Ph.D. Student, Department of Structural Biology, University of Würzburg
Molecular design of novel therapeutic alternatives against Chlamydia infections
Have you ever thought about the journey a medicine makes in your body before fulfilling its healing task? In the case of orally administered drugs like the ones we use to treat our headaches, the active pharmaceutical ingredient (the molecule, which possesses a therapeutic effect) must be absorbed and transported into the blood stream. Once there, it is free to be distributed across the body. But then, how does this molecule manage to specifically address your headache? Why does it predominantly act in certain parts of your body? And more importantly, why do some molecules have the ability to relieve pain while others are able to, for instance, cure an infection?
The easiest way to understand this is to think of your body as a complex system of “locks” and that a drug is nothing more than a “key” with the right shape complementarity to fit into one of them. Addressing the right key hole can cure a specific health disorder.
Of course, in the biochemical world there are no locks but rather an army of molecular entities known as proteins which perform different tasks to sustain life. Such tasks or activities are intimately dependent on the protein’s 3D structure (in analogy to the lock’s internal geometry). As many proteins play major roles in the progression of life threating conditions, elucidating the structure-activity relationship of proteins is essential for understanding the dynamics of human disease.
The same way a locksmith could reproduce the fine details of a key with the knowledge of the lock’s internal geometry as a template, a drug designer makes specific molecules to address the clefts and cavities of a protein given its 3D structure.
My research focuses on the structural characterization of ChlaDUB1, a protein from the pathogen Chlamydia trachomatis, which is responsible of severe sexually transmitted diseases. This protease (a protein that cleaves other proteins) is used by the bacterium as a molecular weapon to overcome the immune response of human cells upon infection, successfully increasing its harming potential.
Based on ChlaDUB1 structure similarity to the Adenovirus protease, the very first drug-like molecules able to target this protein have been identified. Our results revealed the desirable structural framework for ChlaDUB1 inhibition, which readily enables the path for structure-based drug discovery of molecules with the potential to become novel therapeutic alternatives for the treatment of Chlamydia trachomatis infections.
Abstract: Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense.
Pub.: 30 Mar '17, Pinned: 23 Aug '17
Abstract: Adenoviral infections are associated with a wide range of acute diseases, among which ocular viral conjunctivitis (EKC) and disseminated disease in immunocompromised patients. To date, no approved specific anti-adenoviral drug is available, but there is a growing need for an effective treatment of such infections. The adenoviral protease, adenain, plays a crucial role for the viral lifecycle and thus represents an attractive therapeutic target. Structure-guided design with the objective to depeptidize tetrapeptide nitrile 1 led to the novel chemotype 2. Optimization of scaffold 2 resulted in picomolar adenain inhibitors 3a and 3b. In addition, a complementary series of irreversible vinyl sulfone containing inhibitors were rationally designed, prepared and evaluated against adenoviral protease. High resolution X-ray co-crystal structures of representatives of each series proves the successful design of these inhibitors and provides an excellent basis for future medicinal chemistry optimization of these compounds.
Pub.: 13 Jan '15, Pinned: 23 Aug '17
Abstract: Chlamydia trachomatis is an obligate intracellular bacterium that causes a variety of diseases in humans. C. trachomatis has a complex developmental cycle that depends on host cells for replication, during which gene expression is tightly regulated. Here we identify two C. trachomatis proteases that possess deubiquitinating and deneddylating activities. We have designated these proteins ChlaDub1 and ChlaDub2. The genes encoding ChlaDub1 and ChlaDub2 are present in all Chlamydia species except for Chlamydia pneumoniae, and their catalytic domains bear similarity to the catalytic domains of other eukaryotic ubiquitin-like proteases (Ulp). The C. trachomatis DUBs react with activity-based probes and hydrolyse ubiquitinated and neddylated substrates. ChlaDub1 and ChlaDub2 represent the first known bacterial DUBs that possess both deubiquitinating and deneddylating activities.
Pub.: 11 Jul '06, Pinned: 24 Aug '17
Abstract: The cysteine protease adenain is the essential protease of adenovirus and, as such, represents a promising target for the treatment of ocular and other adenoviral infections. Through a concise two-pronged hit discovery approach we identified tetrapeptide nitrile 1 and pyrimidine nitrile 2 as complementary starting points for adenain inhibition. These hits enabled the first high-resolution X-ray cocrystal structures of adenain with inhibitors bound and revealed the binding mode of 1 and 2. The screening hits were optimized by a structure-guided medicinal chemistry strategy into low nanomolar drug-like inhibitors of adenain.
