Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered “undruggable” 1,2. Fragment-based ligand discovery (FBLD) can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries 1,3. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes 4–10, including those that can access regions of proteins that are difficult to access through binding affinity alone 5,10,11. In this manuscript, we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T-cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and −10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.

Recent reports of increased tolerance to artemisinin derivatives-the last widely effective class of antimalarials -bolster the medical need for new treatments. The spirotetrahydro-β-carbolines, or spiroindolones, are a new class of fast-acting and potent schizonticidal drugs displaying low nanomolar potency against Plasmodium falciparum and Plasmodium vivax clinical isolates. Spiroindolones rapidly diminish protein synthesis in P. falciparum, an effect that is ablated in parasites bearing non-synonymous mutations in the gene encoding the P-type cation-transporter ATPase4 (PfATP4). The optimized spiroindolone NITD609 shows an acceptable safety profile and pharmacokinetic properties compatible with once-daily oral dosing; and demonstrates singledose efficacy in a rodent malaria model. Collectively, these data demonstrate that NITD609 possesses a pharmacological profile suitable for a new drug candidate for the treatment of malaria.Globally, 3.3 billion people are exposed to malaria, a devastating disease that causes over 800,000 deaths each year and kills more under five-year-olds than any other infectious agent (1). Fifty years ago, malaria had been eliminated from many areas of the world through effective antimalarial drug treatments, vector control interventions and disease prevention # Corresponding authors (Winzeler@scripps.edu and Thierry.diagana@novartis.com). * These authors equally contributed to this work One-sentence summary We describe the pharmacological profile of a new antimalarial drug candidate-the spiroindolone NITD609-which through a novel mechanism of action rapidly clears a Plasmodium infection upon administration of a single oral dose in a malaria mouse model. NIH Public Access Author ManuscriptScience. Author manuscript; available in PMC 2011 September 3. (2). However, the global spread of drug resistance resulted, by the 1980s, in a substantial increase in disease incidence and mortality. Today, some encouraging epidemiological data suggest that the introduction of new drugs (notably the artemisinin-based combination therapies or ACTs) may have reversed that trend (3). Derivatives of the endoperoxide artemisinin constitute the only antimalarial drugs that remain effective in all malariaendemic regions, but recent reports suggest that decades of continuous use as monotherapies might have fostered the emergence of resistance (4-6). This realization has triggered a concerted search for new drugs that could be deployed if artemisinin resistance were to spread.Many of the therapies currently in development utilize known antimalarial pharmacophores (e.g. aminoquinolines and/or peroxides) chemically modified to overcome the liabilities of their predecessors (7). While these compounds may prove to be important in the treatment of malaria, it would be preferable to discover novel chemotypes with a distinct mechanism of action (8). However, despite significant advances in our understanding of Plasmodium genome biology, the identification and validation of new drug targets has proven challengi...

The most advanced P. falciparum circumsporozoite protein-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective antibodies and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. In this study, antibody apparent affinity correlates best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I β- and Asn pseudo 310 turns; such insights can be incorporated into design of more effective immunogens and antibodies for passive immunization.

Caspases are required for essential biological functions, most notably apoptosis and pyroptosis, but also cytokine production, cell proliferation, and differentiation. One of the most well studied members of this cysteine protease family includes executioner caspase-3, which plays a central role in cell apoptosis and differentiation. Unfortunately, there exists a dearth of chemical tools to selectively monitor caspase-3 activity under complex cellular and in vivo conditions due to its close homology with executioner caspase-7. Commercially available activity-based probes and substrates rely on the canonical DEVD tetrapeptide sequence, which both caspases-3 and -7 recognize with similar affinity, and thus the individual contributions of caspase-3 and/or -7 toward important cellular processes are irresolvable. Here, we analyzed a variety of permutations of the DEVD peptide sequence in order to discover peptides with biased activity and recognition of caspase-3 versus caspases-6, -7, -8, and -9. Through this study, we identify fluorescent and biotinylated probes capable of selective detection of caspase-3 using key unnatural amino acids. Likewise, we determined the X-ray crystal structures of caspases-3, -7, and -8 in complex with our lead peptide inhibitor to elucidate the binding mechanism and active site interactions that promote the selective recognition of caspase-3 over other highly homologous caspase family members.

Caspases are a family of cysteine-aspartyl proteases that are well recognized for their essential roles in apoptosis and inflammation. Recently, caspases have also been linked to the promotion of other biologically important phenomena, such as cellular differentiation and proliferation. Dysregulation of the multifaceted and indispensable activities of caspases has been globally linked to several diseases, including cancer and neurodegenerative disorders; however, the specific caspase members responsible for these diseases have yet to be assigned. Activity-based probes (ABPs) and peptide-based inhibitors are instrumental in the detection and control of protease activity and serve as alternative methods to genetic approaches. Such molecules aid in the interrogation of specific proteases within cellular and animal models as well as help elucidate aberrant proteolytic function correlated to disease phenotypes. No ABPs or inhibitors have been discovered that specifically target one of the eleven human caspases in a cellular context. Therefore, ascribing distinct contributions to an individual caspase activity within naturally occurring biological systems is not possible. Herein, we describe a peptide series optimized for the selective detection and inhibition of active caspase-3 in cells. These compounds exhibit low nanomolar potency against caspase-3 with >120-fold selectivity over caspase-7 which shares 77% active site identity. Our ability to individually target wild-type active caspase-3 for detection and cell permeable inhibition is a valuable proof-of-concept methodology that can be readily employed to probe the significance of caspase-3 in apoptosis, neurological disorders, cardiovascular diseases, and sepsis.