Tubulysin Production by the Dead Cells of Archangium gephyra KYC5002.

Archangium gephyra KYC5002 produces tubulysins during the death phase. In this study, we aimed to determine whether dead cells produce tubulysins. Cells were cultured for three days until the verge of the death phase, disrupted via ultrasonication, incubated for 2 h, and examined for tubulysin production. Non-disrupted cells produced 0.14 mg/L of tubulysin A and 0.11 mg/L of tubulysin B. Notably, tubulysin A production was increased by 4.4-fold to 0.62 mg/L and that of tubulysin B was increased by 6.7-fold to 0.74 mg/L in the disrupted cells. The same increase in tubulysin production was observed when the cells were killed by adding hydrogen peroxide. However, when the enzymes were inactivated via heat treatment of the cultures at 65 °C for 30 min, no significant increase in tubulysin production due to cell death was observed. Reverse transcription-quantitative polymerase chain reaction analysis of tubB mRNA revealed that the expression levels of tubulysin biosynthetic enzyme genes increased during the death phase compared to those during the vegetative growth phase. Our findings suggest that A. gephyra produces biosynthetic enzymes and subsequently uses them for tubulysin production in the cell death phase or during cell lysis by predators.

Tubulysins are Essential for the Preying of Ciliates by Myxobacteria.

Tubulysins are bioactive secondary metabolites produced by myxobacteria that promote microtubule disassembly. Microtubules are required for protozoa such as Tetrahymena to form cilia and flagella. To study the role of tubulysins in myxobacteria, we co-cultured myxobacteria and Tetrahymena. When 4000 Tetrahymena thermophila and 5.0 × 108 myxobacteria were added to 1 ml of CYSE medium and co-cultured for 48 h, the population of T. thermophila increased to more than 75,000. However, co-culturing tubulysin-producing myxobacteria, including Archangium gephyra KYC5002, with T. thermophila caused the population of T. thermophila to decrease from 4000 to less than 83 within 48 h. Almost no dead bodies of T. thermophila were observed in the culture medium. Co-culturing of T. thermophila and the A. gephyra KYC5002 strain with inactivation of the tubulysin biosynthesis gene led to the population of T. thermophila increasing to 46,667. These results show that in nature, most myxobacteria are preyed upon by T. thermophila, but some myxobacteria prey on and kill T. thermophila using tubulysins. Adding purified tubulysin A to T. thermophila changed the cell shape from ovoid to spherical and caused cell surface cilia to disappear.

The Recent Developments of ADCs with the Tubulysins as the Payloads.

As a novel bio-targeting antitumor agent, an antibody-drug conjugate (ADC) combines the high selectivity of monoclonal antibody and potent cytotoxicity of drug or payload. It can expand the scope of clinical application of small molecule drugs. Tubulysin and its bio-precursor pretubulysin (PT) are potent tubulin-binding antitumor drugs. Due to the excellent antitumoral, antimetastatic, antiangiogenic, and anti-multidrug resistance properties, Tubulysins or PT is believed to be a promising cancer therapeutic approach. Currently, the modifications of tubulysin are centering on the C-11 acetoxyl and N,O-acetal groups, and numerous promising payloads are identified. There are at least 5 sites to introduce appropriate drug linkers in tubulysin and PT for connecting the antibodies. The possible sites of attachment are located in Mep, Tuv, or Tup parts. Cleavage and non-cleavage linkers are used in these ADCs. The chemical reactions involved in the final conjugation of antibody and linkerpayload (LP) are cysteine, lysine, site-specific, and click chemistry reactions. In this article, the recent development of ADCs with tubulysins as the payloads is reviewed, with the hope of providing a reference and future strategies for developing new ADSs.

Design, synthesis, and antitumor activity evaluation of pretubulysin analogs.

Pretubulysin, a biosynthetic precursor of the tubulysins, shows potent biological activity in a variety of tumor cell lines. Although there are several total synthesis routes to tubulysin and pretubulysin reported, the commercialization still has been hampered due to the complexity of the structure. To find structurally simpler pretubulysin analogs, a series of 2-(3-(methylamino)propyl)thiazole-4-carboxamides are designed and synthesized, and their anticancer activities are screened using MCF-7 (breast cancer), and NCI-H157 (lung cancer) cell lines. Taxol (IC50  = 0.01 µM) and pretubulysin are used as the control. Compounds 8c (IC50  = 0.05 µM, MCF-7; 0.09 µM, NCI-H157) and 8h (IC50  = 0.01 µM, MCF-7; 0.02 µM, NCI-H157) exhibited certain antitumor activities comparable to those of Taxol. The urea analogs of pretubulysin might represent a promising scaffold for the further development of novel antitumor drugs.

