Mechanism-based inhibition of gut microbial tryptophanases reduces serum indoxyl sulfate.

Indoxyl sulfate is a microbially derived uremic toxin that accumulates in late-stage chronic kidney disease and contributes to both renal and cardiovascular toxicity. Indoxyl sulfate is generated by the metabolism of indole, a compound created solely by gut microbial tryptophanases. Here, we characterize the landscape of tryptophanase enzymes in the human gut microbiome and find remarkable structural and functional similarities across diverse taxa. We leverage this homology through a medicinal chemistry campaign to create a potent pan-inhibitor, (3S) ALG-05, and validate its action as a transition-state analog. (3S) ALG-05 successfully reduces indole production in microbial culture and displays minimal toxicity against microbial and mammalian cells. Mice treated with (3S) ALG-05 show reduced cecal indole and serum indoxyl sulfate levels with minimal changes in other tryptophan-metabolizing pathways. These studies present a non-bactericidal pan-inhibitor of gut microbial tryptophanases with potential promise for reducing indoxyl sulfate in chronic kidney disease.

Discovery of a novel, highly potent and orally bioavailable pyrrolidinone indole series of irreversible Myeloperoxidase (MPO) inhibitors.

Myeloperoxidase (MPO) is a heme-containing peroxidase from phagocytic cells, which plays an important role in the innate immune response. The primary anti-microbial function of MPO is achieved by catalyzing the oxidation of halides by hydrogen peroxide (H2O2). Upon activation of phagocytes, MPO activity is detectable in both phagosomes and extracellularly, where it can remain or transcytose into interstitial compartments. Activated MPO leads to oxidative stress and tissue damage in many inflammatory states, including cardiovascular disease. Starting from a low molecular weight (LMW) high throughput screening (HTS) hit, here we report the discovery of a novel pyrrolidinone indole (IN-4) as a highly potent MPO inhibitor. This compound displays similar in vitro potency across peroxidation, plasma and NETosis assays. In a dilution/dialysis study, <5% of the original MPO activity was detected post-incubation of MPO with IN-4, suggesting irreversible enzyme inhibition. A fast MPO inactivation rate (kinact/Ki) and low partition ratio (k3/k4) make IN-4 kinetic properties attractive for an MPO inhibitor. This compound also displays significant selectivity over the closely related thyroid peroxidase (TPO), and is selective for extracellular MPO over intracellular (neutrophil) MPO. Moreover, IN-4 shows good exposure, low clearance and high oral bioavailability in mice, rats and dogs. The high in vitro MPO activity and high oral exposure observed with IN-4 result in a dose-dependent inhibition of MPO activity in three mouse models of inflammation. In conclusion, IN-4 is a novel, potent, mechanism-based and selective MPO inhibitor, which may be used as superior therapeutic agent to treat multiple inflammatory conditions, including cardiovascular disease.

Difluoromethyl-1,3,4-oxadiazoles are slow-binding substrate analog inhibitors of histone deacetylase 6 with unprecedented isotype selectivity.

Histone deacetylase 6 (HDAC6) is an attractive drug development target because of its role in the immune response, neuropathy, and cancer. Knockout mice develop normally and have no apparent phenotype, suggesting that selective inhibitors should have an excellent therapeutic window. Unfortunately, current HDAC6 inhibitors have only moderate selectivity and may inhibit other HDAC subtypes at high concentrations, potentially leading to side effects. Recently, substituted oxadiazoles have attracted attention as a promising novel HDAC inhibitor chemotype, but their mechanism of action is unknown. Here, we show that compounds containing a difluoromethyl-1,3,4-oxadiazole (DFMO) moiety are potent and single-digit nanomolar inhibitors with an unprecedented greater than 104-fold selectivity for HDAC6 over all other HDAC subtypes. By combining kinetics, X-ray crystallography, and mass spectrometry, we found that DFMO derivatives are slow-binding substrate analogs of HDAC6 that undergo an enzyme-catalyzed ring opening reaction, forming a tight and long-lived enzyme-inhibitor complex. The elucidation of the mechanism of action of DFMO derivatives paves the way for the rational design of highly selective inhibitors of HDAC6 and possibly of other HDAC subtypes as well with potentially important therapeutic implications.

