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A targeted library of small molecules has been prepared to optimize the biological activity of BN82002, our initial lead compound, recently described as an original inhibitor of CDC25 phosphatases. Some of these compounds inhibit CDC25 in the micromolar range and therefore reinforce the interest of CDC25 as an anticancer target.
The advantages of evaporative light-scattering detection over UV detection for the quantitation of combinatorial libraries composed of small organic compounds by HPLC are described. The detector's response is independent of the sample chromophore, which makes it well-suited to chromatographic analyses of mixtures of dissimilar solutes. Thus, HPLC with evaporative light-scattering detection offers to potential for reducing false positive or false negative results in screening assays, because of its ability to detect the presence of impurities that absorb poorly in the UV (e.g., those impurities originating from the polymeric support). Furthermore, the evaporative light-scattering detector exhibits a nearly equivalent response to compounds of similar structural class. Hence, rapid quantitation of compound libraries may be carried out with the use of a single external standard. For example, the quantitation errors, based on a single external standard, for a series of steroids, hydantoins, and BOC- and Fmoc-protected amino acids by normal-phase HPLC with evaporative light-scattering detection average approximately +/-10%. The application of the evaporative light-scattering detector to the quantitation of low-level sample impurities and the detector's compatibility with gradient elution are also described.
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A general approach to the solution phase, parallel synthesis of chemical libraries, which allows the preparation of multi-milligram quantities of each individual member, is exemplified with both a universal and dipeptide mimetic template. In each step of the sequence, the reactants, unreacted starting material, reagents and their byproducts are removed by simple liquid/ liquid or liquid/solid extractions providing the desired intermediates and final compounds in high purities (> or = 90-100%) independent of the reaction yields and without deliberate reaction optimization.
Tyrosine phosphatases (PTPases) dephosphorylate phosphotyrosines while dual-specificity phosphatases (DSPases) dephosphorylate contiguous and semicontiguous phosphothreonine and phosphotyrosine on cyclin dependent kinases and mitogen-activated protein kinases. Consequently, PTPases and DSPases have a central role controlling signal transduction and cell cycle progression. Currently, there are few readily available potent inhibitors of PTPases or DSPases other than vanadate. Using a pharmacophore modeled on natural product inhibitors of phosphothreonine phosphatases, we generated a refined library of novel, phosphate-free, small-molecule compounds synthesized by a parallel, solid-phase combinatorial-based approach. Among the initial 18 members of this targeted diversity library, we identified several inhibitors of DSPases: Cdc25A, -B, and -C and the PTPase PTP1B. These compounds at 100 microM did not significantly inhibit the protein serine/threonine phosphatases PP1 and PP2A. Kinetic studies with two members of this library indicated competitive inhibition for Cdc25 DSPases and noncompetitive inhibition for PTP1B. Compound AC-alphaalpha69 had a Ki of approximately 10 microM for recombinant human Cdc25A, -B, and -C, and a Ki of 0.85 microM for the PTP1B. The marked differences in Cdc25 inhibition as compared to PTP1B inhibition seen with relatively modest chemical modifications in the modular side chains demonstrate the structurally demanding nature of the DSPase catalytic site distinct from the PTPase catalytic site. These results represent the first fundamental advance toward a readily modifiable pharmacophore for synthetic PTPase and DSPase inhibitors and illustrate the significant potential of a combinatorial-based strategy that supplements the rational design of a core structure by a randomized variation of peripheral substituents.
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The selective induction of death in cancer cells is a major challenge in modern medicine. In this communication we describe the synthesis of an 88-membered combinatorial library, and the subsequent evaluation of these compounds for their ability to selectively induce apoptosis in cancerous cells. A compound was identified from the library that induces apoptosis in U-937 and HL-60 cell lines. This compound is a remarkably selective pro-apoptotic agent for these cancer cell lines, as it does not induce significant death in noncancerous white blood cells, even at concentrations as high as 1000 muM.
