ABSTRACT
BACKGROUND: Surgical techniques for Terrible Triad injuries developed 20 years ago. Good and excellent short- and medium-term functional results have been reported. No long-term (over 10 years) functional outcomes have previously been reported. This case-series is the longest follow-up of patients treated for acute, isolated terrible triad injuries using a standard treatment protocol. METHODS: 20 Patients with acute, isolated, surgically managed terrible triad injuries were treated between October 2001 and May 2008. 10 of these patients were seen face-to face for a clinical follow-up and if required a radiological assessment. Mayo Elbow Performance Scores (MEPS) and Disability of the Arm, Shoulder and Hand (DASH) scores, requirement for further surgery and elbow instability were recorded. RESULTS: The average length of follow-up was 18.8 years. The mean MEPS was 88 and the mean DASH score was 12.3. The average loss of pronation was 8 degrees. The average loss of supination was 13 degrees. The re-operation rate was 40%, only one of these was a functionally limiting operation. A trend towards osteoarthritis was observed but there were no conversions to total elbow replacement. DISCUSSION: This is the longest-term follow-up study of these injuries and demonstrates the functional performance that the majority of patients achieve. The low follow-up rate can be expected with such a long interval between treatment and assessment. A relatively high re-operation rate is largely made up of minor procedures (removal of metalwork and cubital tunnel release) which did not impact the patients' functional status. CONCLUSION: This study adds to the evidence that the terrible triad of the elbow is surgically treatable to allow a high functional standard not only in the short-term but also in the long-term. As such this is a useful adjunct to have both when informing patients of what can be expected in their long-term recovery from this injury.
ABSTRACT
Bfl-1 is overexpressed in both hematological and solid tumors; therefore, inhibitors of Bfl-1 are highly desirable. A DNA-encoded chemical library (DEL) screen against Bfl-1 identified the first known reversible covalent small-molecule ligand for Bfl-1. The binding was validated through biophysical and biochemical techniques, which confirmed the reversible covalent mechanism of action and pointed to binding through Cys55. This represented the first identification of a cyano-acrylamide reversible covalent compound from a DEL screen and highlights further opportunities for covalent drug discovery through DEL screening. A 10-fold improvement in potency was achieved through a systematic SAR exploration of the hit. The more potent analogue compound 13 was successfully cocrystallized in Bfl-1, revealing the binding mode and providing further evidence of a covalent interaction with Cys55.
ABSTRACT
CASE: A 14-year-old adolescent boy with SCN1B mutation experienced frequent seizures and recurrent elbow dislocation, occurring up to 30 times per day. Following failed conservative treatment, the decision was made to surgically repair the lateral collateral ligament complex and stabilize the elbow with the internal joint stabilizer (IJS). At more than 3 years postoperatively, the patient has not had a dislocation event and will retain the device for the foreseeable future to maintain predictable elbow stability. CONCLUSION: Although there is scant evidence supporting the use of the IJS in pediatric cases, the current case supports its use in pediatric elbow instability.
Subject(s)
Elbow Joint , Joint Dislocations , Humans , Adolescent , Male , Joint Dislocations/surgery , Joint Dislocations/diagnostic imaging , Elbow Joint/surgery , Elbow Joint/diagnostic imaging , Joint Instability/surgery , Elbow Injuries , RecurrenceABSTRACT
Covalent hit identification is a viable approach to identify chemical starting points against difficult-to-drug targets. While most researchers screen libraries of <2k electrophilic fragments, focusing on lead-like compounds can be advantageous in terms of finding hits with improved affinity and with a better chance of identifying cryptic pockets. However, due to the increased molecular complexity, larger numbers of compounds (>10k) are desirable to ensure adequate coverage of chemical space. Herein, the approach taken to build a library of 12k covalent lead-like compounds is reported, utilizing legacy compounds, robust library chemistry, and acquisitions. The lead-like covalent library was screened against the antiapoptotic protein Bfl-1, and six promising hits that displaced the BIM peptide from the PPI interface were identified. Intriguingly, X-ray crystallography of lead-like compound 8 showed that it binds to a previously unobserved conformation of the Bfl-1 protein and is an ideal starting point for the optimization of Bfl-1 inhibitors.
Subject(s)
Cysteine , Drug Design , Small Molecule Libraries , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Crystallography, X-Ray , Cysteine/chemistry , Humans , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/metabolism , Structure-Activity Relationship , Models, Molecular , Minor Histocompatibility AntigensABSTRACT
Herein, we report the identification and optimization of a series of potent inhibitors of EGFR Exon20 insertions with significant selectivity over wild-type EGFR. A strategically designed HTS campaign, multiple iterations of structure-based drug design (SBDD), and tactical linker replacement led to a potent and wild-type selective series of molecules and ultimately the discovery of 36. Compound 36 is a potent and selective inhibitor of EGFR Exon20 insertions and has demonstrated encouraging efficacy in NSCLC EGFR CRISPR-engineered H2073 xenografts that carry an SVD Exon20 insertion and reduced efficacy in a H2073 wild-type EGFR xenograft model compared to CLN-081 (5), indicating that 36 may have lower EGFR wild-type associated toxicity.
