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Objective Accumulation of mitochondria underlies T-cell dysfunction in systemic lupus erythematosus (SLE). Mitochondrial turnover involves endosomal traffic regulated by HRES-1/Rab4, a small GTPase that is overexpressed in lupus T cells. Therefore, we investigated whether (1) HRES-1/Rab4 impacts mitochondrial homeostasis and (2) Rab geranylgeranyl transferase inhibitor 3-PEHPC blocks mitochondrial accumulation in T cells, autoimmunity and disease development in lupus-prone mice. Methods Mitochondria were evaluated in peripheral blood lymphocytes (PBL) of 38 SLE patients and 21 healthy controls and mouse models by flow cytometry, microscopy and western blot. MRL/lpr mice were treated with 125 μg/kg 3-PEHPC or 1 mg/kg rapamycin for 10 weeks, from 4 weeks of age. Disease was monitored by antinuclear antibody (ANA) production, proteinuria, and renal histology. Results Overexpression of HRES-1/Rab4 increased the mitochondrial mass of PBL (1.4-fold; p=0.019) and Jurkat cells (2-fold; p=0.000016) and depleted the mitophagy initiator protein Drp1 both in human (−49%; p=0.01) and mouse lymphocytes (−41%; p=0.03). Drp1 protein levels were profoundly diminished in PBL of SLE patients (−86±3%; p=0.012). T cells of 4-week-old MRL/lpr mice exhibited 4.7-fold over-expression of Rab4A (p=0.0002), the murine homologue of HRES-1/Rab4, and depletion of Drp1 that preceded the accumulation of mitochondria, ANA production and nephritis. 3-PEHPC increased Drp1 (p=0.03) and reduced mitochondrial mass in T cells (p=0.02) and diminished ANA production (p=0.021), proteinuria (p=0.00004), and nephritis scores of lupus-prone mice (p<0.001). Conclusions These data reveal a pathogenic role for HRES-1/Rab4-mediated Drp1 depletion and identify endocytic control of mitophagy as a treatment target in SLE.

Dysregulated gene expression programs and redox and metabolic adaptations allow cancer cells to survive under high oxidative burden. These mechanisms also represent therapeutic vulnerabilities. Using triple-negative breast cancer (TNBC) as a model, we show that compared to normal human breast epithelial cells, the TNBC cells, MDA-MB-231 and MDA-MB-468 that harbor constitutively active STAT3 also express higher glucose-6-phosphate dehydrogenase (G6PD), thioredoxin reductase (TrxR)1, NADPH, and GSH levels for survival. Present studies discover that the natural product, R001, targets these adaptation mechanisms. Treatment of TNBC cells with R001 inhibited constitutively active STAT3, STAT3-regulated gene expression, and the functions of G6PD and TrxR1. Consequently, in the TNBC, but not normal cells, R001 suppressed GSH levels, but raised NADPH levels, reflective of a loss of mitochondrial respiration and which led to reactive oxygen species (ROS) induction, all of which led to loss of viable cells and inhibition of anchorage-dependent and independent growth. R001 treatment further led to early pyroptosis and late DNA damage, cell cycle arrest, and apoptosis only in the TNBC cells. Oral administration of 5 mg/kg R001 inhibited MDA-MB-468 xenografts growth in mice, with reduced pY705-STAT3, G6PD, TrxR1, and GSH levels. R001 serves as a therapeutic entity that targets the vulnerabilities of TNBC cells to inhibit tumor growth in vivo.