Pub.: 26 Aug '14, Pinned: 23 Aug '17
Abstract: Pathogenic bacteria rely on secreted effector proteins to manipulate host signaling pathways, often in creative ways. CE clan proteases, specific hydrolases for ubiquitin-like modifications (SUMO and NEDD8) in eukaryotes, reportedly serve as bacterial effector proteins with deSUMOylase, deubiquitinase, or, even, acetyltransferase activities. Here, we characterize bacterial CE protease activities, revealing K63-linkage-specific deubiquitinases in human pathogens, such as Salmonella, Escherichia, and Shigella, as well as ubiquitin/ubiquitin-like cross-reactive enzymes in Chlamydia, Rickettsia, and Xanthomonas. Five crystal structures, including ubiquitin/ubiquitin-like complexes, explain substrate specificities and redefine relationships across the CE clan. Importantly, this work identifies novel family members and provides key discoveries among previously reported effectors, such as the unexpected deubiquitinase activity in Xanthomonas XopD, contributed by an unstructured ubiquitin binding region. Furthermore, accessory domains regulate properties such as subcellular localization, as exemplified by a ubiquitin-binding domain in Salmonella Typhimurium SseL. Our work both highlights and explains the functional adaptations observed among diverse CE clan proteins.
Pub.: 19 Jul '16, Pinned: 23 Aug '17
Abstract: Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes various human diseases, including blindness caused by ocular infection and sexually transmitted diseases resulting from urogenital infection. After infecting host cells, Chlamydiae avoid alarming the host's immune system. Among the immune evasion mechanisms, Chlamydiae can inhibit NF-kappaB activation, a crucial pathway for host inflammatory responses. In this study, we show that ChlaDub1, a deubiquitinating and deNeddylating protease from C. trachomatis, is expressed in infected cells. In transfection experiments, ChlaDub1 suppresses NF-kappaB activation induced by several pro-inflammatory stimuli and binds the NF-kappaB inhibitory subunit IkappaBalpha, impairing its ubiquitination and degradation. Thus, we provide further insight into the mechanism by which C. trachomatis may evade the host inflammatory response by demonstrating that ChlaDub1, a protease produced by this microorganism, is capable of inhibiting IkappaBalpha degradation and blocking NF-kappaB activation.
Pub.: 28 May '08, Pinned: 23 Aug '17
Abstract: The diverse roles of deubiquitinating enzymes, or DUBs, in determining the fate of specific proteins continue to unfold. Concurrent with the revelation of DUBs as potential therapeutic targets are publications of small molecule inhibitors of these enzymes. In this review, we summarize these molecules and their associated data and suggest additional experiments to further validate and characterize these compounds. We believe the field of drug discovery against DUBs is still in its infancy, but advances in assay development, biophysical techniques, and screening libraries hold promise for identifying suitable agents that could advance into the clinic.
Pub.: 05 Nov '14, Pinned: 28 Aug '17
Abstract: Publication date: Available online 12 January 2016 Source:Progress in Medicinal Chemistry Author(s): Mark Kemp This review examines the small molecules described over the past decade as inhibitors of any of the approximately 100 human deubiquitinating enzymes (DUBs). Structures from patent publications as well as from the primary literature are included. Inhibitors of two viral DUBs are also described since these proteases share structural similarity with one of the human DUB sub-families. The structure, function and disease associations of certain DUBs are presented. The evolution of the screening assays used to identify and characterise new inhibitors is discussed. Several emerging trends in the series are highlighted and the ‘drug-likeness’ of the various inhibitors is analysed. Large pharmaceutical company collaborations have drawn attention to this field, and these recent advances are discussed in the context of the wider range of therapeutically important DUB targets.
Pub.: 16 Jan '16, Pinned: 28 Aug '17
Abstract: The docking methods used in structure-based virtual database screening offer the ability to quickly and cheaply estimate the affinity and binding mode of a ligand for the protein receptor of interest, such as a drug target. These methods can be used to enrich a database of compounds, so that more compounds that are subsequently experimentally tested are found to be pharmaceutically interesting. In addition, like all virtual screening methods used for drug design, structure-based virtual screening can focus on curated libraries of synthesizable compounds, helping to reduce the expense of subsequent experimental verification. In this review, we introduce the protein-ligand docking methods used for structure-based drug design and other biological applications. We discuss the fundamental challenges facing these methods and some of the current methodological topics of interest. We also discuss the main approaches for applying protein-ligand docking methods. We end with a discussion of the challenging aspects of evaluating or benchmarking the accuracy of docking methods for their improvement, and discuss future directions.
