The Development of the Bengamides as New Antibiotics against Drug-Resistant Bacteria.

The bengamides comprise an interesting family of natural products isolated from sponges belonging to the prolific Jaspidae family. Their outstanding antitumor properties, coupled with their unique mechanism of action and unprecedented molecular structures, have prompted an intense research activity directed towards their total syntheses, analogue design, and biological evaluations for their development as new anticancer agents. Together with these biological studies in cancer research, in recent years, the bengamides have been identified as potential antibiotics by their impressive biological activities against various drug-resistant bacteria such as Mycobacterium tuberculosis and Staphylococcus aureus. This review reports on the new advances in the chemistry and biology of the bengamides during the last years, paying special attention to their development as promising new antibiotics. Thus, the evolution of the bengamides from their initial exploration as antitumor agents up to their current status as antibiotics is described in detail, highlighting the manifold value of these marine natural products as valid hits in medicinal chemistry.

Selective Neutral pH Inhibitor of Cathepsin B Designed Based on Cleavage Preferences at Cytosolic and Lysosomal pH Conditions.

Cathepsin B is a cysteine protease that normally functions within acidic lysosomes for protein degradation, but in numerous human diseases, cathepsin B translocates to the cytosol having neutral pH where the enzyme activates inflammation and cell death. Cathepsin B is active at both the neutral pH 7.2 of the cytosol and the acidic pH 4.6 within lysosomes. We evaluated the hypothesis that cathepsin B may possess pH-dependent cleavage preferences that can be utilized for design of a selective neutral pH inhibitor by (1) analysis of differential cathepsin B cleavage profiles at neutral pH compared to acidic pH using multiplex substrate profiling by mass spectrometry (MSP-MS), (2) design of pH-selective peptide-7-amino-4-methylcoumarin (AMC) substrates, and (3) design and validation of Z-Arg-Lys-acyloxymethyl ketone (AOMK) as a selective neutral pH inhibitor. Cathepsin B displayed preferences for cleaving peptides with Arg in the P2 position at pH 7.2 and Glu in the P2 position at pH 4.6, represented by its primary dipeptidyl carboxypeptidase and modest endopeptidase activity. These properties led to design of the substrate Z-Arg-Lys-AMC having neutral pH selectivity, and its modification with the AOMK warhead to result in the inhibitor Z-Arg-Lys-AOMK. This irreversible inhibitor displays nanomolar potency with 100-fold selectivity for inhibition of cathepsin B at pH 7.2 compared to pH 4.6, shows specificity for cathepsin B over other cysteine cathepsins, and is cell permeable and inhibits intracellular cathepsin B. These findings demonstrate that cathepsin B possesses pH-dependent cleavage properties that can lead to development of a potent, neutral pH inhibitor of this enzyme.

Chemical Inhibition of ENL/AF9 YEATS Domains in Acute Leukemia.

Transcriptional coregulators, which mediate chromatin-dependent transcriptional signaling, represent tractable targets to modulate tumorigenic gene expression programs with small molecules. Genetic loss-of-function studies have recently implicated the transcriptional coactivator, ENL, as a selective requirement for the survival of acute leukemia and highlighted an essential role for its chromatin reader YEATS domain. Motivated by these discoveries, we executed a screen of nearly 300,000 small molecules and identified an amido-imidazopyridine inhibitor of the ENL YEATS domain (IC50 = 7 µM). Improvements to the initial screening hit were enabled by adopting and expanding upon a SuFEx-based approach to high-throughput medicinal chemistry, ultimately demonstrating that it is compatible with cell-based drug discovery. Through these efforts, we discovered SR-0813, a potent and selective ENL/AF9 YEATS domain inhibitor (IC50 = 25 nM). Armed with this tool and a first-in-class ENL PROTAC, SR-1114, we detailed the biological response of AML cells to pharmacological ENL disruption for the first time. Most notably, we discovered that ENL YEATS inhibition is sufficient to selectively suppress ENL target genes, including HOXA9/10, MYB, MYC, and a number of other leukemia proto-oncogenes. Cumulatively, our study establishes YEATS domain inhibition as a viable approach to disrupt the pathogenic function of ENL in acute leukemia and provides the first thoroughly characterized chemical probe for the ENL YEATS domain.