HRES-1/Rab4 is a small GTPase that regulates endocytic recycling. It has been colocalized to mitochondria and the mechanistic target of rapamycin (mTOR), a suppressor of autophagy. Since the autophagosomal membrane component microtubule-associated protein light chain 3 (LC3) is derived from mitochondria, we investigated the impact of HRES-1/Rab4 on the formation of LC3(+) autophagosomes, their colocalization with HRES-1/Rab4 and mitochondria, and the retention of mitochondria during autophagy induced by starvation and rapamycin. HRES-1/Rab4 exhibited minimal baseline colocalization with LC3, which was enhanced 22-fold upon starvation or 6-fold upon rapamycin treatment. Colocalization of HRES-1/Rab4 with mitochondria was increased >2-fold by starvation or rapamycin. HRES-1/Rab4 overexpression promoted the colocalization of mitochondria with LC3 upon starvation or rapamycin treatment. A dominant-negative mutant, HRES-1/Rab4(S27N) had reduced colocalization with LC3 and mitochondria upon starvation but not rapamycin treatment. A constitutively active mutant, HRES-1/Rab4(Q72L) showed diminished colocalization with LC3 but promoted the partitioning of mitochondria with LC3 upon starvation or rapamycin treatment. Phosphorylation-resistant mutant HRES-1/Rab4(S204Q) showed diminished colocalization with LC3 but increased partitioning to mitochondria. A newly discovered C-terminally truncated native isoform, HRES-1/Rab4(1-121), showed enhanced localization to LC3 and mitochondria without starvation or rapamycin treatment. HRES-1/Rab4(1-121) increased the formation of LC3(+) autophagosomes in resting cells, while other isoforms promoted autophagosome formation upon starvation. HRES-1/Rab4, HRES-1/Rab4(1-121), HRES-1/Rab4(Q72L) and HRES-1/Rab4(S204Q) promoted the accumulation of mitochondria during starvation. The specificity of HRES-1/Rab4-mediated mitochondrial accumulation is indicated by its abrogation by dominant-negative HRES-1/Rab4(S27N) mutation. The formation of interconnected mitochondrial tubular networks was markedly enhanced by HRES-1/Rab4(Q72L) upon starvation, which may contribute to the retention of mitochondria during autophagy. The present study thus indicates that HRES-1/Rab4 regulates autophagy through promoting the formation of LC3(+) autophagosomes and the preservation of mitochondria.

Key Points Considerable evidence supports the crucial roles of the signal transducer and activator of transcription (STAT) family of proteins in human diseases, particularly in immune and inflammatory disorders, infection and cancer Increasing emphasis is being placed on developing direct STAT inhibitors for clinical application, mainly through the discovery of small molecules, oligonucleotides and natural product derivatives. A large part of the ongoing STATs drug discovery research for therapeutics is focused on targeting STAT3, of which the efforts to develop small-molecule STAT3 inhibitors is extensive. While research into oligodeoxynucleotide (ODN) decoys and antisense oligonucleotides (ASOs) as STAT-inhibitory approaches is not as widespread, these efforts appear to be advancing as a STAT3 ODN has progressed to clinical trials (Phase 0). Tyrosine kinase inhibitors (TKIs) as therapeutic modalities are widely explored, and this approach is highly established. TKI agents may be therapeutic considerations in STAT-associated diseases in so far as a causal link could be established between the target tyrosine kinase and dysregulated STAT signalling that is prevalent in the disease. The precedence of natural product-based therapeutics for many diseases and the number of reports on natural product inhibitors of STAT3 signalling together highlight the potential of this resource as an important source of leads for developing STAT inhibitors. There is a clinical trial to evaluate ISIS-STAT3Rx ASO against advanced cancers. Other clinical trials are focusing on therapeutic modalities that can affect STAT function and STAT-associated diseases, including the evaluation of curcumin for pancreatic cancer, resveratrol for colorectal cancer and 3,3′-diindolylmethane for breast cancer, and TKIs against many cancer types. Members of the signal transducer and activator of transcription (STAT) protein family are implicated in a variety of diseases. In particular, aberrant activation of STAT3 is known to promote malignant transformation. In this Review, Turkson and colleagues discuss the various therapeutic approaches used to modulate the activation of the different STAT family members, which include dimerization inhibitors, tyrosine kinase inhibitors and DNA decoys. The signal transducer and activator of transcription (STAT) proteins have important roles in biological processes. The abnormal activation of STAT signalling pathways is also implicated in many human diseases, including cancer, autoimmune diseases, rheumatoid arthritis, asthma and diabetes. Over a decade has passed since the first inhibitor of a STAT protein was reported and efforts to discover modulators of STAT signalling as therapeutics continue. This Review discusses the outcomes of the ongoing drug discovery research endeavours against STAT proteins, provides perspectives on new directions for accelerating the discovery of drug candidates, and highlights the noteworthy candidate therapeutics that have progressed to clinical trials.