Subject(s)
ErbB Receptors , Exons , Protein Kinase Inhibitors , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , ErbB Receptors/metabolism , Humans , Animals , Structure-Activity Relationship , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/therapeutic use , Cell Line, Tumor , Mice , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/therapeutic use , Drug Discovery , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Mutagenesis, Insertional , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Xenograft Model Antitumor Assays , MutationABSTRACT
The structure-based design of small-molecule inhibitors targeting protein-protein interactions (PPIs) remains a huge challenge as the drug must bind typically wide and shallow protein sites. A PPI target of high interest for hematological cancer therapy is myeloid cell leukemia 1 (Mcl-1), a prosurvival guardian protein from the Bcl-2 family. Despite being previously considered undruggable, seven small-molecule Mcl-1 inhibitors have recently entered clinical trials. Here, we report the crystal structure of the clinical-stage inhibitor AMG-176 bound to Mcl-1 and analyze its interaction along with clinical inhibitors AZD5991 and S64315. Our X-ray data reveal high plasticity of Mcl-1 and a remarkable ligand-induced pocket deepening. Nuclear Magnetic Resonance (NMR)-based free ligand conformer analysis demonstrates that such unprecedented induced fit is uniquely achieved by designing highly rigid inhibitors, preorganized in their bioactive conformation. By elucidating key chemistry design principles, this work provides a roadmap for targeting the largely untapped PPI class more successfully.
Subject(s)
Apoptosis , Naphthalenes , Models, Molecular , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , LigandsABSTRACT
Using a relatively small training set of ~16 thousand images from macromolecular crystallisation experiments, we compare classification results obtained with four of the most widely-used convolutional deep-learning network architectures that can be implemented without the need for extensive computational resources. We show that the classifiers have different strengths that can be combined to provide an ensemble classifier achieving a classification accuracy comparable to that obtained by a large consortium initiative. We use eight classes to effectively rank the experimental outcomes, thereby providing detailed information that can be used with routine crystallography experiments to automatically identify crystal formation for drug discovery and pave the way for further exploration of the relationship between crystal formation and crystallisation conditions.
Subject(s)
Deep Learning , Neural Networks, ComputerABSTRACT
ROS1 rearrangements account for 1-2% of non-small cell lung cancer patients, yet there are no specifically designed, selective ROS1 therapies in the clinic. Previous knowledge of potent ROS1 inhibitors with selectivity over TrkA, a selected antitarget, enabled virtual screening as a hit finding approach in this project. The ligand-based virtual screening was focused on identifying molecules with a similar 3D shape and pharmacophore to the known actives. To that end, we turned to the AstraZeneca virtual library, estimated to cover 1015 synthesizable make-on-demand molecules. We used cloud computing-enabled FastROCS technology to search the enumerated 1010 subset of the full virtual space. A small number of specific libraries were prioritized based on the compound properties and a medicinal chemistry assessment and further enumerated with available building blocks. Following the docking evaluation to the ROS1 structure, the most promising hits were synthesized and tested, resulting in the identification of several potent and selective series. The best among them gave a nanomolar ROS1 inhibitor with over 1000-fold selectivity over TrkA and, from the preliminary established SAR, these have the potential to be further optimized. Our prospective study describes how conceptually simple shape-matching approaches can identify potent and selective compounds by searching ultralarge virtual libraries, demonstrating the applicability of such workflows and their importance in early drug discovery.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Cloud Computing , Drug Evaluation, Preclinical , Humans , Molecular Docking Simulation , Prospective Studies , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases , Proto-Oncogene Proteins , Receptor Protein-Tyrosine KinasesABSTRACT
Although monoclonal antibodies have greatly improved cancer therapy, they can trigger side effects due to on-target, off-tumor toxicity. Over the past decade, strategies have emerged to successfully mask the antigen-binding site of antibodies, such that they are only activated at the relevant site, for example, after proteolytic cleavage. However, the methods for designing an ideal affinity-based mask and what parameters are important are not yet well understood. Here, we undertook mechanistic studies using three masks with different properties and identified four critical factors: binding site and affinity, as well as association and dissociation rate constants, which also played an important role. HDX-MS was used to identify the location of binding sites on the antibody, which were subsequently validated by obtaining a high-resolution crystal structure for one of the mask-antibody complexes. These findings will inform future designs of optimal affinity-based masks for antibodies and other therapeutic proteins.