Antitumor activity of a systemic STING-activating non-nucleotide cGAMP mimetic.

Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed that SR-717 functions as a direct cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) mimetic that induces the same "closed" conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8+ T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-dependent manner.

An Irreversible Inhibitor to Probe the Role of Streptococcus pyogenes Cysteine Protease SpeB in Evasion of Host Complement Defenses.

Members of the CA class of cysteine proteases have multifaceted roles in physiology and virulence for many bacteria. Streptococcal pyrogenic exotoxin B (SpeB) is secreted by Streptococcus pyogenes and implicated in the pathogenesis of the bacterium through degradation of key human immune effector proteins. Here, we developed and characterized a clickable inhibitor, 2S-alkyne, based on X-ray crystallographic analysis and structure-activity relationships. Our SpeB probe showed irreversible enzyme inhibition in biochemical assays and labeled endogenous SpeB in cultured S. pyogenes supernatants. Importantly, application of 2S-alkyne decreased S. pyogenes survival in the presence of human neutrophils and supports the role of SpeB-mediated proteolysis as a mechanism to limit complement-mediated host defense. We posit that our SpeB inhibitor will be a useful chemical tool to regulate, label, and quantitate secreted cysteine proteases with SpeB-like activity in complex biological samples and a lead candidate for new therapeutics designed to sensitize S. pyogenes to host immune clearance.

Sulfur(VI) Fluoride Exchange (SuFEx)-Enabled High-Throughput Medicinal Chemistry.

Optimization of small-molecule probes or drugs is a synthetically lengthy, challenging, and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible sulfur(VI) fluoride exchange (SuFEx) click chemistry. A high-throughput screening hit benzyl (cyanomethyl)carbamate (Ki = 8 µM) against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN═S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products were directly screened to yield drug-like inhibitors with 480-fold higher potency (Ki = 18 nM). We showed that the improved molecule is active in a bacteria-host coculture. Since this SuFEx linkage reaction succeeds on picomole scale for direct screening, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.

Genetic basis for the cooperative bioactivation of plant lignans by Eggerthella lenta and other human gut bacteria.

Plant-derived lignans, consumed daily by most individuals, are thought to protect against cancer and other diseases1; however, their bioactivity requires gut bacterial conversion to enterolignans2. Here, we dissect a four-species bacterial consortium sufficient for all five reactions in this pathway. A single enzyme (benzyl ether reductase, encoded by the gene ber) was sufficient for the first two biotransformations, variable between strains of Eggerthella lenta, critical for enterolignan production in gnotobiotic mice and unique to Coriobacteriia. Transcriptional profiling (RNA sequencing) independently identified ber and genomic loci upregulated by each of the remaining substrates. Despite their low abundance in gut microbiomes and restricted phylogenetic range, all of the identified genes were detectable in the distal gut microbiomes of most individuals living in northern California. Together, these results emphasize the importance of considering strain-level variations and bacterial co-occurrence to gain a mechanistic understanding of the bioactivation of plant secondary metabolites by the human gut microbiome.

N-Terminomics/TAILS Profiling of Proteases and Their Substrates in Ulcerative Colitis.