Mechanism-based inhibition of GH127/146 cysteine glycosidases by stereospecifically functionalized l-arabinofuranosides.

To understand the precise mechanism of the glycoside hydrolase (GH) family 127, a cysteine ß-l-arabinofuranosidase (Arafase) - HypBA1 - has been isolated from Bifidobacterium longum in the human Gut microbiota, and the design and synthesis of the mechanism-based inhibitors such as l-Araf-haloacetamides have been carried out. The α-l-Araf-azide derivative was used as the monoglycosylamine equivalent to afford the l-Araf-chloroacetamides (α/ß-1-Cl) as well as bromoacetamides (α/ß-1-Br) in highly stereoselective manner through Staudinger reaction followed by amide formation with/without anomerization. Against HypBA1, the probes 1, especially in the case of α/ß-1-Br inhibited the hydrolysis. Conformational implications of these observations are discussed in this manuscript. Additional examinations using l-Araf-azides (α/ß-5) resulted in further mechanistic observations of the GH127/146 cysteine glycosidases, including the hydrolysis of ß-5 as the substrate and oxidative inhibition by α-5 using the GH127 homologue.

The Mechanism-Based Inactivation of CYP3A4 by Ritonavir: What Mechanism?

Ritonavir is the most potent cytochrome P450 (CYP) 3A4 inhibitor in clinical use and is often applied as a booster for drugs with low oral bioavailability due to CYP3A4-mediated biotransformation, as in the treatment of HIV (e.g., lopinavir/ritonavir) and more recently COVID-19 (Paxlovid or nirmatrelvir/ritonavir). Despite its clinical importance, the exact mechanism of ritonavir-mediated CYP3A4 inactivation is still not fully understood. Nonetheless, ritonavir is clearly a potent mechanism-based inactivator, which irreversibly blocks CYP3A4. Here, we discuss four fundamentally different mechanisms proposed for this irreversible inactivation/inhibition, namely the (I) formation of a metabolic-intermediate complex (MIC), tightly coordinating to the heme group; (II) strong ligation of unmodified ritonavir to the heme iron; (III) heme destruction; and (IV) covalent attachment of a reactive ritonavir intermediate to the CYP3A4 apoprotein. Ritonavir further appears to inactivate CYP3A4 and CYP3A5 with similar potency, which is important since ritonavir is applied in patients of all ethnicities. Although it is currently not possible to conclude what the primary mechanism of action in vivo is, it is unlikely that any of the proposed mechanisms are fundamentally wrong. We, therefore, propose that ritonavir markedly inactivates CYP3A through a mixed set of mechanisms. This functional redundancy may well contribute to its overall inhibitory efficacy.

Roles of human cytochrome P450 1A2 in coumarin 3,4-epoxidation mediated by untreated hepatocytes and by those metabolically inactivated with furafylline in previously transplanted chimeric mice.

Coumarin is a naturally occurring component of food products but is of clinical interest for its potential hepatotoxicity in humans. In the current study, the pharmacokinetics of coumarin in humanized-liver mice after oral and intravenous administrations (30 mg/kg) were investigated for its transformations to metabolically active coumarin 3,4-epoxide (as estimated by the levels of o-hydroxyphenylacetic acid) and to excretable 7-hydroxycoumarin. After oral administration, control mice metabolized coumarin to o-hydroxyphenylacetic acid at roughly the same rate as that to 7-hydroxycoumarin (total of unconjugated and conjugated forms). In contrast, the in vivo biotransformation of coumarin to o-hydroxyphenylacetic acid by humanized-liver mice was around two orders of magnitude less than that to conjugated and unconjugated 7-hydroxycoumarin. After intravenous administrations of coumarin, differences were observed in the plasma concentrations of o-hydroxyphenylacetic acid between humanized-liver mice treated with furafylline (daily oral doses of 13 mg/kg for 3 days) and untreated humanized-liver mice. The mean values of the areas under the plasma concentration versus time curves and the maximum concentrations for o-hydroxyphenylacetic acid were significantly lower in the group treated with furafylline (45% and 57% of the untreated values, respectively). These results suggested that the metabolic activation of coumarin in humans was mediated mainly by P450 1A2, which was suppressed by furafylline, and that humanized-liver mice orally treated with furafylline might constitute an in vivo model for metabolically inactivated P450 1A2 in human hepatocytes transplanted into chimeric mice.

