Chemical methodology as a source of small-molecule checkpoint inhibitors and heat shock protein 70 (Hsp70) modulators

Abstract

Unique chemical methodology enables the synthesis of innovative and diverse scaffolds and chemotypes and allows access to previously unexplored "chemical space." Compound collections based on such new synthetic methods can provide small-molecule probes of proteins and/or pathways whose functions are not fully understood. We describe the identification, characterization, and evolution of two such probes. In one example, a pathway-based screen for DNA damage checkpoint inhibitors identified a compound, MARPIN (ATM and ATR pathway inhibitor) that sensitizes p53-deficient cells to DNA-damaging agents. Modification of the small molecule and generation of an immobilized probe were used to selectively bind putative protein target(s) responsible for the observed activity. The second example describes a focused library approach that relied on tandem multicomponent reaction methodologies to afford a series of modulators of the heat shock protein 70 (Hsp70) molecular chaperone. The synthesis of libraries based on the structure of MAL3-101 generated a collection of chemotypes, each modulating Hsp70 function, but exhibiting divergent pharmacological activities. For example, probes that compromise the replication of a disease-associated polyomavirus were identified. These projects highlight the importance of chemical methodology development as a source of small-molecule probes and as a drug discovery starting point.

Fig. 1.

Cyclocarbonylation and cycloisomerization reactions of allene-ynes and ene-allenes.

Fig. 2.

Representative compounds from libraries based on scaffolds 2–6. Compounds 7–9,11,12,14 originating from chemistries similar to that used to obtain scaffold 4, compound 13 originating from scaffold 6 and compound 10 from scaffold 2.

Fig. 3.

Derivatization of MARPIN. (A) Library design strategy. (B) Structure activity data for a series of MARPIN derivatives. IC₅₀ values indicate the concentrations at which compounds provide 50% inhibition of phosphorylation of Chk1 at Ser345 in cells treated with hydroxyurea.

Fig. 4.

Bioactivities of MARPIN derivatives and their use for detection of MARPIN-binding proteins. (A) Inhibitory effects of MARPIN derivatives on hydroxyurea (HU)-induced phosphorylation of Chk1. 293T cells were treated with 3 mM HU and the indicated concentrations of MARPIN (15) and its derivatives (17, 18, or 26) or with no hydroxyurea (N) for 2 h. Immunoblots quantitated phospho-Chk1 (pChk1) at Ser345, total Chk1, and GAPDH. AffiGel (AG) loaded with pegylated MARPIN was used for pull-down assays. (B) MARPIN affinity matrix studies. Cell lysates of 293T cells were preincubated with vehicle (DMSO) or MARPIN derivatives (active compound 18, 26 or inactive compound 17) as fivefold excess free soluble competitor. Capped AG or MARPIN-loaded AG (M-AG) were then mixed with the preincubated lysates for affinity enrichment. Samples (lanes 2–6) and 1 μg of total protein (lane 1) were analyzed by SDS-PAGE/silver stain. Arrowheads indicate proteins of interest that bound to M-AG (lane 3) and could be competed from binding to M-AG matrix by excess soluble MARPIN derivatives that were active (lanes 4 and 6) but not inactive (lane 5).

Fig. 5.

Synthesis of pegylated and immobilized MARPIN derivatives.

Fig. 6.

MAL3-101 and three analogs with differential activities in models of cancer, malaria, trypanosome infection, and polyomavirus infection.

Fig. 7.

General synthesis of MAL3-101 libraries, MAL2-11B, and MAL2-11B isosteres. (a) (i) TEA, ClCO₂Et; (ii) NH₂OH; (b) SOCl₂, MeOH; (c) TMSN₃, TBAF, microwave irradiation.

Fig. 8.

Effect of MAL2-11B and 32 on T-antigen and viral replication. Steady-state ATPase assays with purified T-antigen were performed as described (, SI Appendix: S2) in the presence of the indicated concentrations of (A) MAL2-11B or (B) 32. Data represent the means of at least three determinations ± SD. (C) The replication of the BK virus and viral DNA content were assessed in the presence of the indicated supplements as outlined (SI Appendix: S2). MAL2-11B and 32 were used at a final concentration of 100 μM. Data represent the means of two independent infections, each performed in triplicate, ± SEM; p ≤ 0.007 for DMSO vs. the compound treated samples.

Fig. 9.

Synthesis of tetrazole library.