Dysregulated protease activity is often implicated in the initiation of inflammation and immune cell recruitment in gastrointestinal inflammatory diseases. Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compared proteases, along with their substrates and inhibitors, between colonic mucosal biopsies of healthy patients and those with ulcerative colitis (UC). Among the 1642 N-termini enriched using TAILS, increased endogenous processing of proteins was identified in UC compared to healthy patients. Changes in the reactome pathways for proteins associated with metabolism, adherens junction proteins (E-cadherin, liver-intestinal cadherin, catenin alpha-1, and catenin delta-1), and neutrophil degranulation were identified between the two groups. Increased neutrophil infiltration and distinct proteases observed in ulcerative colitis may result in extensive break down, altered processing, or increased remodeling of adherens junctions and other cellular functions. Analysis of the preferred proteolytic cleavage sites indicated that the majority of proteolytic activity and processing comes from host proteases, but that key microbial proteases may also play a role in maintaining homeostasis. Thus, the identification of distinct proteases and processing of their substrates improves the understanding of dysregulated proteolysis in normal intestinal physiology and ulcerative colitis.

X-ray structure of an inactive zymogen clostripain-like protease from Parabacteroides distasonis.

The clostripain-like (C11) family of cysteine proteases are ubiquitously produced by the vast majority of the bacterial strains that make up the human distal gut microbiome. Recent reports show that some C11 proteases promote host immune responses and bacterial pathogenesis, including the induction of neutrophil phagocytosis and the activation of bacterial pathogenic toxins, respectively. The crystal structure of distapain, the only C11 protease predicted within the genome of the commensal bacterium Parabacteroides distasonis, was determined in the inactive zymogen state to 1.65 Šresolution. This is the first C11 protease structure of a zymogen, and the structure helped to uncover key unique conformations among critical active-site residues that are likely to assist in preserving the inactive protease. His135, a member of the catalytic dyad, is repositioned approximately 5.5 Šfrom the orientation found in active C11 structures and forms a hydrogen bond to Asp180 and a π-stacking interaction with Trp133. The structure sheds light on the potential importance of Asp180 and Trp133, as these residues are highly conserved across C11 proteases. Structure elucidation of C11 proteases will ultimately help to identify new ways to chemically and/or biologically regulate this family of enzymes, which represent potential drug-discovery targets in microbiome-related gastrointestinal diseases.

X-ray Structures of Two Bacteroides thetaiotaomicron C11 Proteases in Complex with Peptide-Based Inhibitors.

Commensal bacteria secrete proteins and metabolites to influence host intestinal homeostasis, and proteases represent a significant constituent of the components at the host:microbiome interface. Here, we determined the structures of the two secreted C11 cysteine proteases encoded by the established gut commensal Bacteroides thetaiotaomicron. We employed mutational analysis to demonstrate the two proteases, termed "thetapain" and "iotapain", undergo in trans autoactivation after lysine and/or arginine residues, as observed for other C11 proteases. We determined the structures of the active forms of thetapain and iotapain in complex with irreversible peptide inhibitors, Ac-VLTK-AOMK and biotin-VLTK-AOMK, respectively. Structural comparisons revealed key active-site interactions important for peptide recognition are more extensive for thetapain; however, both proteases employ a glutamate residue to preferentially bind small polar residues at the P2 position. Our results will aid in the design of protease-specific probes to ultimately understand the biological role of C11 proteases in bacterial fitness, elucidate their host and/or microbial substrates, and interrogate their involvement in microbiome-related diseases.

Nanobody-based binding assay for the discovery of potent inhibitors of CFTR inhibitory factor (Cif).