Subject(s)
Antibodies, Monoclonal , Antibodies, Monoclonal/chemistry , Antibody Affinity , Binding SitesABSTRACT
The epidermal growth factor receptor (EGFR) harboring activating mutations is a clinically validated target in non-small-cell lung cancer, and a number of inhibitors of the EGFR tyrosine kinase domain, including osimertinib, have been approved for clinical use. Resistance to these therapies has emerged due to a variety of molecular events including the C797S mutation which renders third-generation C797-targeting covalent EGFR inhibitors considerably less potent against the target due to the loss of the key covalent-bond-forming residue. We describe the medicinal chemistry optimization of a biochemically potent but modestly cell-active, reversible EGFR inhibitor starting point with sub-optimal physicochemical properties. These studies culminated in the identification of compound 12 that showed improved cell potency, oral exposure, and in vivo activity in clinically relevant EGFR-mutant-driven disease models, including an Exon19 deletion/T790M/C797S triple-mutant mouse xenograft model.
Subject(s)
Antineoplastic Agents/therapeutic use , ErbB Receptors/antagonists & inhibitors , Neoplasms/drug therapy , Organophosphorus Compounds/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Humans , Mice, Nude , Mice, SCID , Mutation , Organophosphorus Compounds/chemical synthesis , Organophosphorus Compounds/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/metabolism , Pyrimidines/chemical synthesis , Pyrimidines/metabolism , Rats , Xenograft Model Antitumor AssaysABSTRACT
Background Midcarpal instability is a term for a collection of poorly understood conditions where the proximal row of the carpus is unstable. The most common type of midcarpal instability is palmar midcarpal instability (PMCI). Treatment for PMCI includes nonoperative proprioceptive retraining of the wrist, splints, and strengthening. If this fails, various authors have suggested several different fusions, tenodesis procedures, or capsular shrinkage. There are no long-term case series in the literature. Objective The aim of this study is to assess the long-term results of arthroscopic capsular shrinkage when used for PMCI of the wrist. Methods A prospective cohort study of patients who underwent arthroscopic capsular shrinkage for PMCI was performed. Ethical board approval was given for this study. All patients were followed up and reviewed independently from the operating surgeon. Assessment included a structured questionnaire, disabilities of the arm, shoulder and hand (DASH) questionnaire, and clinical examination using a goniometer. PMCI was assessed objectively with the anterior drawer test and radiological imaging was only performed if clinically relevant to the residual symptoms. Results Thirteen patients (15 wrists) underwent arthroscopic capsular shrinkage for PMCI. Twelve patients (14 wrists) were available for clinical review with a follow-up rate of 92.3%. The mean time from index procedure to final review was 12 years (range: 10-14years). The symptoms of instability had completely resolved in nine wrists (7 patients). Only 2 of the 14 wrists had symptoms that were reproduced with a positive anterior drawer test. All other wrists were stable on objective assessment. The mean DASH score had improved from pre op of 34 to post op of 12.1 and at 12-year follow-up this had deteriorated minimally to 15.3. Assessment of the range of motion showed an average increase in range of flexion/extension by 22 degrees. Patient satisfaction was excellent. The patients rated that nine wrists were much better than presurgery, three as better, one unchanged, and one worse. Discussion/Conclusion There are no studies looking at the long-term natural history of treatments for PMCI. The lead author proposes a grading system for symptomatic PMCI that has been retrospectively applied to this cohort. It is a grading system from 1 to 4 and is based on a treatment algorithm. This is the first long-term study from any joint, where the results of capsular shrinkage have been maintained over time. In this series, we have not seen any deleterious effect from possible mechanoreceptor injury. We suspect that functioning mechanoreceptors are more relevant in the unstable joint, than the structurally stable joint. The authors propose that thermal capsular shrinkage is an effective and durable option for use in mild-to-moderate forms of PMCI.
ABSTRACT
In macromolecular crystallization, success is often dependent on the pH of the experiment. However, little is known about the pH of reagents used, and it is generally assumed that the pH of the experiment will closely match that of any buffering chemical in the solution. We use a large dataset of experimentally measured solution pH values to show that this assumption can be very wrong and generate a model that can be used to successfully predict the overall solution pH of a crystallization experiment. Furthermore, we investigate the time dependence of the pH of some polyethylene glycol polymers widely used in protein crystallization under different storage conditions.