Improving Antibody-Tubulysin Conjugates through Linker Chemistry and Site-Specific Conjugation.

Tubulysins have emerged in recent years as a compelling drug class for delivery to tumor cells via antibodies. The ability of this drug class to exert bystander activity while retaining potency against multidrug-resistant cell lines differentiates them from other microtubule-disrupting agents. Tubulysin M, a synthetic analogue, has proven to be active and well tolerated as an antibody-drug conjugate (ADC) payload, but has the liability of being susceptible to acetate hydrolysis at the C11 position, leading to attenuated potency. In this work, we examine the ability of the drug-linker and conjugation site to preserve acetate stability. Our findings show that, in contrast to a more conventional protease-cleavable dipeptide linker, the ß-glucuronidase-cleavable glucuronide linker protects against acetate hydrolysis and improves ADC activity in vivo. In addition, site-specific conjugation can positively impact both acetate stability and in vivo activity. Together, these findings provide the basis for a highly optimized delivery strategy for tubulysin M.

Synthesis and Evaluation of Biological Properties of 2-Amino-thiazole-4-carboxamides: Amide Linkage Analogues of Pretubulysin.

Pretubulysin is a bio-precursor of highly toxic tetrapeptide tubulysins. Although pretubulysin has a much simpler chemical structure, it has similar anti-mitotic potency. A series of 2-amino-thiazole-4-carboxamides were designed and synthesized based on the structure of cemadotin. These are all novel compounds and their structures are characterized by 1H-NMR, 13C-NMR, and high resolution (HR)MS. The antitumor activities of these compounds were screened using the methyl thiazolyl tetrazolium colorimetric (MTT) cell viability method in MCF7 (breast cancer) and NCI-H1650 (lung cancer) cells. All the synthesized compounds 6a-n showed moderate anti-proliferation activities. Compounds 6m exhibited antitumor activity with the IC50 value of 0.47 and 1.1 µM in MCF7 and NCI-H1650 cells, respectively.

Design and validation of fibroblast activation protein alpha targeted imaging and therapeutic agents.

Background: Cancer-associated fibroblasts (CAFs) comprise a major cell type in the tumor microenvironment where they support tumor growth and survival by producing extracellular matrix, secreting immunosuppressive cytokines, releasing growth factors, and facilitating metastases. Because tumors with elevated CAFs are characterized by poorer prognosis, considerable effort is focused on developing methods to quantitate, suppress and/or eliminate CAFs. We exploit the elevated expression of fibroblast activation protein (FAP) on CAFs to target imaging and therapeutic agents selectively to these fibroblasts in solid tumors. Methods: FAP-targeted optical imaging, radioimaging, and chemotherapeutic agents were synthesized by conjugating FAP ligand (FL) to either a fluorescent dye, technetium-99m, or tubulysin B hydrazide. In vitro and in vivo studies were performed to determine the specificity and selectivity of each conjugate for FAP in vitro and in vivo. Results: FAP-targeted imaging and therapeutic conjugates showed high binding specificity and affinity in the low nanomolar range. Injection of FAP-targeted 99mTc into tumor-bearing mice enabled facile detection of tumor xenografts with little off-target uptake. Optical imaging of malignant lesions was also readily achieved following intravenous injection of FAP-targeted near-infrared fluorescent dye. Finally, systemic administration of a tubulysin B conjugate of FL promoted complete eradication of solid tumors with no evidence of gross toxicity to the animals. Conclusion: In view of the near absence of FAP on healthy cells, we conclude that targeting of FAP on cancer-associated fibroblasts can enable highly specific imaging and therapy of solid tumors.

Structure-activity relationships of tubulysin analogues.