Lead identification and optimization are essential steps in the development of a new drug. It requires cost-effective, selective and sensitive chemical tools. Here, we report a novel method using nanobodies that allows the efficient screening for potent ligands. The method is illustrated with the cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), a virulence factor secreted by the opportunistic pathogen Pseudomonas aeruginosa. 18 nanobodies selective to Cif were isolated by bio-panning from nanobody-phage library constructed from immunized llama. 8 out of 18 nanobodies were identified as potent inhibitors of Cif enzymatic activity with IC50s in the range of 0.3-6.4 µM. A nanobody VHH219 showed high affinity (KD = 0.08 nM) to Cif and the highest inhibitory potency, IC50 = 0.3 µM. A displacement sandwich ELISA (dsELISA) with VHH219 was then developed for classification of synthetic small molecule inhibitors according their inhibitory potency. The developed assay allowed identification of new inhibitor with highest potency reported so far (0.16 ±â€¯0.02 µM). The results from dsELISA assay correlates strongly with a conventional fluorogenic assay (R = 0.9998) in predicting the inhibitory potency of the tested compounds. However, the novel dsELISA is an order of magnitude more sensitive and allows the identification and ranking of potent inhibitors missed by the classic fluorogenic assay method. These data were supported with Octet biolayer interferometry measurements. The novel method described herein relies solely on the binding properties of the specific neutralizing nanobody, and thus is applicable to any pharmacological target for which such a nanobody can be found, independent of any requirement for catalytic activity.

ComPIL 2.0: An Updated Comprehensive Metaproteomics Database.

We designed a metaproteomic analysis method (ComPIL) to accommodate the ever-increasing number of sequences against which experimental shotgun proteomics spectra could be accurately and rapidly queried. Our objective was to create these large databases for the analysis of complex metasamples with unknown composition, including those derived from human, animal, and environmental microbiomes. The amount of high-throughput sequencing data has substantially increased since our original database was assembled in 2014. Here, we present a rebuild of the ComPIL libraries comprised of updated publicly disseminated sequence data as well as a modified version of the search engine ProLuCID-ComPIL optimized for querying experimental spectra. ComPIL 2.0 consists of 113 million protein records and roughly 4.8 billion unique tryptic peptide sequences and is 2.3 times the size of our original version. We searched a data set collected on a healthy human gut microbiome proteomic sample and compared the results to demonstrate that ComPIL 2.0 showed a substantial increase in the number of unique identified peptides and proteins compared to the first ComPIL version. The high confidence of protein identification and accuracy demonstrated by the use of ComPIL 2.0 may encourage the method's application for large-scale proteomic annotation of complex protein systems.

A Commensal Dipeptidyl Aminopeptidase with Specificity for N-Terminal Glycine Degrades Human-Produced Antimicrobial Peptides in Vitro.

Proteases within the C1B hydrolase family are encoded by many organisms. We subjected a putative C1B-like cysteine protease secreted by the human gut commensal Parabacteroides distasonis to mass spectrometry-based substrate profiling to find preferred peptide substrates. The P. distasonis protease, which we termed Pd_dinase, has a sequential diaminopeptidase activity with strong specificity for N-terminal glycine residues. Using the substrate sequence information, we verified the importance of the P2 glycine residue with a panel of fluorogenic substrates and calculated kcat and KM for the dipeptide glycine-arginine-AMC. A potent and irreversible dipeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, glycine-arginine- AOMK, was then synthesized and demonstrated that the Pd_dinase active site requires a free N-terminal amine for potent and rapid inhibition. We next determined the homohexameric Pd_dinase structure in complex with glycine-arginine- AOMK and uncovered unexpected active site features that govern the strict substrate preferences and differentiate this protease from members of the C1B and broader papain-like C1 protease families. We finally showed that Pd_dinase hydrolyzes several human antimicrobial peptides and therefore posit that this P. distasonis enzyme may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.

Lipoprotein Signal Peptidase Inhibitors with Antibiotic Properties Identified through Design of a Robust In Vitro HT Platform.

As resistance to antibiotics increases, the exploration of new targets and strategies to combat pathogenic bacteria becomes more urgent. Ideal protein targets are required for viability across many species, are unique to prokaryotes to limit effects on the host, and have robust assays to quantitate activity and identify inhibitors. Lipoprotein signal peptidase (Lsp) is a transmembrane aspartyl protease required for lipoprotein maturation and comprehensively fits these criteria. Here, we have developed the first in vitro high-throughput assay to monitor proteolysis by Lsp. We employed our high-throughput screen assay against 646,275 compounds to discover inhibitors of Lsp and synthesized a range of analogs to generate molecules with nanomolar half maximal inhibitory concentration values. Importantly, our inhibitors are effective in preventing the growth of E. coli cultures in the presence of outer-membrane permeabilizer PMBN and should facilitate development of antibacterial agents with a novel mechanism of action to treat antibiotic-resistant bacteria.

