Combinatorial Chemistry: A new dimension of Drug Discovery

Introduction :

Combinatorial chemistry has emerged as a new technology for rapid synthesis
of large number of compounds. Combinatorial chemistry is a method for generating
vast numbers of molecular substances, then rapidly screening them for desirable
properties. Now a days this technology is used for synthesis of library of compounds
having therapeutical activities. Combinatorial technologies offer significant
advances over traditional scientific research methodologies. In particular,
their high-speed approach promises faster results at considerably lower costs
than conventional techniques.

The use of High Throughput Screening robotic technology allows up to 1000
samples to be tested per day using a single robot. The conventional techniques
of synthesis consumes a lot of time and money etc. In the combinatorial chemistry
a library of thousands of compounds can be prepared and which can be screened
for the therapeutic activities.

Combinatorial chemistry dates back to 1963, when Rockefeller University biochemist R. Bruce Merrifield developed a solid-phase method of synthesizing peptides. However, the field had little impact until the mid-1980s.  In 1991, the seminal description of the practical synthesis and use of combinatorial libraries was published ¹. The solid phase synthesis of a combinatorial library containing 64,000,000 hexapeptides made from the 20 proteogenic amino acids was described. This library was synthesized as 400 subsets, in which the first two amino acid positions for each subset were known, while the remaining four amino acid positions contained all possible combinations of amino acids.

The combinatorial library was synthesized using the "divide, couple, and recombine"
(DCR) technique in which the resin is divided into aliquots, reactions are carried
out on individual aliquots, and the resin is then recombined to yield a close
to equimolar mixture of compounds. Active compounds within this combinatorial
library were determined based on an iterative deconvolution of the screening
data in which progressively smaller subsets of the original library were synthesized.
In 1992, the power of combinatorial synthesis was extended with the introduction
of combinatorial libraries in a positional scanning format ². In
this format, six orthogonal libraries were synthesized in which a single amino
acid position was defined while the remaining five positions contained all possible
combinations of amino acids. Each of the six libraries contained the same 64,000,000
peptides, divided into 20 subsets corresponding to each of the 20 amino acids
at the known position. Following screening of each library, the importance of
an amino acid at each position was determined. Subsequently, individual peptides
were synthesized and shown to have biological activity.

Due to the practical difficulties of synthesizing positional scanning combinatorial
libraries using the "divide, couple, recombine" method, mixture positions in
this library were incorporated using a predetermined mixture of protected amino
acids ³. The techniques used to determine the proper ratios for equimolar
incorporation and was published in 1994.Many libraries, including peptide, peptidomimetic,
and heterocyclic libraries, have now been synthesized using these techniques.

Methods :

A combinatorial chemistry or biology approach to discovery begins with a large
collection of unique substances, often molecules, which is the source
of molecular diversity. By the process of screening, the members
of this library that have "desirable properties" (as determined by
the screen) are culled out. Combinatorial chemistry enables chemists to examine
a large volume of compounds for biological screening, with increased chemical
diversity. Combinatorial Chemistry synthetic methods have the potential to generate
a very large number of molecules to be tested, either in mixtures or as individual
molecules⁴. As with traditional drug design, combinatorial chemistry
relies on organic synthesis methodologies. The difference is the scope instead
of synthesizing a single compound, combinatorial chemistry exploits automation
and miniaturization to synthesize large libraries of compounds. But because
large libraries do not produce active compounds independently, scientists also
need a straightforward way to find the active components within these enormous
populations. Thus, combinatorial organic synthesis (COS) is not random, but
systematic and repetitive, using sets of chemical "building blocks" to form
a diverse set of molecular entities. Scientists have developed several different
COS strategies, to determine active compounds within populations, either spatially,
through chemical encoding, or by systematic, successive synthesis and biological
evaluation (deconvolution).

There are three common approaches to COS. During arrayed, spatially addressable
synthesis, building blocks are reacted systematically in individual reaction
wells or positions to form separated "discrete molecules." Active compounds
are identified by their location on the grid. This method has been applied in
scale as well as in miniature. The second technique, known as encoded mixture
synthesis, uses nucleotide, peptide, or other types of more inert chemical tags
to identify each compound.

During deconvolution, the third approach, a series of compound mixtures is
synthesized combinatorially, each time fixing some specific structural feature.
Each mixture is assayed as a mixture and the most active combination is processed.
Further rounds systematically fix other structural features until a manageable
number of discrete structures can be synthesized and screened. Scientists working
with peptides, for example, can use deconvolution to optimize, or locate, the
most active peptide sequence from millions of possibilities. The whole idea
is to remove the guesswork and instead, to create and test as many compounds
or mixtures as possible logically and systematically to obtain a viable set
of active leads.

Encoding Methods in combinatorial chemistry

1.Positional Encoding

        J. Ellman described the synthesis of various organic compound libraries⁵. In his work, the solid-phase support for synthesis begins with a one-piece polypropylene rack of pins arrayed in a 12 × 8 matrix. The synthetic method is designed so that each pin bears a different, single organic compound (assuming the synthetic method worked). Because the arrangement of each pin in the rack cannot change, the physical position serves as the code for each library member. That is, after a screen is complete one might find that the compound on pin E-7 was a "winner." By checking back on how the synthesis was conducted, one can know the chemical identity of the compound on pin E-7. Because it is an assumption that all reactions worked, the very first next step is to resynthesize the compound you expect to be on pin E-7, to thoroughly characterize that compound, and to verify that it indeed is a "winner."