DNA-encoded chemical libraries are collections of compounds individually coupled to unique DNA tags serving as amplifiable identification barcodes. By bridging split-and-pool combinatorial synthesis with the ligation of unique encoding DNA oligomers, million- to billion-member libraries can be synthesized for use in hundreds of healthcare target screens. Although structural diversity and desirable molecular property ranges generally guide DNA-encoded chemical library design, recent reports have highlighted the utility of focused DNA-encoded chemical libraries that are structurally biased for a class of protein targets. Herein, a protease-focused DNA-encoded chemical library was designed that utilizes chemotypes known to engage conserved catalytic protease residues. The three-cycle library features functional moieties such as guanidine, which interacts strongly with aspartate of the protease catalytic triad, as well as mild electrophiles such as sulfonamide, urea, and carbamate. We developed a DNA-compatible method for guanidinylation of amines and reduction of nitriles. Employing these optimized reactions, we constructed a 9.8-million-membered DNA-encoded chemical library. Affinity selection of the library with thrombin, a common protease, revealed a number of enriched features which ultimately led to the discovery of a 1 nM inhibitor of thrombin. Thus, structurally focused DNA-encoded chemical libraries have tremendous potential to find clinically useful high-affinity hits for the rapid discovery of drugs for targets (e.g., proteases) with essential functions in infectious diseases (e.g., severe acute respiratory syndrome coronavirus 2) and relevant healthcare conditions (e.g., male contraception).

TGFβ family ligands, which include the TGFβs, BMPs, and activins, signal by forming a ternary complex with type I and type II receptors. For TGFβs and BMPs, structures of ternary complexes have revealed differences in receptor assembly. However, structural information for how activins assemble a ternary receptor complex is lacking. We report the structure of an activin class member, GDF11, in complex with the type II receptor ActRIIB and the type I receptor Alk5. The structure reveals that receptor positioning is similar to the BMP class, with no interreceptor contacts; however, the type I receptor interactions are shifted toward the ligand fingertips and away from the dimer interface. Mutational analysis shows that ligand type I specificity is derived from differences in the fingertips of the ligands that interact with an extended loop specific to Alk4 and Alk5. The study also reveals differences for how TGFβ and GDF11 bind to the same type I receptor, Alk5. For GDF11, additional contacts at the fingertip region substitute for the interreceptor interactions that are seen for TGFβ, indicating that Alk5 binding to GDF11 is more dependent on direct contacts. In support, we show that a single residue of Alk5 (Phe84), when mutated, abolishes GDF11 signaling, but has little impact on TGFβ signaling. The structure of GDF11/ActRIIB/Alk5 shows that, across the TGFβ family, different mechanisms regulate type I receptor binding and specificity, providing a molecular explanation for how the activin class accommodates low-affinity type I interactions without the requirement of cooperative receptor interactions.

The signal transducer and activator of transcription (STAT) proteins have important roles in biological processes. The abnormal activation of STAT signalling pathways is also implicated in many human diseases, including cancer, autoimmune diseases, rheumatoid arthritis, asthma and diabetes. Over a decade has passed since the first inhibitor of a STAT protein was reported and efforts to discover modulators of STAT signalling as therapeutics continue. This Review discusses the outcomes of the ongoing drug discovery research endeavours against STAT proteins, provides perspectives on new directions for accelerating the discovery of drug candidates, and highlights the noteworthy candidate therapeutics that have progressed to clinical trials.