The tubulysins are an emerging antibody-drug conjugate (ADC) payload that maintain potent anti-proliferative activity against cells that exhibit the multi-drug resistant (MDR) phenotype. These drugs possess a C-11 acetate known to be hydrolytically unstable in plasma, and loss of the acetate significantly attenuates cytotoxicity. Structure-activity relationship studies were undertaken to identify stable C-11 tubulysin analogues that maintain affinity for tubulin and potent cytotoxicity. After identifying several C-11 alkoxy analogues that possess comparable biological activity to tubulysin M with significantly improved plasma stability, additional analogues of both the Ile residue and N-terminal position were synthesized. These studies revealed that minor changes within the tubulin binding site of tubulysin can profoundly alter the activity of this chemotype, particularly against MDR-positive cell types.

HER2 antibody-drug conjugate controls growth of breast cancer brain metastases in hematogenous xenograft models, with heterogeneous blood-tumor barrier penetration unlinked to a passive marker.

BACKGROUND: Brain metastases of HER2+ breast cancer persist as a clinical challenge. Many therapeutics directed at human epidermal growth factor receptor 2 (HER2) are antibodies or antibody-drug conjugates (ADCs), and their permeability through the blood-tumor barrier (BTB) is poorly understood. We investigated the efficacy of a biparatopic anti-HER2 antibody-tubulysin conjugate (bHER2-ATC) in preclinical models of brain metastases. METHODS: The compound was evaluated in 2 hematogenous HER2+ brain metastasis mouse models, SUM190-BR and JIMT-1-BR. Endpoints included metastasis count, compound brain penetration, cancer cell proliferation, and apoptosis. RESULTS: Biparatopic HER2-ATC 3 mg/kg prevented metastasis outgrowth in the JIMT-1-BR model. At 1 mg/kg bHER2-ATC, a 70% and 92% reduction in large and micrometastases was observed. For the SUM190-BR model, an 85% and 53% reduction, respectively, in large and micrometastases was observed at 3 mg/kg, without statistical significance. Proliferation was reduced in both models at the highest dose. At the endpoint, bHER2-ATC uptake covered a median of 4-6% and 7-17% of metastasis area in the JIMT-1-BR and SUM190-BR models, respectively. Maximal compound uptake in the models was 19% and 86% in JIMT-1-BR and SUM190-BR, respectively. Multiple lesions in both models demonstrated ADC uptake in the absence or low diffusion of Texas Red Dextran, a marker of paracellular permeability. Using in vitro BTB assays, the ADC was endocytosed into brain endothelial cells, identifying a potentially new mechanism of antibody permeability. CONCLUSIONS: Biparatopic HER2-ATC significantly prevented JIMT-1-BR brain metastasis outgrowth and showed activity in the SUM190-BR model. The bHER2-ATC penetration into metastases that are impermeable to fluorescent dye suggested an endocytic mechanism of brain penetration.

Multiplex LC-MS/MS Assays for Clinical Bioanalysis of MEDI4276, an Antibody-Drug Conjugate of Tubulysin Analogue Attached via Cleavable Linker to a Biparatopic Humanized Antibody against HER-2.

Bioanalysis of complex biotherapeutics, such as antibody-drug conjugates (ADCs), is challenging and requires multiple assays to describe their pharmacokinetic (PK) profiles. To enable exposure-safety and exposure-efficacy analyses, as well as to understand the metabolism of ADC therapeutics, three bioanalytical methods are typically employed: Total Antibody, Antibody Conjugated Toxin or Total ADC and Unconjugated Toxin. MEDI4276 is an ADC comprised of biparatopic humanized antibody attached via a protease-cleavable peptide-based maleimidocaproyl linker to a tubulysin toxin (AZ13599185) with an approximate average drug-antibody ratio of 4. The conjugated payload of MEDI4276 can undergo ester hydrolysis to produce the conjugated payload AZ13687308, leading to the formation of MEDI1498 (de-acetylated MEDI4276). In this report, we describe the development, validation and application of three novel multiplex bioanalytical methods. The first ligand-binding liquid chromatography coupled with tandem mass spectrometry (LBA-LC-MS/MS) method was developed and validated for simultaneous measurement of total antibody and total ADC (antibody-conjugated AZ13599185) from MEDI4276. The second LBA-LC-MS/MS assay quantified total ADC (antibody-conjugated AZ13687308) from MEDI1498. The third multiplex LC-MS/MS assay was used for simultaneous quantification of unconjugated AZ13599185 and AZ13687308. Additional stability experiments confirmed that quantification of the released warhead in the presence of high concentrations of MEDI4276 was acceptable. Subsequently, the assays were employed in support of a first-in-human clinical trial (NCT02576548).