Natural products have been a critically important source of clinically relevant small molecule therapeutics. However, the discovery rate of novel structural classes of antimicrobial molecules has declined. Recently, increasing evidence has shown that the number of species cultivated from soil represents less than 1% of the total population, opening up the exciting possibility that these uncultured species may provide a large untapped pool from which novel natural products can be discovered. We have constructed and expressed in E. coli a BAC (bacterial artificial chromosome) library containing genomic fragments of DNA (5-120kb) isolated directly from soil organisms (S-DNA). Screening of the library resulted in the identification of several antimicrobial activities expressed by different recombinant clones. One clone (mg1.1) has been partially characterized and found to express several small molecules related to and including indirubin. These results show that genes involved in natural product synthesis can be cloned directly from S-DNA and expressed in a heterologous host, supporting the idea that this technology has the potential to provide novel natural products from the wealth of environmental microbial diversity and is a potentially important new tool for drug discovery.
In eukaryotes, phosphorylation of serine, threonine, and tyrosine residues on proteins is a fundamental posttranslational regulatory process for such functions as signal transduction, gene transcription, RNA splicing, cellular adhesion, apoptosis, and cell cycle control. Based on functional groups present in natural product serine/threonine protein phosphatase (PSTPase) inhibitors, we have designed pharmacophore model 1 and demonstrated the feasibility of a combinatorial chemistry approach for the preparation of functional analogues of 1. Preliminary biological testing of 18 structural variants of 1 has identified two compounds with growth inhibitory activity against cultured human breast cancer cells. In vitro inhibition of the PSTPase PP2A was demonstrated with compound 1d. Using flow cytometry we observed that compound f1 caused prominent inhibition in the G1 phase of the cell cycle. Thus, the combinatorial modifications of the minimal pharmacophore 1 can generate biologically interesting antiproliterative agents.
Combinatorial libraries were screened for molecules that induce mouse myogenic lineage committed cells to dedifferentiate in vitro. A 2,6-disubstituted purine, reversine, was discovered that induces lineage reversal of C2C12 cells to become multipotent progenitor cells which can redifferentiate into osteoblasts and adipocytes. This and other such molecules are likely to provide new insights into the molecular mechanisms that control cellular dedifferentiation and may ultimately be useful to in vivo stem cell biology and therapy.
The relatively new field of combinatorial chemistry has enabled researchers to create large mixtures of compounds that can be screened for leads in developing potential drug candidates. The new synthetic method has also created a need for better procedures to analyze the complex mixtures that are generated. The immediate goal in most cases is to verify the synthetic procedure and to determine the purity and completeness of the library sample before binding studies are initiated. We report here a method to rapidly characterize small-molecule combinatorial libraries in solution. All combinatorial library samples were synthesized by combining a core molecule bearing two acid chloride functionalities with various amino acids to generate libraries of 36, 78 and 120 components. Using electrospray ionization fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) we were able to identify 70-80% of the library components. All samples were analyzed as mixtures by direct infusion without chromatographic separation. Furthermore, nominally isobaric components could be resolved and identified through exact mass assignments without tandem mass spectrometery. ESI-FTICR-MS is a rapid and convenient tool for the characterization of small-molecule libraries. The method is especially useful for the analysis of larger libraries that contain many nominally isobaric components and impurities.
A new strategy for monovalently displaying small molecules on phage surfaces was developed and applied to high throughput screening for molecules with high binding affinity to the target protein. Peptidyl carrier protein (PCP) excised from nonribosomal peptide synthetase was monovalently displayed on the surface of M13 phage as pIII fusion proteins. Small molecules of diverse structures were conjugated to coenzyme A (CoA) and then covalently attached to the phage displayed PCP by Sfp phosphopantetheinyl transferase. Because Sfp is broadly promiscuous for the transfer of small molecule linked phosphopantetheinyl moieties to apo PCP domains, this approach will enable displaying libraries of small molecules on phage surfaces. Unique 20-base-pair (bp) DNA sequences were also incorporated into the phagemid DNA so that each compound displayed on the phage surface was encoded by a DNA bar code encapsulated inside the phage coat protein. Single round selection of phage displayed small molecules achieved more than 2000-fold enrichment of small molecules with nM binding affinity to the target protein. The selection process is further accelerated by the use of DNA decoding arrays for identifying the selected small molecules.