ABSTRACT
Apoptosis is a crucial process by which multicellular organisms control tissue growth, removal and inflammation. Disruption of the normal apoptotic function is often observed in cancer, where cell death is avoided by the overexpression of anti-apoptotic proteins of the Bcl-2 (B-cell lymphoma 2) family, including Mcl-1 (myeloid cell leukaemia 1). This makes Mcl-1 a potential target for drug therapy, through which normal apoptosis may be restored by inhibiting the protective function of Mcl-1. Here, the discovery and biophysical properties of an anti-Mcl-1 antibody fragment are described and the utility of both the scFv and Fab are demonstrated in generating an Mcl-1 crystal system amenable to iterative structure-guided drug design.
Subject(s)
Drug Discovery , Immunoglobulin Fab Fragments/chemistry , Myeloid Cell Leukemia Sequence 1 Protein/chemistry , Single-Chain Antibodies/chemistry , Animals , Apoptosis , CHO Cells , Cloning, Molecular , Cricetulus , Escherichia coli/genetics , HumansABSTRACT
Over the last ten years, targeted covalent inhibition has become a key discipline within medicinal chemistry research, most notably in the development of oncology therapeutics. One area where this approach is underrepresented, however, is in targeting protein-protein interactions. This is primarily because these hydrophobic interfaces lack appropriately located cysteine residues to allow for standard conjugate addition chemistry. Herein, we report our development of the first covalent inhibitors of the antiapoptotic protein B-cell lymphoma extra-large (Bcl-xL), utilizing a sulfonyl fluoride (SF) warhead to selectively covalently modify tyrosine 101 of the BH3 domain-binding groove. These compounds display time-dependent inhibition in a biochemical assay and are cellularly active (U266B1). In addition, compound 7 was further elaborated to generate a chemical-biology probe molecule, which may find utility in understanding the intricacies of Bcl-xL biology.
Subject(s)
bcl-X Protein/antagonists & inhibitors , Humans , Models, Molecular , Protein BindingABSTRACT
Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic molecule with high selectivity and affinity for Mcl-1 currently in clinical development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clinical trial has been launched for evaluation of AZD5991 in patients with hematological malignancies (NCT03218683).
Subject(s)
Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Leukemia, Myeloid, Acute/drug therapy , Multiple Myeloma/drug therapy , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Animals , Bortezomib/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Humans , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred C57BL , Mice, SCID , Multiple Myeloma/pathology , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Rats , Rats, Nude , Sulfonamides/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
The ultimate goal of protein design is to introduce new biological activity. We propose a computational approach for designing functional antibodies by focusing on functional epitopes, integrating large-scale statistical analysis with multiple structural models. Machine learning is used to analyze these models and predict specific residue-residue contacts. We use this approach to design a functional antibody to counter the proinflammatory effect of the cytokine interleukin-17A (IL-17A). X-ray crystallography confirms that the designed antibody binds the targeted epitope and the interaction is mediated by the designed contacts. Cell-based assays confirm that the antibody is functional. Importantly, this approach does not rely on a high-quality 3D model of the designed complex or even a solved structure of the target. As demonstrated here, this approach can be used to design biologically active antibodies, removing some of the main hurdles in antibody design and in drug discovery.
Subject(s)
Antibodies/immunology , Antibody Specificity/immunology , Computational Biology/methods , Epitopes/chemistry , Algorithms , Amino Acid Sequence , Antibodies/chemistry , Humans , Immunoglobulin Fab Fragments/chemistry , Models, MolecularABSTRACT
B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.
Subject(s)
Antineoplastic Agents/pharmacology , Proto-Oncogene Proteins c-bcl-6/antagonists & inhibitors , Quinolones/pharmacology , Thalidomide/analogs & derivatives , Thalidomide/pharmacology , Adaptor Proteins, Signal Transducing , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Cell Line, Tumor , HEK293 Cells , Humans , Ligands , Lymphoma, Large B-Cell, Diffuse/drug therapy , Peptide Hydrolases/metabolism , Protein Binding , Proteolysis , Proto-Oncogene Proteins c-bcl-6/chemistry , Proto-Oncogene Proteins c-bcl-6/metabolism , Quinolones/chemical synthesis , Quinolones/metabolism , Thalidomide/chemical synthesis , Thalidomide/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
[This corrects the article DOI: 10.1021/acsmedchemlett.6b00464.].
ABSTRACT
The evidence behind management options for midcarpal instability (MCI) is scarce, relying solely on case series. Established treatments cause significant loss of wrist motion. As understanding of the condition has progressed, surgeons have been trying soft tissue techniques. The treatment option should be chosen for the appropriate type and grade of MCI. The Hargreaves grading system for palmar MCI aids treatment decision-making. A possible role for arthroscopy in treatment of MCI has been developed using arthroscopic thermal capsular shrinkage, appropriate for cases with dynamic instabilities. Static deformities require a soft tissue reconstruction or a partial wrist fusion.