Pharmacokinetics, metabolism and off-target effects in the rat of 8-[(1H- benzotriazol-1-yl)amino]octanoic acid, a selective inhibitor of human cytochrome P450 4Z1: ß-oxidation as a potential augmenting pathway for inhibition.

8-[(1H-1,2,3-benzotriazol-1-yl)amino]octanoic acid (8-BOA) was recently identified as a selective and potent mechanism-based inactivator (MBI) of breast cancer-associated CYP4Z1 and exhibited favourable inhibitory activity in vitro, thus meriting in vivo characterization.The pharmacokinetics and metabolism of 8-BOA in rats was examined after a single IV bolus dose of 10 mg/kg. A biphasic time-concentration profile resulted in relatively low clearance and a prolonged elimination half-life.The major circulating metabolites identified in plasma were products of ß-oxidation; congeners losing two and four methylene groups accounted for >50% of metabolites by peak area. The -(CH2)2 product was characterized previously as a CYP4Z1 MBI and so represents an active metabolite that may contribute to the desired pharmacological effect.Ex vivo analysis of total CYP content in rat liver and kidney microsomes showed that off-target CYP inactivation was minimal; liver microsomal probe substrate metabolism also demonstrated low off-target inactivation. Standard clinical chemistries provided no indication of acute toxicity.In silico simulations using the free concentration of 8-BOA in plasma suggested that the in vivo dose used here may effectively inactivate CYP4Z1 in a xenografted tumour.

Development of Non-Hydrolysable Oligosaccharide Activity-Based Inactivators for Endoglycanases: A Case Study on α-1,6 Mannanases.

There is a vast genomic resource for enzymes active on carbohydrates. Lagging far behind, however, are functional chemical tools for the rapid characterization of carbohydrate-active enzymes. Activity-based probes (ABPs) offer one chemical solution to these issues with ABPs based upon cyclophellitol epoxide and aziridine covalent and irreversible inhibitors representing a potent and widespread approach. Such inhibitors for enzymes active on polysaccharides are potentially limited by the requirement for several glycosidic bonds, themselves substrates for the enzyme targets. Here, it is shown that non-hydrolysable trisaccharide can be synthesized and applied even to enzymes with challenging subsite requirements. It was found that incorporation of carbasugar moieties, which was accomplished by cuprate-assisted regioselective trans-diaxial epoxide opening of carba-mannal synthesised for this purpose, yields inactivators that act as powerful activity-based inhibitors for α-1,6 endo-mannanases. 3-D structures at 1.35-1.47 Šresolutions confirm the design rationale and binding to the enzymatic nucleophile. Carbasugar oligosaccharide cyclophellitols offer a powerful new approach for the design of robust endoglycosidase inhibitors, while the synthesis procedures presented here should allow adaptation towards activity-based endoglycosidase probes as well as configurational isosteres targeting other endoglycosidase families.

Discovery of a novel indole pharmacophore for the irreversible inhibition of myeloperoxidase (MPO).

Myeloperoxidase (MPO) activity and subsequent generation of hypochlorous acid has been associated with the killing of host-invading microorganisms (e.g. bacteria, viruses, and fungi). However, during oxidative stress, high MPO activity can damage host tissue and is linked to several chronic inflammatory conditions. Herein, we describe the development of a novel biaryl, indole-pyrazole series of irreversible mechanism-based inhibitors of MPO. Derived from an indole-containing high-throughput screen hit, optimization efforts resulted in potent and selective 6-substituted indoles with good oral bioavailability and in vivo activity.

Dasotraline as a selective cytochrome 2B6 inhibitor for reaction phenotyping.