The synthetic tubulysin derivative, tubugi-1, improves the innate immune response by macrophage polarization in addition to its direct cytotoxic effects in a murine melanoma model.

Synthetic tubugis are equally potent but more stable than their natural forms. Their anticancer potential was estimated on a solid melanoma in vitro and in vivo. Tubugi-1 induced the apoptosis in B16 cells accompanied with strong intracellular production of reactive species, subsequently imposing glutathione and thiol group depletion. Paradoxically, membrane lipids were excluded from the cascade of intracellular oxidation, according to malondialdehyde decrease. Although morphologically apoptosis was typical, externalization of phosphatidylserine (PS) as an early apoptotic event was not detected. Even their exposition is pivotal for apoptotic cell eradication, primary macrophages successfully eliminated PS-deficient tubugi-1 induced apoptotic cells. The tumor volume in animals exposed to the drug in therapeutic mode was reduced in comparison to control as well as to paclitaxel-treated animals. Importantly, macrophages isolated from tubugi-1 treated animals possessed conserved phagocytic activity and were functionally and phenotypically recognized as M1. The cytotoxic effect of tubugi-1 is accomplished through its ability to polarize the macrophages toward M1, probably by PS independent apoptotic cell engulfment. The unique potential of tubugi-1 to prime the innate immune response through the induction of a specific pattern of tumor cell apoptosis can be of extraordinary importance from fundamental and applicable aspects.

Synthesis of a tubugi-1-toxin conjugate by a modulizable disulfide linker system with a neuropeptide Y analogue showing selectivity for hY1R-overexpressing tumor cells.

Tubugi-1 is a small cytotoxic peptide with picomolar cytotoxicity. To improve its cancer cell targeting, it was conjugated using a universal, modular disulfide derivative. This allowed conjugation to a neuropeptide-Y (NPY)-inspired peptide [K4(C-ßA-),F7,L17,P34]-hNPY, acting as NPY Y1 receptor (hY1R)-targeting peptide, to form a tubugi-1-SS-NPY disulfide-linked conjugate. The cytotoxic impacts of the novel tubugi-1-NPY peptide-toxin conjugate, as well as of free tubugi-1, and tubugi-1 bearing the thiol spacer (liberated from tubugi-1-NPY conjugate), and native tubulysin A as reference were investigated by in vitro cell viability and proliferation screenings. The tumor cell lines HT-29, Colo320 (both colon cancer), PC-3 (prostate cancer), and in conjunction with RT-qPCR analyses of the hY1R expression, the cell lines SK-N-MC (Ewing`s sarcoma), MDA-MB-468, MDA-MB-231 (both breast cancer) and 184B5 (normal breast; chemically transformed) were investigated. As hoped, the toxicity of tubugi-1 was masked, with IC50 values decreased by ca. 1,000-fold compared to the free toxin. Due to intracellular linker cleavage, the cytotoxic potency of the liberated tubugi-1 that, however, still bears the thiol spacer (tubugi-1-SH) was restored and up to 10-fold higher compared to the entire peptide-toxin conjugate. The conjugate shows toxic selectivity to tumor cell lines overexpressing the hY1R receptor subtype like, e.g., the hard to treat triple-negative breast cancer MDA-MB-468 cells.

Synthesis and antitumor activities of 3-substituted-analine derivatives: structure modifications of Tuv part of tubulysins.

BACKGROUND: Tubulysins family is a kind of natural compound with potent, antitumor activity. To simplify the synthesis route and find new antitumor compounds is becoming a hotspot of research recent years. RESULTS: Starting from 3-nitrobenzoic acid, after 7 steps transformations, 12 new tubulysin analogues were synthesized by the conformational restraint and bioisostere principle. These structures are featuring 3-substituted analine moieties. All these compounds are new compounds, and the structures were characterized by 1H NMR, 13C NMR, and HRMS. The antitumor activities were screened by the MTT method using MDA-MB-231and MCF7 cells. CONCLUSIONS: Compound IIb exhibited certain antitumor activity with the IC50 value of 7.6 and 11.8 µM against MDA-MB-231 and MCF7 cells respectively. Compounds IIa-IIe had moderate antitumor activities suggested that the thiazole ring in the Tuv could be replaced by the phenyl ring. However, Compounds Ia-Ie lose antitumor activity dramatically suggested that the conformation of the Tuv was crucial for the tubulysin analogues to maintain the biological activity.

