light-activated drugs
Medical Hypotheses (1999) 53(6), 504–506 © 1999 Harcourt Publishers Ltd Article No. mehy.1998.0799
Directed evolution of light-activated drugs K. Hall Center for Nonlinear Dynamics in Physiology and Medicine, McGill University, Montreal, Quebec, Canada
Summary Photodynamic therapy is a new technology that uses light-activated molecules to target drug action to diseased areas while sparing healthy tissue. Unfortunately, the present arsenal of photosensitive molecules is limited in both number and scope. We hypothesize that new photosensitive molecules could be developed using directed molecules evolution. This paper outlines a possible selection mechanism to evolve molecules activated by a desired wavelength of light. © 1999 Harcourt Publishers Ltd
PHOTODYNAMIC THERAPY
DIRECTED MOLECULAR EVOLUTION
Physicians typically give systemic doses of drugs even when pathology can be localized to a specific region of the body. This can upset the normal function of healthy tissue and cause unwanted side effects. For example, chemotherapy not only suppresses tumor growth, but also inhibits bone marrow production of platelets and granulocytes resulting in bleeding and infection. What if we could localize drug action to diseased areas and leave healthy tissue unaffected? A new technology called photodynamic therapy addresses this question by using molecules that are activated only when exposed to a specific wavelength of light (1–3). This allows the physician to activate drugs by directing light to a desired region while sparing the surrounding tissue. Presently, the only light-activated molecules available for photodynamic therapy have a single mode of action – they produce free radicals leading to cell death (1–3). Since these photosensitive molecules have been observed to selectively accumulate in hyperproliferative cells, they are primarily useful for cancer treatment (1–3). However, photodynamic therapy could have broader applications if new photosensitive molecules could be created.
Biochemists have recently harnessed the power of evolution to invent new molecules with properties prescribed in advance by the experimenter (4–6). This technology, called directed molecular evolution, has already been used to create molecules that catalyze certain reactions (7) or bind to specific targets (8). The biotechnology industry has recognized the promise of ‘test tube evolution’ for drug development and has already evolved novel anticoagulants to treat heart disease (8). We hypothesize that directed molecular evolution can create new light-activated drugs. Directed molecular evolution involves the same three processes as Darwinian evolution: selection, reproduction, and mutation. Starting with a large pool of molecular variants, biochemists can select the ‘fittest’ molecules by identifying those that best perform some prescribed task, e.g. the most efficient catalysts or the molecules with the highest binding affinities to a target. These molecules are selected and amplified and the unwanted molecules are discarded. The amplification process is designed to introduce mutations and create a large new generation of molecular variants. While most of the mutants will perform worse than their predecessors, some will perform better and be amplified in the next generation. After several generations, this process evolves molecules that perform orders of magnitude better than any in the original pool. Directed molecular evolution has primarily been applied to DNA and RNA molecules since they are easily amplified and mutated using a ‘sloppy’ version of the polymerase chain reaction. This limitation is not funda-
Received 1 April 1998 Accepted 25 August 1998 Correspondence to: Kevin Hall, Entelos, Inc., 4040 Campbell Avenue, Suite 200, Menlo Park, CA 94040, USA. Phone: +1 650 330 5209; Fax: +1 650 330 5201
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Directed evolution of light-activated drugs
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Fig. 1 Possible two-stage selection mechanism for the directed evolution of photosensitive ligands. Candidate molecules are first subjected to an affinity purification process in the presence of light. Next, the light is shut off and we collect the molecules whose binding affinities are weakened in the absence of the light. This collection of photosensitive ligands undergoes the amplification and mutation process to obtain new molecules with enhanced photosensitivily.