Substrate Profiling and High Resolution Co-complex Crystal Structure of a Secreted C11 Protease Conserved across Commensal Bacteria.

Cysteine proteases are among the most abundant hydrolytic enzymes produced by bacteria, and this diverse family of proteins have significant biological roles in bacterial viability and environmental interactions. Members of the clostripain-like (C11) family of cysteine proteases from commensal gut bacterial strains have recently been shown to mediate immune responses by inducing neutrophil phagocytosis and activating bacterial pathogenic toxins. Development of substrates, inhibitors, and probes that target C11 proteases from enteric bacteria will help to establish the role of these proteins at the interface of the host and microbiome in health and disease. We employed a mass spectrometry-based substrate profiling method to identify an optimal peptide substrate of PmC11, a C11 protease secreted by the commensal bacterium Parabacteroides merdae. Using this substrate sequence information, we synthesized a panel of fluorogenic substrates to calculate kcat and KM and to evaluate the importance of the P2 amino acid for substrate turnover. A potent and irreversible tetrapeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, Ac-VLTK-AOMK, was then synthesized. We determined the crystal structure of PmC11 in complex with this inhibitor and uncovered key active-site interactions that govern PmC11 substrate recognition and specificity. This is the first C11 protease structure in complex with a substrate mimetic and is also the highest resolution crystal structure of a C11 protease to date at 1.12 Å resolution. Importantly, subjecting human epithelial cell lysates to PmC11 hydrolysis in combination with subtiligase-based N-terminal labeling and tandem mass spectrometry proteomics complemented the stringent substrate specificity observed in the in vitro substrate profiling experiment. The combination of chemical biological, biophysical, and biochemical techniques presented here to elucidate and characterize PmC11 substrate selectivity can be expanded to other proteases and the development of chemical tools to study these essential proteins in biologically relevant samples, such as the highly complex distal gut microbiome.

Quantitative Metaproteomics and Activity-Based Probe Enrichment Reveals Significant Alterations in Protein Expression from a Mouse Model of Inflammatory Bowel Disease.

Tandem mass spectrometry based shotgun proteomics of distal gut microbiomes is exceedingly difficult due to the inherent complexity and taxonomic diversity of the samples. We introduce two new methodologies to improve metaproteomic studies of microbiome samples. These methods include the stable isotope labeling in mammals to permit protein quantitation across two mouse cohorts as well as the application of activity-based probes to enrich and analyze both host and microbial proteins with specific functionalities. We used these technologies to study the microbiota from the adoptive T cell transfer mouse model of inflammatory bowel disease (IBD) and compare these samples to an isogenic control, thereby limiting genetic and environmental variables that influence microbiome composition. The data generated highlight quantitative alterations in both host and microbial proteins due to intestinal inflammation and corroborates the observed phylogenetic changes in bacteria that accompany IBD in humans and mouse models. The combination of isotope labeling with shotgun proteomics resulted in the total identification of 4434 protein clusters expressed in the microbial proteomic environment, 276 of which demonstrated differential abundance between control and IBD mice. Notably, application of a novel cysteine-reactive probe uncovered several microbial proteases and hydrolases overrepresented in the IBD mice. Implementation of these methods demonstrated that substantial insights into the identity and dysregulation of host and microbial proteins altered in IBD can be accomplished and can be used in the interrogation of other microbiome-related diseases.

A comprehensive and scalable database search system for metaproteomics.