Development of a Small Molecule Tubulysin B Conjugate for Treatment of Carbonic Anhydrase IX Receptor Expressing Cancers.

Carbonic anhydrase IX (CAIX) is a membrane-spanning zinc metalloenzyme that catalyzes the reversible consumption of CO2 and water to form H+ + HCO3-. Many human cancers upregulate CAIX to help control the pH in their hypoxic microenvironments. The consequent overexpression of CAIX on malignant cells and low expression on normal tissues render CAIX a particularly attractive target for small molecule inhibitors, antibody-drug conjugates, and ligand-targeted drugs. In this study, CAIX-targeted fluorescent reporter molecules were initially exploited to investigate CAIX-specific binding to multiple cancer cell lines, where they were shown to display potent and selective binding to CAIX positive cells. A small molecule CAIX-targeted tubulysin B conjugate was then synthesized and examined for its ability to kill CAIX-expressing tumor cells in vitro. Potent therapeutic conjugates were subsequently tested in vivo and demonstrated to eliminate solid human tumor xenografts in murine tumor models without exhibiting overt signs of toxicity. Because most solid tumors contain hypoxic regions where CAIX is overexpressed, development of a method to selectively deliver drugs to these hypoxic regions could aid in the therapy of otherwise difficult to treat tumors.

Stabilizing a Tubulysin Antibody-Drug Conjugate To Enable Activity Against Multidrug-Resistant Tumors.

The tubulysins are promising anticancer cytotoxic agents due to the clinical validation of their mechanism of action (microtubule inhibition) and their particular activity against multidrug-resistant tumor cells. Yet their high potency and subsequent systemic toxicity make them prime candidates for targeted therapy, particularly in the form of antibody-drug conjugates (ADCs). Here we report a strategy to prepare stable and bioreversible conjugates of tubulysins to antibodies without loss of activity. A peptide trigger along with a quaternary ammonium salt linker connection to the tertiary amine of tubulysin provided ADCs that were potent in vitro. However, we observed metabolism of a critical acetate ester of the drug in vivo, resulting in diminished conjugate activity. We were able to circumvent this metabolic liability with the judicious choice of a propyl ether replacement. This modified tubulysin ADC was stable and effective against multidrug-resistant lymphoma cell lines and tumors.

Development of a Ligand-Targeted Therapeutic Agent for Neurokinin-1 Receptor Expressing Cancers.

The neurokinin-1 receptor (NK1R) plays a significant role in the progression and metastasis of several neuroendocrine tumors. Due to its upregulation in these cancers, NK1R constitutes an attractive receptor for development of ligand-targeted imaging and therapeutic agents. In this report, we present the design and synthesis of an NK1R targeting ligand conjugated to the chemotherapeutic agent, tubulysin B hydrazide (TubBH), via a self-immolative linker. We then explore the ability of this low molecular weight tubulysin conjugate to kill NK1R overexpressing cancer cells both in vitro and in vivo without killing receptor negative healthy cells. Because similar studies in mice bearing NK1-negative tumors reveal no therapeutic impact, we conclude that our NK1R targeting ligand is specific for NK1R-expressing cells. Taken together, the data suggest a possible new approach for the treatment of NK1R-positive neuroendocrine cancers.

KEMTUB012-NI2, a novel potent tubulysin analog that selectively targets hypoxic cancer cells and is potentiated by cytochrome p450 reductase downregulation.

PURPOSE: There is an urgent need to develop effective therapies and treatment strategies to treat hypoxic tumors, which have a very poor prognosis and do not respond well to existing therapies. METHODS: A novel hypoxia-targeting agent, KEMTUB012-NI2, was synthesized by conjugating a 2-nitroimidazole hypoxia-targeting moiety to a synthetic tubulysin, a very potent antimitotic. Its hypoxic selectivity and mode of action were studied in breast cancer cell lines. RESULTS: KEMTUB012-NI2 exhibited a similar selectivity for hypoxic cells to that of tirapazamine, a well-established hypoxia-targeting agent, but was >1,000 times more potent in cell cytotoxicity assays. The hypoxia-targeting mechanism for both KEMTUB012-NI2 and tirapazamine was selective and mediated by one-electron reductases. However, while cytochrome p450 reductase (POR) downregulation could inhibit tirapazamine cytotoxicity, it actually sensitized hypoxic cells to KEMTUB012-NI2. CONCLUSION: KEMTUB012-NI2 is a potent new agent that can selectively target hypoxic cancer cells. The hypoxia selectivity of KEMTUB012-NI2 and tirapazamine appears to be differentially activated by reductases. Since reductases are heterogeneously expressed in tumors, the different activation mechanisms will allow these agents to complement each other. Combining POR downregulation with KEMTUB012-NI2 treatment could be a new treatment strategy that maximizes efficacy toward hypoxic tumor cells while limiting systemic toxicity.