Reaction phenotyping using human liver microsomes or hepatocytes with chemical inhibitors is one of the most commonly applied methods to assess the fraction metabolized (fm ) of drug candidates by enzymes. The fm information is critical to understanding the risk of victim drug-drug interactions in the clinic. Inhibitor selectivity is essential in order to generate reliable data and irreversible inhibitors are often preferred over reversible inhibitors to minimize the impact of inhibitor depletion. Although many selective cytochrome P450 (CYP) inhibitors are available, the identification of selective CYP2B6 inhibitors has been challenging due to cross inhibition to the other enzymes. In this study, dasotraline was evaluated as a selective inactivator of CYP2B6 under reaction phenotyping conditions with human hepatocytes. The results show that dasotraline is a very selective inactivator for CYP2B6 with minimal inhibition to other enzymes. A concentration of 0.1 µM dasotraline is recommended for reaction phenotyping with a hepatocyte cell density of 0.5 million cells/ml or 0.5 µM for 2 million cells/ml, when using a 15 minute preincubation, as well as the protocol of inactivator removal before the addition of substrates.

Structural Insights into the Interaction of Cytochrome P450 3A4 with Suicide Substrates: Mibefradil, Azamulin and 6',7'-Dihydroxybergamottin.

Human cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme. Some drugs and natural compounds can act as suicide (mechanism-based) inactivators of CYP3A4, leading to unanticipated drug-drug interactions, toxicity and therapeutic failures. Despite significant clinical and toxicological implications, the mechanism-based inactivation remains incompletely understood. This study provides the first direct insights into the interaction of CYP3A4 with three suicide substrates: mibefradil, an antihypertensive drug quickly withdrawn from the market; a semi-synthetic antibiotic azamulin; and a natural furanocoumarin, 6',7'-dihydroxybergamottin. Novel structural findings help better understand the suicide substrate binding and inhibitory mechanism, and can be used to improve the predictability of the binding ability, metabolic sites and inhibitory/inactivation potential of newly developed drugs and other chemicals relevant to public health.

Late-Stage Conversion of Diphenylphosphonate to Fluorophosphonate Probes for the Investigation of Serine Hydrolases.

Diphenylphosphonates (DPPs) have been used for 50 years to inactivate serine hydrolases, creating adducts representative of tetrahedral intermediates of this important class of enzymes. Failure to react at active site serine residues, however, can thwart their usefulness. Here, we describe a facile route and allied mechanistic studies to highly reactive, structurally complex organofluorophosphonates (FPs) by direct fluorinative hydrolysis of DPPs. Advantages over current preparations of FPs are exemplified by the synthesis of a ß-lactam-containing peptide substrate analog capable of modifying the C-terminal, dual-function thioesterase involved in nocardicin A biosynthesis. Although this serine hydrolase was found to resist modification by classic DPP inhibitors, active site selective phosphonylation by the corresponding FP occurs rapidly to form a stable adduct. This simple, late-stage method enables the ready preparation of FP probes that retain important structural motifs of native substrates, thus promoting efforts in mechanistic enzymology by accessing biologically relevant enzyme-inhibitor co-structures.

Direct and quantitative evaluation of the major human CYP contribution (fmCYP) to drug clearance using the in vitro Silensomes™ model.

1. We have applied the concept of using MBIs to produce CYP-Silensomes to quantify the contribution of the major CYPs to drug metabolism (fmCYP). 2. The target CYPs were extensively and selectivity inhibited by the selected MBIs, while non-target CYPs were inhibited by less than 20% of the homologous control activities. Only CYP2D6-Silensomes exhibited a CYP2B6 inhibition that could be easily and efficiently encountered by subtracting the fmCYP2B6 measured using CYP2B6-Silensomes to adjust the fmCYP2D6. 3. To validate the use of a panel of 6 CYP-Silensomes, we showed that the fmCYP values of mono- and multi-CYP metabolised drugs were well predicted, with 70% within ± 15% accuracy. Moreover, the correlation with observed fmCYP values was higher than that for rhCYPs, which were run in parallel using the same drugs (<45% within ±15% accuracy). Moreover, the choice of the RAF substrate in rhCYP predictions was shown to affect the accuracy of the fmCYP measurement. 4. These results support the use of CYP1A2-, CYP2B6-, CYP2C8-, CYP2C9-, CYP2D6 and CYP3A4-Silensomes to accurately predict fmCYP values during the in vitro enzyme phenotyping assays in early, as well as in development, phases of drug development.

Biochemical Profiling of DMSP Lyases.

