Discovery of Ligands from Combinatorial Libraries

The ALIS platform has been used to successfully screen a variety of therapeuti-cally valuable proteins against combinatorial libraries of small, drug-like compounds, yielding novel ligands to a number of targets, including targets of unknown function identified by genomic and proteomic profiling, well established targets in the pharmaceutical industry, and popular but notoriously intractable (or ''hard'') targets for which the discovery of small molecule drugs has proven difficult [41]. Mixture-based combinatorial libraries are designed using software algorithms [42] to minimize the amount of mass redundancy present at both the library synthesis and library pooling stages, while insuring that each member is constructed from building blocks chosen to maximize the diversity of shape and functionality [43]. As such, each library member is self-encoded by its molecular weight [44, 45].

As an example, the bifunctional epoxy ester core (+)-2 was reacted with building blocks 3-18 to yield solution-phase library NGL127A443 containing nominally 512 substitutionally and stereochemically unique compounds (Figs. 3.3, 3.4). Of these, 82% have a molecular weight unique to 0.050 amu. This library was combined with four other 500-member libraries to form a 2500-member primary library that was screened against the important antibacterial target dihydro-folate reductase (DHFR, also known as Fol-A).

This screening experiment yielded the monochlorinated DHFR ligand 1 at m/z 515.24, corresponding to an [M+H]+ ion with a monoisotopic molecular weight of 514.23 amu. No signal for this ion was evident in an ALIS control experiment with DHFR in the absence of the screening library (Fig. 3.5). Table 3.1 shows a

Fig. 3.3 Synthetic scheme for mass-encoded library NGL127A443 containing isobaric positional isomers 1 and 19. Reprinted from [40] with permission from Elsevier.
Fig. 3.4 Amine building blocks used in the synthesis of library NGL127A443. Reprinted from [40] with permission from Elsevier.

Fig. 3.5 (A) Extracted ion chromatogram (XIC) of m/z 515.2 (M+H)+ from an ALIS experiment with DHFR and NGL127A443. (B) XIC of m/z 515.2 from control experiment (no library). (C) Mass spectrum of the region near m/z 515.2 underlying the XIC peak in A. Reprinted from [40] with permission from Elsevier.

Fig. 3.5 (A) Extracted ion chromatogram (XIC) of m/z 515.2 (M+H)+ from an ALIS experiment with DHFR and NGL127A443. (B) XIC of m/z 515.2 from control experiment (no library). (C) Mass spectrum of the region near m/z 515.2 underlying the XIC peak in A. Reprinted from [40] with permission from Elsevier.

portion of the membership of the 2500-member screening library; only one of the five combined libraries contains a monochlorinated member within 0.05 amu of the measured molecular weight.

An independent affinity selection experiment confirmed that the ligand originated from library NGL127A443. Compound 1, also known as NGD-157, and its positional isomer 19 were then synthesized as purified, discrete compounds for ALIS binding confirmation and measurement of their binding affinity, competition profiles versus other, known DHFR ligands, and their biological activity. As demonstrated in detail below, NGD-157 was found to bind specifically to the active site of DHFR with a Kd of 3.5 + 1.7 mM. Isomer 19 was found to be inactive in ALIS binding experiments and in bacterial growth inhibition assays.

The discovery of DHFR inhibitor NGD-157 demonstrates that ALIS is an efficient system for identifying novel, bioactive lead compounds from large combinatorial libraries. A single ALIS experiment containing over 2500 compounds is complete in under 10 min, allowing more than 250 000 compounds to

Table 3.1 A portion of the membership of the ALIS screening library, composed of NGL127A443 (library 3 in this table) and four other libraries, which yields DHFR ligand 1 (NGD-157, entry 11). Compounds of similar exact molecular weight (EMW) are distributed among the five pooled libraries to minimize mass overlap and simplify hit deconvolution. Reprinted from [40] with permission from Elsevier.

Entry EMW Formula Library

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