             The method of positional encoding was utilized in the first synthetic combinatorial library, in which peptides were synthesized on such a rack of pins and screened for antibody binding ability ⁶. Such a two-dimensional matrix approach is also the encoding method currently in use for the "DNA chip" technologies.

2. Chemical Encoding

        Using the in vitro selection method, M. Famulok described the generation of vast numbers (billions and up) of RNA sequences in a small volume of solution. The solution was passed through an affinity column, and RNA sequences that bound tightly to the immobilized ligands eluted more slowly than others. By necessity, only minuscule amounts of each sequence are present in the starting mixture. Polymerase chain reaction (PCR) technology permits the amplification of such minuscule amounts of nucleic acid sequences. After the amount of RNA available for analysis has been amplified, the RNA sequence can be determined by using existing analytical methods. By establishing the RNA sequence of the "winner," its chemical identity becomes known. While this thinking may sound circuitous, the determination of chemical identity may also be thought of as simply the most direct method of chemical encoding. If the "winner" of a screening experiment had been 10 mg of a small organic molecule, its structure could have been determined by using standard characterization methods (NMR, mass spectrometry, microanalysis, etc.) and the sample would have encoded its own identity by way of direct chemical determination.

           It is unlikely that any "winner" from a synthesized library would ever be obtained in 10-mg amount. (A 10,000-member library would have to contain 100 g, and that would be very hard to screen indeed.) While it is possible to synthesize mixtures of small molecules each in a tiny amount, determining the identity of "winners" is accomplished by a process of  deconvolution rather than encoding. [This is because the only code in such a library is chemical identity, and at such tiny amount the only tool useful to establish identity is mass spectrometry     ^7-10. Instead, such small molecule libraries are produced on polymer beads, each bead possessing only one type of molecule¹¹. Encoding of each bead can then be accomplished with a different type of chemical encoding, a chemical tag. The first chemical tags were nucleic acids, because their sequence can be established with an exceedingly tiny amount of the tag¹². Rather recently, more robust forms of chemical tags based on abiotic molecules have been described ¹³.

      This form of encoding is today accomplished
by using a microelectronic device called a radiofrequency (rf) memory
tag¹⁴. The tag, measuring 13 × 3 mm, is
encased in heavy-walled glass and contains the following: (i)
a silicon chip, on to which is laser-etched a unique binary code
(with 2⁴⁰ possible codes, no two chips need ever be manufactured
with the same code); (ii) a rectifying circuit, with which
absorbed rf energy is converted to D.C. electrical energy; (iii)
a transmitter/receiver circuit; and (iv) an antenna, through
which energy is received and rf signals are both received and sent.
No external power source is required. Placing this chip near a computer-interfaced
transceiver causes the chip to power up, read its identification
tag, and send that code to the transceiver in an rf pulse. A reaction
platform (e.g., a bead, tube, bag, or can) bearing an rf chip is
thus uniquely tagged in a machine-readable fashion. Automated handling
is facilitated by the near nondirectionality of the reading frame.
A sorting machine designed to send rf-tagged reaction platforms to
vessels for reaction or cleavage has been described

Other Encoding Methods like graphical, optical, NMR-based and physical methods
of encoding. are also available but less extensively used.

Conclusion :

Combinatorial chemistry involves use of sophisticated instruments and it has
advantage over conventional methods that it can generate large number of compounds
which can be screened for their biological activities. The traditional methods
of drug discovery are time consuming. With the help of combinatorial chemistry
we can save time, money and the process of drug discovery can be speed up to
explore new drug molecules. No doubt that combinatorial chemistry has evolved
as a future of drug discovery and will change the scenario of drug discovery
in the new millennium.

 References  :

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Dr. Parakh S.R.  Jagdale  S.C. ^* and  
Chabukswar A.R.

MAEER's Maharashtra Institute of Pharmacy, S. No. 124, Ex-Serviceman colony,
MIT campus Paud road, Kothrud, Pune – 411038

* Corresponding author Ms. Swati Changdeo Jagdale is
presently working as Assistant Professor in Pharmaceutics at MAEER's
Maharashtra Institute of Pharmacy, Pune, India.She is first University rank
holder at M.Pharm ( Pharmaceutics) in Pune University.She has also qualified
GATE examination at National level with 99.09%.Besides her academic excellance
and experience, she also has Industrial experience in Research and Development
department .She has National and International publications to her credit.

Dr. Parakh S.R. is Principal and Professor in Pharmaceutics
at MAEER'S Maharashtra Institute of Pharmacy.
He is a member of Senate, University of Pune. He is working in board of studies
of Pharmaceutics of University of Pune. He has several National and International
publications to his credit. He had eight years of Industrial experience and
twenty one years of Academic experience. He is active member of Indian
Pharmaceutical Association, Association
of Pharmaceuticals Teachers of India.

Chabukswar A.R.. is working as Assistant Professor in Pharmaceutical
Chemistry at MAEER’S Maharashtra
Institute of Pharmacy, Pune-400038 . He had completed his M.Pharm in Pharmaceutical
Chemistry from K.M.Kundanani college of Pharmacy, Bombay.