Dimethyl sulfide (DMS) is released at rates of >107 tons annually and plays a key role in the oceanic sulfur cycle and ecology. Marine bacteria, algae, and possibly other organisms release DMS via cleavage of dimethylsulfoniopropionate (DMSP). DMSP lyases have been identified in various organisms, including bacteria, coral, and algae, thus comprising a range of gene families putatively assigned as DMSP lyases. Metagenomics may therefore provide insight regarding the presence of DMSP lyases in various marine environments, thereby promoting a better understanding of global DMS emission. However, gene counts, and even mRNA levels, do not necessarily reflect the level of DMSP cleavage activity in a given environmental sample, especially because some of the families assigned as DMSP lyases may merely exhibit promiscuous lyase activity. Here, we describe a range of biochemical profiling methods that can assign an observed DMSP lysis activity to a specific gene family. These methods include selective inhibitors and DMSP substrate analogues. Combined with genomics and metagenomics, biochemical profiling may enable a more reliable identification of the origins of DMS release in specific organisms and in crude environmental samples.

Synthesis and inhibitory activity of mechanism-based 4-coumaroyl-CoA ligase inhibitors.

4-Coumaroyl-CoA ligase (4CL) is ubiquitous in the plant kingdom, and plays a central role in the biosynthesis of phenylpropanoids such as lignins, flavonoids, and coumarins. 4CL catalyzes the formation of the coenzyme A thioester of cinnamates such as 4-coumaric, caffeic, and ferulic acids, and the regulatory position of 4CL in the phenylpropanoid pathway renders the enzyme an attractive target that controls the composition of phenylpropanoids in plants. In this study, we designed and synthesized mechanism-based inhibitors for 4CL in order to develop useful tools for the investigation of physiological functions of 4CL and chemical agents that modulate plant growth with the ultimate goal to produce plant biomass that exhibits features that are beneficial to humans. The acylsulfamide backbone of the inhibitors in this study was adopted as a mimic of the acyladenylate intermediates in the catalytic reaction of 4CL. These acylsulfamide inhibitors and the important synthetic intermediates were fully characterized using two-dimensional NMR spectroscopy. Five 4CL proteins with distinct substrate specificity from four plant species, i.e., Arabidopsis thaliana, Glycine max (soybean), Populus trichocarpa (poplar), and Petunia hybrida (petunia), were used to evaluate the inhibitory activity, and the half-maximum inhibitory concentration (IC50) of each acylsulfamide in the presence of 4-coumaric acid (100 µM) was determined as an index of inhibitory activity. The synthetic acylsulfamides used in this study inhibited the 4CLs with IC50 values ranging from 0.10 to 722 µM, and the IC50 values of the most potent inhibitors for each 4CL were 0.10-2.4 µM. The structure-activity relationship observed in this study revealed that both the presence and the structure of the acyl group of the synthetic inhibitors strongly affect the inhibitory activity, and indicates that 4CL recognizes the acylsulfamide inhibitors as acyladenylate mimics.

Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop.

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (RP/SP). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (kon=174M-1s-1) was ca. 8-fold more potent than the d-isomer (kon=21.5M-1s-1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be SP. The SP-isomers inhibited human GGT (kon=21.5-174M-1s-1), while the RP-isomers were inactive even at concentrations of 0.1mM.

Impact of Mutations at Arg220 and Thr237 in PER-2 ß-Lactamase on Conformation, Activity, and Susceptibility to Inhibitors.

PER-2 accounts for up to 10% of oxyimino-cephalosporin resistance in Klebsiella pneumoniae and Escherichia coli in Argentina and hydrolyzes both cefotaxime and ceftazidime with high catalytic efficiencies (kcat/Km ). Through crystallographic analyses, we recently proposed the existence of a hydrogen bond network connecting Ser70-Gln69-oxyanion water-Thr237-Arg220 that might be important for the activity and inhibition of the enzyme. Mutations at Arg244 in most class A ß-lactamases (such as TEM and SHV) reduce susceptibility to mechanism-based inactivators, and Arg220 in PER ß-lactamases is equivalent to Arg244. Alterations in the hydrogen bond network of the active site in PER-2, through modifications in key residues such as Arg220 and (to a much lesser extent) Thr237, dramatically impact the overall susceptibility to inactivation, with up to ∼300- and 500-fold reductions in the rate constant of inactivation (kinact)/Ki values for clavulanic acid and tazobactam, respectively. Hydrolysis on cephalosporins and aztreonam was also affected, although to different extents compared to with wild-type PER-2; for cefepime, only an Arg220Gly mutation resulted in a strong reduction in the catalytic efficiency. Mutations at Arg220 entail modifications in the catalytic activity of PER-2 and probably local perturbations in the protein, but not global conformational changes. Therefore, the apparent structural stability of the mutants suggests that these enzymes could be possibly selected in vivo.