A small-molecule synthetic combinatorial library was designed and synthesized that features potential pharmacophores attached to a variety of small cyclic scaffolds. The synthesis of the library involved randomization of three types of building blocks: 20 amino acids, 10 aromatic hydroxy acids and 21 alcohols, totaling a library complexity of 4200 compounds. Mitsunobu polymer-supported etherification was used in the last randomization. The library compounds were attached to beads via an ester-bond linkage enabling both on-bead as well as in-solution screening. When the library was tested against a model target, streptavidin, specific binders were found. The structures of the most active compounds were determined from the fragmentation pattern in MS/MS experiments.
An improved forward chemical genetics approach was successfully demonstrated using a tagged library concept. A small-molecule triazine library with linkers was used to screen for brain/eye developmental phenotypes in a zebrafish embryo system. This approach enabled the rapid isolation of the target proteins by facile affinity matrix preparation and elucidated the first small-molecule inhibitors for several ribosomal accessory proteins or their complex as the target.
Roberts and Li break the standard library mold by generating hybrid libraries of small molecules tethered to peptides. Hybrid libraries harness larger chemical and structural diversities and thus represent a new frontier in lead drug discovery.
A quantitation study using reversed phase HPLC with UV and evaporative light-scattering detector (ELSD) was conducted on 90 library standards selected from 15 small molecule combinatorial libraries (six standards from each library). This study assessed the quantitation errors using a single calibration curve for rapid purity analysis of combinatorial libraries. The average quantitation error of six standards from one library at 200 microM by UV was 13. 4%, 20.6%, and 60.3%, at 214, 220, and 254 nm, respectively. By ELSD, the average quantitation error of these six standards at 200 micro was only 7.7%. Applying this ELSD calibration curve to 84 standards from 14 structurally diverse libraries, an average quantitation error of 16.4% was obtained. The average quantitation error of all 90 standards from 15 libraries using 15 calibration curves was 18.5%.
Amyotrophic lateral sclerosis (ALS) has become an increasingly attractive area for the pharmaceutical industry, the most experimentally tractable of the neurodegenerative diseases. Mechanisms underlying cell death in ALS are likely to be important in more common but more complex disorders. Riluzole, the only drug launched for treatment ALS is currently undergoing industrial trials for Alzheimer's, Parkinson's, Huntington disease, stroke and head injury. Other compounds in Phase III testing for ALS (mecamserin, xaliproden, gabapentin) are also in trials for other neurodegenerative disorders. Mechanisms of action of these advanced compounds are limited to glutamate antagonism, direct or indirect growth factor activity, as well as GABA agonism and interaction with calcium channels. A broader range of mechanisms is represented by compounds in Phase I trials: glutamate antagonism (dextramethorphan/p450 inhibitor; talampanel), growth factors (leukemia inhibiting factor; IL-1 receptor; encapsulated cells secreting CNTF) and antioxidants (TR500, a glutathione-repleting agent; recombinant superoxide dismutase; procysteine.) An even broader range of mechanisms is being explored in preclinical discovery programs. Recognition of the difficulties associated with delivery of protein therapeutics to the CNS has led to development of small molecules interacting either with neurotrophin receptors or with downstream intracellular signalling pathways. Other novel drug targets include caspaces, protein kinases and other molecules influencing apoptosis. High-throughput screens of large libraries of small molecules yield lead compounds that are subsequently optimized by chemists, screened for toxicity, and validated before a candidate is selected for clinical trials. The net is cast wide in early discovery efforts, only about 1% of which result in useful drugs at the end of a decade-long process. Successful discovery and development of novel drugs will increasingly depend on collaborative efforts between the academy and industry.
A novel and highly diverse tagged triazine library incorporating a triethylene glycol-based linker was synthesized using an orthogonal combinatorial approach on the solid phase and covalently immobilized on a glass substrate as a small molecule microarray (SMM). The SMM was screened with a fluorophore-conjugated human IgG, and 4 novel binders from a library of 2688 compounds were identified from the fully spatially addressable array without the need for compound decoding. Using surface plasmon resonance (SPR) analysis, binding seen on the array was confirmed, and a binding constant as low as Kd = 2.02 x10(-6) M was measured.