Biology subjects

Lessene, G.

Publications and source records attributed to Lessene, G..

7 recordsLinked to original sources

bioRxiv2024

Differential regulation of BAX and BAK apoptotic activity revealed by a novel small molecule

Defective apoptosis mediated by BAK or BAX underlies various human pathologies including autoimmune and degenerative conditions. The mitochondrial channel protein VDAC2 interacts with BAK and BAX through a common interface to either inhibit BAK or to facilitate BAX apoptotic activity. Using a newly developed small molecule (WEHI-3773) that inhibits the interaction between VDAC2 and BAK or BAX, we reveal contrasting effects on BAX and BAK apoptotic activity. WEHI-3773 inhibits apoptosis mediated by BAX by blocking VDAC2-mediated BAX recruitment to mitochondria. Conversely, WEHI-3773 primes BAK for apoptosis by impairing its inhibitory sequestration by VDAC2 on the mitochondrial membrane. In cells expressing both BAX and BAK, repressing their association with VDAC2 promotes apoptosis, because once BAK is activated, it further activates BAX through a feed-forward mechanism. In some leukemias, mutation or loss of BAX is a key driver of resistance to the BH3-mimetic anti-cancer drug venetoclax. Strikingly, promoting BAK-mediated killing by small molecule dissociation of the VDAC2 interaction can overcome this resistance in different leukemia models. These data reveal a hitherto unappreciated level of coordination of BAX and BAK apoptotic activity through their interaction with VDAC2 that may be targeted therapeutically.

cell biology↗

bioRxiv2024

Mutational Profiling of SARS-CoV-2 PLpro in human cells reveals requirements for function, structure, and drug escape

SARS-CoV-2, the causative agent of COVID-19, is responsible for the recent global pandemic and remains a major source of mortality. Papain-like protease (PLpro) is a target for SARS-CoV-2 inhibitor development, as it is not only essential for viral replication through cleavage of the viral poly-proteins pp1a and pp1ab, but also has de-ubiquitylation and de-ISGylation activities, which can affect innate immune responses. To understand the features of PLpro that dictate activity and anticipate how emerging PLpro variants will affect function, we employed Deep Mutational Scanning to evaluate the mutational effects on enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify all mutations in neighboring residues that support or ablate activity. We characterize residues responsible for substrate binding and demonstrate that although the blocking loop is remarkably tolerant to nearly all mutations, its flexibility is important for enzymatic function. We additionally find a connected network of mutations affecting function but not structure that extends far from the active site. Using our DMS libraries we were able to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process.

biochemistry↗

bioRxiv2023

Molecular definition of the BAK:VDAC2 interaction as a target to manipulate apoptosis

BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial porin VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, whilst interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK, and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease, or to promote BAK-mediated apoptosis for cancer therapy.

cell biology↗

bioRxiv2021

Dual Drug Targeting to Kill Colon Cancer Cells

Colorectal cancer (CRC) is driven by a small set of oncogenic and tumor suppressor mutations. However, different combinations of mutations often lead to poor tumor responses to individual anti-cancer drugs. We have investigated the anti-proliferative and in vitro cytotoxic activity of pair-wise combinations of inhibitors which target specific signalling pathways. Colon cancer cells in non-adherent cultures were killed more effectively by combinations of pyrvinium pamoate (a Wnt pathway inhibitor) and ABT263 (a pro-apoptotic Bcl-2 family inhibitor) or Ly29004 (a PI3kinase inhibitor). However, in a mouse xenograft model, the formulation and toxicity of the ABT737/PP combination prevent the use of these drugs for treatment of tumors. Fortunately, oral analogues of PP (pyrvinium phosphate, PPh) and ABT737(ABT263) have equivalent activity and can be used for treatment of mice carrying SW620 colorectal cancer xenografts. The PPh/ABT263 induced SW620 tumor cell apoptosis and reduced the rate of SW620 tumor growth. Combinations of Wnt signaling inhibitors and specific inhibitor of pro-survival proteins should be considered for the treatment of precancerous colon adenomas and advanced colorectal cancers with APC mutations.

cancer biology↗

bioRxiv2020

Mechanism and inhibition of SARS-CoV-2 PLpro

Coronaviruses, including SARS-CoV-2, encode multifunctional proteases that are essential for viral replication and evasion of host innate immune mechanisms. The papain-like protease PLpro cleaves the viral polyprotein, and reverses inflammatory ubiquitin and anti-viral ubiquitin-like ISG15 protein modifications1,2. Drugs that target SARS-CoV-2 PLpro (hereafter, SARS2 PLpro) may hence be effective as treatments or prophylaxis for COVID-19, reducing viral load and reinstating innate immune responses3. We here characterise SARS2 PLpro in molecular and biochemical detail. SARS2 PLpro cleaves Lys48-linked polyubiquitin and ISG15 modifications with high activity. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. We further exploit two strategies to target PLpro. A repurposing approach, screening 3727 unique approved drugs and clinical compounds against SARS2 PLpro, identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors were able to inhibit SARS2 PLpro with high potency and excellent antiviral activity in SARS-CoV-2 infection models.

biochemistry↗

bioRxiv2020

BCL-XL is essential for the protection from secondary anemia caused by radiation-induced fatal kidney damage

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis, but less is known about the roles of these proteins in adults, including in the response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL-deficiency in erythroid cells. Unexpectedly, the combination of total-body {gamma}-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combinations with DNA damage-inducing drugs for cancer therapy. SummaryThe inducible loss of BCL-XL in all cells of adult mice causes primary anemia due to apoptosis of erythroid and megakaryocytic cell populations. In contrast {gamma}-radiation plus loss of BCL-XL in all cells except hematopoietic cells causes secondary anemia resulting from kidney damage.

pharmacology and toxicology↗

bioRxiv2018

Humanized Mcl-1 mice enable accurate pre-clinical evaluation of MCL-1 inhibitors destined for clinical use

MCL-1 is a pro-survival BCL-2 protein required for the sustained growth of many cancers. Recently a highly specific MCL-1-inhibitor, S63845, showing 6-fold higher affinity to human compared to mouse MCL-1 has been described. To accurately test efficacy and tolerability of this BH3 mimetic drug in pre-clinical cancer models, we developed a humanized Mcl-1 (huMcl-1) mouse in which MCL-1 was replaced with its human homologue. HuMcl-1 mice are phenotypically indistinguishable from wild-type mice but are more sensitive to MCL-1 inhibition. Importantly, non-transformed cells and lymphomas from huMcl-1;E-Myc mice are more sensitive to S63845 in vitro than their control counterparts. When huMcl-1;E-Myc lymphoma cells are transplanted into huMcl-1 mice, treatment with S63845 alone or alongside cyclophosphamide leads to long-term remission in ~60% or almost 100% of mice, respectively. These results demonstrate the potential of our huMCL-1 mouse model to test MCL-1 inhibitors, allowing precise predictions of efficacy and tolerability for clinical translation.

cancer biology↗