HRES-1/Rab4 is a small GTPase that regulates endocytic recycling. It has been colocalized to mitochondria and the mechanistic target of rapamycin (mTOR), a suppressor of autophagy. Since the autophagosomal membrane component microtubule-associated protein light chain 3 (LC3) is derived from mitochondria, we investigated the impact of HRES-1/Rab4 on the formation of LC3.sup.+ autophagosomes, their colocalization with HRES-1/Rab4 and mitochondria, and the retention of mitochondria during autophagy induced by starvation and rapamycin. HRES-1/Rab4 exhibited minimal baseline colocalization with LC3, which was enhanced 22-fold upon starvation or 6-fold upon rapamycin treatment. Colocalization of HRES-1/Rab4 with mitochondria was increased >2-fold by starvation or rapamycin. HRES-1/Rab4 overexpression promoted the colocalization of mitochondria with LC3 upon starvation or rapamycin treatment. A dominant-negative mutant, HRES-1/Rab4.sup.S27N had reduced colocalization with LC3 and mitochondria upon starvation but not rapamycin treatment. A constitutively active mutant, HRES-1/Rab4.sup.Q72L showed diminished colocalization with LC3 but promoted the partitioning of mitochondria with LC3 upon starvation or rapamycin treatment. Phosphorylation-resistant mutant HRES-1/Rab4.sup.S204Q showed diminished colocalization with LC3 but increased partitioning to mitochondria. A newly discovered C-terminally truncated native isoform, HRES-1/Rab4.sup.1-121, showed enhanced localization to LC3 and mitochondria without starvation or rapamycin treatment. HRES-1/Rab4.sup.1-121 increased the formation of LC3.sup.+ autophagosomes in resting cells, while other isoforms promoted autophagosome formation upon starvation. HRES-1/Rab4, HRES-1/Rab4.sup.1-121, HRES-1/Rab4.sup.Q72L and HRES-1/Rab4.sup.S204Q promoted the accumulation of mitochondria during starvation. The specificity of HRES-1/Rab4-mediated mitochondrial accumulation is indicated by its abrogation by dominant-negative HRES-1/Rab4.sup.S27N mutation. The formation of interconnected mitochondrial tubular networks was markedly enhanced by HRES-1/Rab4.sup.Q72L upon starvation, which may contribute to the retention of mitochondria during autophagy. The present study thus indicates that HRES-1/Rab4 regulates autophagy through promoting the formation of LC3.sup.+ autophagosomes and the preservation of mitochondria.

A doktori értekezésemben bemutatott munkáimban célul tűztük ki egy nagy áteresztőképességű és gyors mikrotiter lemez alapú fluorometriás módszer kidolgozását, mely alkalmas különböző retrovirális proteázok aktivitásának és gátlási állandóinak közvetlen összehasonlítására, illetve részben ezen módszerre alapozva, új típusú makromolekuláris HIV-1 PR inhibitorok tervezését, előállítását és tesztelését.Összefoglalásul elmondhatjuk, hogy a munkánk során kidolgozott fluorometriás módszer egy jó alternatíva lehet a hagyományos HPLC-alapú módszer mellett, mivel gyors és egyszerre nagy számú minta kezelését teszi lehetővé. Módszerünket a hagyományos HPLC-alapú módszerrel validáltuk vad típusú HIV-1 és HTLV-1 proteázok, valamint EDANS és DABCYL csoportokat hordozó új tervezésű szubsztrátok felhasználásával. Kidolgoztunk egy új eljárást a fluoreszcens donor és akceptor csoportokat egyszerre hordozó szubsztrátok mérése esetén fellépő belső szűrő hatás korrekciójára. Az új módszer segítségével összehasonlítottuk a HIV-1 és a HTLV-1 proteázok gátlási profilját néhány, már klinikai használatban lévő HIV-1 PR ellenes és néhány, a munkacsoportunk által tervezett HTLV-1 PR inhibitor felhasználásával. Módszerünket későbbiekben sikerrel alkalmaztuk más vad típusú és mutáns retrovirális proteázok esetében is (Kádas és mtsai, 2004; Fehér és mtsai, 2006; Sperka és mtsai, 2007).A továbbiakban a szubsztrátkötő helyen hidrofil, hidrofób és töltött aminosavakat hordozó mutáns HIV-1 proteázokat terveztünk és vizsgáltunk. A mutációk in silico megtervezése után elkészítettük és megtisztítottuk a fehérjéket, majd teszteltük azok in vitroés sejtkultúrás kísérletekben mutatott gátló hatását. A töltött aminosavakat hordozó mutánsaink, amelyek a HIV-1 proteáz ellen tervezett makromolekuláris inhibitorok egy új generációját képviselik, dózisfüggő, specifikus, transz domináns gátló hatást mutattak kísérleteinkben. Elsőként detektáltuk egy vad típusú retrovirális proteáz és egy transz domináns negatív mutáns heterodimerizációját NMR spektroszkópiával. Az Asp25Arg és a Gly49Glu csere egyéb retrovirális proteázokban is gátló hatást eredményezhet a vad típusú proteázzal szemben, mivel ezen aminosavcserék célpontjai a hidrolitikus aktivitáshoz elengedhetetlen katalitikus aszpartátok.