Direct and quantitative evaluation of the human CYP3A4 contribution (fm) to drug clearance using the in vitro SILENSOMES model.

1. Among the different in vitro studies recommended by the regulatory agencies, no gold-standard model can easily and directly measure the quantitative CYP450 contributions to drug biotransformation. In this article, we propose an original strategy, called SilensomesTM, to produce human liver microsomes silenced for one specific CYP450, thanks to specific mechanism-based inhibitors (MBI). 2. Using azamulin as a specific CYP3A4 MBI, we demonstrated the proof of concept that CYP3A4 can be totally, specifically (even against 3A5) and permanently (at least for six years) inhibited by our process. Thus, comparing clearance in control and CYP3A4-SilensomesTM, CYP3A4 contributions were determined for 11 CYP3A4 substrates which correlated with known in vivo contributions and revealed accuracy with less than 10% error. In comparison, contributions determined using recombinant human CYP450 (rhCYP450s) were less accurate (more than 10% error for 30% of the tested CYP3A4 substrates). 3. This easy and ready-to-use in vitro method combines the advantages of existing models (specificity of rhCYP450s and representativeness of HLM) without their drawbacks. The same strategy could be used to silence other major CYP450s one-by-one to provide a complete direct CYP450 quantitative phenotyping kit.

Phosphonate-based irreversible inhibitors of human γ-glutamyl transpeptidase (GGT). GGsTop is a non-toxic and highly selective inhibitor with critical electrostatic interaction with an active-site residue Lys562 for enhanced inhibitory activity.

γ-Glutamyl transpeptidase (GGT, EC 2.3.2.2) that catalyzes the hydrolysis and transpeptidation of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione metabolism and is an attractive pharmaceutical target. We report here the evaluation of a phosphonate-based irreversible inhibitor, 2-amino-4-{[3-(carboxymethyl)phenoxy](methoyl)phosphoryl}butanoic acid (GGsTop) and its analogues as a mechanism-based inhibitor of human GGT. GGsTop is a stable compound, but inactivated the human enzyme significantly faster than the other phosphonates, and importantly did not inhibit a glutamine amidotransferase. The structure-activity relationships, X-ray crystallography with Escherichia coli GGT, sequence alignment and site-directed mutagenesis of human GGT revealed a critical electrostatic interaction between the terminal carboxylate of GGsTop and the active-site residue Lys562 of human GGT for potent inhibition. GGsTop showed no cytotoxicity toward human fibroblasts and hepatic stellate cells up to 1mM. GGsTop serves as a non-toxic, selective and highly potent irreversible GGT inhibitor that could be used for various in vivo as well as in vitro biochemical studies.

Development of new Malt1 inhibitors and probes.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) is a promising therapeutic target for the treatment of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL). Several research groups have reported on the development of Malt1 inhibitors and activity-based probes for in vitro and in situ monitoring and modulating Malt1 activity. In this paper, we report on two activity-based Malt1 probes (6 and 7) and a focused library of 19 new Malt1 inhibitors. Our peptide-based probe 6 labels Malt1 in an activity-based manner. In contrast, probe 7, derived from the known covalent inhibitor MI-2, labels both wild type and catalytically inactive Cys to Ala mutant Malt1, suggesting that MI-2 inhibits Malt1 by reacting with a nucleophilic residue other than the active site cysteine. Furthermore, two of our inhibitors (9, apparent IC50 3.0µM, and 13, apparent IC50 2.1µM) show good inhibitory activity against Malt1 and outperform MI-2 (apparent IC50 7.8µM) in our competitive activity-based protein profiling assay.