Pub.: 16 Jul '14, Pinned: 28 Aug '17
Abstract: Deubiquitinating-enzymes (DUBs) are key components of the ubiquitin-proteasome system (UPS). The fundamental role of DUBs is specific removal of ubiquitin from substrates. DUBs contribute to activation/deactivation, recycling and localization of numerous regulatory proteins, and thus play major roles in diverse cellular processes. Altered DUB activity is associated with a multitudes of pathologies including cancer. Therefore, DUBs represent novel candidates for target-directed drug development.The article is a thorough review/accounting of patented compounds targeting DUBs and stratifying/classifying the patented compounds based on: chemical-structures, nucleic-acid compositions, modes-of-action, and targeting sites. The review provides a brief background on the UPS and the involvement of DUBs. Furthermore, methods for assessing efficacy and potential pharmacological utility of DUB inhibitor (DUBi) are discussed.The FDA's approval of the 20S proteasome inhibitors (PIs): bortezomib and carfilzomib for treatment of hematological malignancies established the UPS as an anti-cancer target. Unfortunately, many patients are inherently resistant or develop resistance to PIs. One potential strategy to combat PI resistance is targeting upstream components of the UPS such as DUBs. DUBs represent a promising potential therapeutic target due to their critical roles in various cellular processes including protein turnover, localization and cellular homeostasis. While considerable efforts have been undertaken to develop DUB modulators, significant advancements are necessary to move DUBis into the clinic.
Pub.: 17 Jun '15, Pinned: 28 Aug '17
Abstract: This review summarizes the recently published data on the molecular mechanisms of Chlamydiae-host cell interaction, first of all, on chlamydial effector proteins. Such proteins, along with type III transport system proteins, which transfer many effector proteins into the host cytoplasm, are attractive targets for drug therapy of chlamydial infections. The majority of the data concerns two species, Chlamydia trachomatis and Chlamydophila pneumoniae. The C. trachomatis protein TARP, which is presynthesized in elementary bodies, plays an essential role in the initial stages of infection. The pathogen proteins that are involved in the next stage, which is the intracellular inclusion traffic to the centrosome, are C. trachomatis CT229 and C. pneumoniae Cpn0585, which interact with cell Rab GTPases. In C. trachomatis, IncA plays a key role in the fusion of chlamydial inclusions, CT847 modulates the life cycle of the host cell, and LDA3 is essential for the acquisition of nutrients. The protease CPAF and the inclusion membrane proteins IncG and CADD are involved in suppressing apoptosis of infected cells. The proteases CPAF and CT441 and the deubiquitinating protein ChlaDub1 help the pathogen to evade the immune response.
Pub.: 04 Dec '09, Pinned: 28 Aug '17
Abstract: The proteolytic enzymes that depend upon a cysteine residue for activity have come from at least seven different evolutionary origins, each of which has produced a group of cysteine peptidases with distinctive structures and properties. We show here that the characteristic molecular topologies of the peptidases in each evolutionary line can be seen not only in their three-dimensional structures, but commonly also in the two-dimensional structures. Clan CA contains the families of papain (C1), calpain (C2), streptopain (C10) and the ubiquitin-specific peptidases (C12, C19), as well as many families of viral cysteine endopeptidases. Clan CD contains the families of clostripain (C11), gingipain R (C25), legumain (C13), caspase-1 (C14) and separin (C50). These enzymes have specificities dominated by the interactions of the S1 subsite. Clan CE contains the families of adenain (C5) from adenoviruses, the eukaryotic Ulp1 protease (C48) and the bacterial YopJ proteases (C55). Clan CF contains only pyroglutamyl peptidase I (C15). The picornains (C3) in clan PA have probably evolved from serine peptidases, which still form the majority of enzymes in the clan. The cysteine peptidase activities in clans PB and CH are autolytic only. In conclusion, we suggest that although almost all the cysteine peptidases depend for activity on catalytic dyads of cysteine and histidine, it is worth noting some important differences that they have inherited from their distant ancestral peptidases.
Pub.: 24 Aug '01, Pinned: 23 Aug '17
Abstract: Protein ubiquitylation controls many cellular pathways, and timely removal of ubiquitin by deubiquitylating enzymes (DUBs) is essential to govern these different functions. To map endogenous expression of individual DUBs as well as that of any interacting proteins, we developed a catch-and-release ubiquitin probe. Ubiquitin was equipped with an activity-based warhead and a cleavable linker attached to a biotin affinity-handle through tandem site-specific modification, in which we combined intein chemistry with sortase-mediated ligation. The resulting probe is cell-impermeable and was therefore delivered to the cytosol of perfringolysin O (PFO)-permeabilized cells. This allowed us to retrieve and identify 34 DUBs and their interacting partners. We also noted the expression, in host cells infected with Chlamydia trachomatis, of two additional DUBs. Furthermore, we retrieved and identified chlamydial DUB1 (ChlaDUB1) and DUB2 (ChlaDUB2), demonstrating by experiment that ChlaDUB2, the presence and activity of which had not been detected in infected cells, is in fact expressed during the course of infection.
Pub.: 22 Jan '13, Pinned: 28 Aug '17