mental and researchers are developing techniques to amplify and mutate other molecules (9,10). EVOLVING LIGHT-ACTIVATED MOLECULES In order to create photosensitive molecules using directed molecular evolution, the selection mechanism must somehow test for the ability to activate molecules with light. This could be accomplished by dividing the selection procedure into two steps. The first step selects the fittest molecules when the system is exposed to a desired wavelength of light. The second step involves turning off the light and selecting the molecules that survived the first step but no longer perform the required task. Thus, the two-stage selection mechanism chooses molecules whose performance is enhanced by the light. Figure 1 illustrates how this selection mechanism could be used to evolve ligands that bind to a given target only in the presence of light. First, the starting population of © 1999 Harcourt Publishers Ltd
molecules is added to a vessel with target molecules immobilized on its surface. The entire system is exposed to light at a desired wavelength. The molecules which do not bind to the targets are washed away and discarded. Molecules whose binding affinities are weakened by shutting off the light can be washed away and collected. This collection is subjected to the amplification and mutation process creating the next generation of molecules with photo-enhanced binding properties. The two-stage selection mechanism is equally applicable to the evolution of molecules that can be turned on and off by other external stimuli. Perhaps it would be desirable to activate molecules using a magnetic field or ultrasound instead of light. However, the choice of the activating stimulus is limited by the likelihood that the stimulus will modify performance. For example, energy considerations would prevent the evolution of molecules activated by audible sound. However, light has an excellent chance to enhance binding affinity or catalyzing efficiency. Medical Hypotheses (1999) 53(6), 504–506
506
Hall
The above two-stage selection mechanism is sufficiently simple that it could be easily incorporated in existing molecular evolution protocols. If successful, directed evolution of clinically useful photosensitive molecules could be just around the corner – and the future of photodynamic therapy will indeed be bright. REFERENCES 1. Fisher A. M. R., Murphree A. L., Gomer C. J. Clinical and preclinical photodynamic therapy. Lasers Surg Med 1995; 17: 2–31. 2. Levy J. G. Photodynamic therapy. TIB Tech 1995; 13: 14–18. 3. Ochsner M. Photophysical and photobiological processes in the photodynamic therapy of tumors. J Photochem Photobiol 1997; 39: 1–18.
Medical Hypotheses (1999) 53(6), 504–506
4. Joyce G. F. Directed molecular evolution. Sci Am 1992; 267: 89–97. 5. Kauffman S. A. Applied molecular evolution. J Theor Biol 1992; 157: 1–7. 6. Flam F. Co-opting a blind watchmaker. Science 1994; 265: 1032–1033. 7. Breaker R. R., Joyce G. F. Inventing and improving ribozyme function: rational design versus iterative selection methods. TIB Tech 1994; 12: 268–275. 8. Brock L. C., Griffin L. C. Latham J. A. et al. Selection of singlestranded DNA molecules that bind and inhibit human thrombin. Nature 1992; 355: 564–566. 9. Brenner S., Lerner R. A. Encoded combinatorial chemistry. Proc Natl Acad Sci USA 1992; 89: 5381–5383. 10. Roberts R. W., Szostak J. W. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci USA 1997; 94: 12297–12302.
© 1999 Harcourt Publishers Ltd
Directed evolution of light-activated drugs K. Hall Center for Nonlinear Dynamics in Physiology and Medicine, McGill University, Montreal, Quebec, Canada
Summary Photodynamic therapy is a new technology that uses light-activated molecules to target drug action to diseased areas while sparing healthy tissue. Unfortunately, the present arsenal of photosensitive molecules is limited in both number and scope. We hypothesize that new photosensitive molecules could be developed using directed molecules evolution. This paper outlines a possible selection mechanism to evolve molecules activated by a desired wavelength of light. © 1999 Harcourt Publishers Ltd
PHOTODYNAMIC THERAPY
DIRECTED MOLECULAR EVOLUTION
Physicians typically give systemic doses of drugs even when pathology can be localized to a specific region of the body. This can upset the normal function of healthy tissue and cause unwanted side effects. For example, chemotherapy not only suppresses tumor growth, but also inhibits bone marrow production of platelets and granulocytes resulting in bleeding and infection. What if we could localize drug action to diseased areas and leave healthy tissue unaffected? A new technology called photodynamic therapy addresses this question by using molecules that are activated only when exposed to a specific wavelength of light (1–3). This allows the physician to activate drugs by directing light to a desired region while sparing the surrounding tissue. Presently, the only light-activated molecules available for photodynamic therapy have a single mode of action – they produce free radicals leading to cell death (1–3). Since these photosensitive molecules have been observed to selectively accumulate in hyperproliferative cells, they are primarily useful for cancer treatment (1–3). However, photodynamic therapy could have broader applications if new photosensitive molecules could be created.
