Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic molecule with high selectivity and affinity for Mcl-1 currently in clinical development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clinical trial has been launched for evaluation of AZD5991 in patients with hematological malignancies (NCT03218683).

Summary Clamp loaders load sliding clamps onto primer-template DNA. The structure of the E. coli clamp loader bound to DNA reveals the formation of an ATP-dependent spiral of ATPase domains that tracks only the template strand, allowing recognition of both RNA and DNA primers. Unlike hexameric helicases, in which DNA translocation requires distinct conformations of the ATPase domains, the clamp loader spiral is symmetric and is set up to trigger release upon DNA recognition. Specificity for primed DNA arises from blockage of the end of the primer and accommodation of the emerging template along a surface groove. A related structure reveals how the ψ protein, essential for coupling the clamp loader to single-stranded DNA binding protein (SSB), binds to the clamp loader. By stabilizing a conformation of the clamp loader that is consistent with the ATPase spiral observed upon DNA binding, ψ binding promotes the clamp loading activity of the complex.

Previous experimental and theoretical studies have produced highresolution descriptions of the native and folding transition states of chymotrypsin inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation-condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding͞ unfolding pathway of CI2 at atomic resolution.CI2 ͉ nuclear magnetic resonance ͉ molecular dynamics simulations ͉ conformational transitions ͉ nucleation-condensation P rotein folding is a rapid and complex process that is difficult to characterize. To add to this difficulty, the denatured state consists of a large ensemble of conformations interconverting at a rapid rate. The denatured state is often assumed to be devoid of intramolecular interactions, such that the stability of a protein can be explained purely in terms of interactions in the native state. In recent years, it has become apparent that many proteins contain residual structure in the denatured state (ref. 1 and refs. therein). However, detailed characterization of this structure is very challenging, if not impossible in many cases. As such, during folding one follows the transition of a diverse system from an unknown starting point to a well-ordered native state. Further information about the diversity, dynamics, and structure of the denatured state is necessary to characterize and understand better this process.The simplest folding pathway to define is two state, i.e., involving only the denatured and native states, which are separated by the energetically unfavorable transition state. Chymotrypsin inhibitor 2 (CI2) was the first protein shown to fold by a two-state mechanism, and it has since been the focus of a number of experimental and theoretical studies. It is a 64-residue protein that consists of an ␣-helix and a three-stranded ␤-sheet (Fig. 1). The main hydrophobic core is formed by the packing of the ␣-helix against the ␤-sheet.Experimentally, the structure of the transition state has been studied by the protein engineering (⌽-value) method (2). In combination with molecular dynamics (MD) simulations, an atomic-resolution model of the transition state has been proposed (3-7) and verified (8). The rate-limiting step for the folding of CI2 involves the final expulsion of water molecules from the exposed nonpolar side chains and the tight packing of the hydrophobic core. The transition state is similar to an expanded native state with some disruption of the secondary structure.In contrast, the denatured state of CI2 appears to be largely unstructured as probed by NMR studies of variou...