BACKGROUND: Mass spectrometry-based shotgun proteomics experiments rely on accurate matching of experimental spectra against a database of protein sequences. Existing computational analysis methods are limited in the size of their sequence databases, which severely restricts the proteomic sequencing depth and functional analysis of highly complex samples. The growing amount of public high-throughput sequencing data will only exacerbate this problem. We designed a broadly applicable metaproteomic analysis method (ComPIL) that addresses protein database size limitations. RESULTS: Our approach to overcome this significant limitation in metaproteomics was to design a scalable set of sequence databases assembled for optimal library querying speeds. ComPIL was integrated with a modified version of the search engine ProLuCID (termed "Blazmass") to permit rapid matching of experimental spectra. Proof-of-principle analysis of human HEK293 lysate with a ComPIL database derived from high-quality genomic libraries was able to detect nearly all of the same peptides as a search with a human database (~500x fewer peptides in the database), with a small reduction in sensitivity. We were also able to detect proteins from the adenovirus used to immortalize these cells. We applied our method to a set of healthy human gut microbiome proteomic samples and showed a substantial increase in the number of identified peptides and proteins compared to previous metaproteomic analyses, while retaining a high degree of protein identification accuracy and allowing for a more in-depth characterization of the functional landscape of the samples. CONCLUSIONS: The combination of ComPIL with Blazmass allows proteomic searches to be performed with database sizes much larger than previously possible. These large database searches can be applied to complex meta-samples with unknown composition or proteomic samples where unexpected proteins may be identified. The protein database, proteomic search engine, and the proteomic data files for the 5 microbiome samples characterized and discussed herein are open source and available for use and additional analysis.

Synthesis and Sulfur Electrophilicity of the Nuphar Thiaspirane Pharmacophore.

We describe a general method to synthesize the iminium tetrahydrothiophene embedded in the dimeric Nuphar alkaloids. In contrast to prior studies, the sulfur atom of the thiaspirane pharmacophore is shown to be electrophilic. This α-thioether reacts with thiophenol or glutathione at ambient temperature to cleave the C-S bond and form a disulfide. Rates of conversion are proportional to the corresponding ammonium ion pK a and exhibit half-lives less than 5 h at a 5 mM concentration of thiol. A simple thiophane analogue of the Nuphar dimers causes apoptosis at single-digit micromolar concentration and labels reactive cysteines at similar levels as the unsaturated iminium "warhead". Our experiments combined with prior observations suggest the sulfur of the Nuphar dimers can react as an electrophile in cellular environments and that sulfur-triggered retrodimerization can occur in the cell.

Proteome-wide covalent ligand discovery in native biological systems.

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'. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. 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.

Identification and Co-complex Structure of a New S. pyogenes SpeB Small Molecule Inhibitor.

The secreted Streptococcus pyogenes cysteine protease SpeB is implicated in host immune system evasion and bacterial virulence. We present a small molecule inhibitor of SpeB 2477 identified from a high-throughput screen based on the hydrolysis of a fluorogenic peptide substrate Ac-AIK-AMC. 2477 inhibits other SpeB-related proteases but not human caspase-3, suggesting that the molecule targets proteases with the papain-like structural fold. A 1.59 Å X-ray crystal structure of 2477 bound to the SpeB active site reveals the mechanism of inhibition and the essential constituents of 2477 necessary for binding. An assessment against a panel of 2477 derivatives confirms our structural findings and shows that a carbamate and nitrile on 2477 are required for SpeB inhibition, as these moieties provide an extensive network of electrostatic and hydrogen-bonding interactions with SpeB active site residues. Surprisingly, despite 2477 having a reduced inhibitory potential against papain, the majority of 2477-related compounds inhibit papain to a much greater and broader extent than SpeB. These findings indicate that SpeB is more stringently selective than papain for this panel of small molecule inhibitors. On the basis of our structural and biochemical characterization, we propose modifications to 2477 for subsequent rounds of inhibitor design that will impart specificity to SpeB over other papain-like proteases, including alterations of the compound to exploit the differences in CA protease active site pocket sizes and electrostatics.