Biochemists have recently harnessed the power of evolution to invent new molecules with properties prescribed in advance by the experimenter (4–6). This technology, called directed molecular evolution, has already been used to create molecules that catalyze certain reactions (7) or bind to specific targets (8). The biotechnology industry has recognized the promise of ‘test tube evolution’ for drug development and has already evolved novel anticoagulants to treat heart disease (8). We hypothesize that directed molecular evolution can create new light-activated drugs. Directed molecular evolution involves the same three processes as Darwinian evolution: selection, reproduction, and mutation. Starting with a large pool of molecular variants, biochemists can select the ‘fittest’ molecules by identifying those that best perform some prescribed task, e.g. the most efficient catalysts or the molecules with the highest binding affinities to a target. These molecules are selected and amplified and the unwanted molecules are discarded. The amplification process is designed to introduce mutations and create a large new generation of molecular variants. While most of the mutants will perform worse than their predecessors, some will perform better and be amplified in the next generation. After several generations, this process evolves molecules that perform orders of magnitude better than any in the original pool. Directed molecular evolution has primarily been applied to DNA and RNA molecules since they are easily amplified and mutated using a ‘sloppy’ version of the polymerase chain reaction. This limitation is not funda-
Received 1 April 1998 Accepted 25 August 1998 Correspondence to: Kevin Hall, Entelos, Inc., 4040 Campbell Avenue, Suite 200, Menlo Park, CA 94040, USA. Phone: +1 650 330 5209; Fax: +1 650 330 5201
504
Directed evolution of light-activated drugs
505
Fig. 1 Possible two-stage selection mechanism for the directed evolution of photosensitive ligands. Candidate molecules are first subjected to an affinity purification process in the presence of light. Next, the light is shut off and we collect the molecules whose binding affinities are weakened in the absence of the light. This collection of photosensitive ligands undergoes the amplification and mutation process to obtain new molecules with enhanced photosensitivily.
mental and researchers are developing techniques to amplify and mutate other molecules (9,10). EVOLVING LIGHT-ACTIVATED MOLECULES In order to create photosensitive molecules using directed molecular evolution, the selection mechanism must somehow test for the ability to activate molecules with light. This could be accomplished by dividing the selection procedure into two steps. The first step selects the fittest molecules when the system is exposed to a desired wavelength of light. The second step involves turning off the light and selecting the molecules that survived the first step but no longer perform the required task. Thus, the two-stage selection mechanism chooses molecules whose performance is enhanced by the light. Figure 1 illustrates how this selection mechanism could be used to evolve ligands that bind to a given target only in the presence of light. First, the starting population of © 1999 Harcourt Publishers Ltd
molecules is added to a vessel with target molecules immobilized on its surface. The entire system is exposed to light at a desired wavelength. The molecules which do not bind to the targets are washed away and discarded. Molecules whose binding affinities are weakened by shutting off the light can be washed away and collected. This collection is subjected to the amplification and mutation process creating the next generation of molecules with photo-enhanced binding properties. The two-stage selection mechanism is equally applicable to the evolution of molecules that can be turned on and off by other external stimuli. Perhaps it would be desirable to activate molecules using a magnetic field or ultrasound instead of light. However, the choice of the activating stimulus is limited by the likelihood that the stimulus will modify performance. For example, energy considerations would prevent the evolution of molecules activated by audible sound. However, light has an excellent chance to enhance binding affinity or catalyzing efficiency. Medical Hypotheses (1999) 53(6), 504–506
506
Hall
The above two-stage selection mechanism is sufficiently simple that it could be easily incorporated in existing molecular evolution protocols. If successful, directed evolution of clinically useful photosensitive molecules could be just around the corner – and the future of photodynamic therapy will indeed be bright. REFERENCES 1. Fisher A. M. R., Murphree A. L., Gomer C. J. Clinical and preclinical photodynamic therapy. Lasers Surg Med 1995; 17: 2–31. 2. Levy J. G. Photodynamic therapy. TIB Tech 1995; 13: 14–18. 3. Ochsner M. Photophysical and photobiological processes in the photodynamic therapy of tumors. J Photochem Photobiol 1997; 39: 1–18.
Medical Hypotheses (1999) 53(6), 504–506
4. Joyce G. F. Directed molecular evolution. Sci Am 1992; 267: 89–97. 5. Kauffman S. A. Applied molecular evolution. J Theor Biol 1992; 157: 1–7. 6. Flam F. Co-opting a blind watchmaker. Science 1994; 265: 1032–1033. 7. Breaker R. R., Joyce G. F. Inventing and improving ribozyme function: rational design versus iterative selection methods. TIB Tech 1994; 12: 268–275. 8. Brock L. C., Griffin L. C. Latham J. A. et al. Selection of singlestranded DNA molecules that bind and inhibit human thrombin. Nature 1992; 355: 564–566. 9. Brenner S., Lerner R. A. Encoded combinatorial chemistry. Proc Natl Acad Sci USA 1992; 89: 5381–5383. 10. Roberts R. W., Szostak J. W. RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci USA 1997; 94: 12297–12302.
© 1999 Harcourt Publishers Ltd