Enhancing the therapeutic range of a targeted small-molecule tubulysin conjugate for folate receptor-based cancer therapy.

PURPOSE: EC0305 represents a folate-tubulysin B construct capable of specifically eradicating folate receptor (FR)-positive subcutaneous tumors from mice (Leamon et al., Cancer Res 68:9839-9844, 8). Herein we report on the use of multiple polar carbohydrate segments (e.g. 1-amino-1-deoxy-glucitolyl-γ-glutamate) placed in-between the folate and tubulysin B moieties of EC0305 creating a new conjugate, herein referred to as EC0531, with more desirable biological properties. METHODS: The synthesis of EC0531 and its tritium-labeled counterpart are described. EC0531's affinity for FR binding and specific cytotoxic activity was assessed using standard in vitro assays. Human tumor xenografts were used to directly compare EC0305 and EC0531's antitumor activity. Finally, bile duct cannulated, female Sprague-Dawley rats were used to compare hepatobiliary clearance of these two targeted chemotherapeutic agents. RESULTS: EC0531 tightly binds to the FR with an affinity about half that of folic acid. It was found to specifically inhibit the growth of FR+ cells (IC50 of ~2 nM) in a dose-dependent manner. Using 3H-labeled compounds, more than a 12-fold higher amount of tubulysin was measured in a FR + human tumor xenograft compared to the unconjugated drug, a finding that explains, in part, why EC0531 displays curative activity, whereas the unconjugated tubulysin agent is essentially inactive. EC0531 was found to produce greater FR-specific anti-tumor activity at lower dose levels than EC0305; furthermore, EC0531's maximum tolerated dose level was significantly higher than that of EC0305, likely because EC0531's saccharopeptidic-based spacer allows for ~sixfold reduction in hepatic clearance. CONCLUSIONS: These data provide additional evidence that the therapeutic range of targeted small-molecule drug conjugates can be favorably increased using molecular spacers constructed with 1-amino-1-deoxy-glucitolyl-γ-glutamate residues.

Optimization of Tubulysin Antibody-Drug Conjugates: A Case Study in Addressing ADC Metabolism.

As part of our efforts to develop new classes of tubulin inhibitor payloads for antibody-drug conjugate (ADC) programs, we developed a tubulysin ADC that demonstrated excellent in vitro activity but suffered from rapid metabolism of a critical acetate ester. A two-pronged strategy was employed to address this metabolism. First, the hydrolytically labile ester was replaced by a carbamate functional group resulting in a more stable ADC that retained potency in cellular assays. Second, site-specific conjugation was employed in order to design ADCs with reduced metabolic liabilities. Using the later approach, we were able to identify a conjugate at the 334C position of the heavy chain that resulted in an ADC with considerably reduced metabolism and improved efficacy. The examples discussed herein provide one of the clearest demonstrations to-date that site of conjugation can play a critical role in addressing metabolic and PK liabilities of an ADC. Moreover, a clear correlation was identified between the hydrophobicity of an ADC and its susceptibility to metabolic enzymes. Importantly, this study demonstrates that traditional medicinal chemistry strategies can be effectively applied to ADC programs.

Design, Synthesis, and Cytotoxic Evaluation of Novel Tubulysin Analogues as ADC Payloads.

The tubulysin class of natural products has attracted much attention from the medicinal chemistry community due to its potent cytotoxicity against a wide range of human cancer cell lines, including significant activity in multidrug-resistant carcinoma models. As a result of their potency, the tubulysins have become an important tool for use in targeted therapy, being widely pursued as payloads in the development of novel small molecule drug conjugates (SMDCs) and antibody-drug conjugates (ADCs). A structure-based and parallel medicinal chemistry approach was applied to the synthesis of novel tubulysin analogues. These efforts led to the discovery of a number of novel and potent cytotoxic tubulysin analogues, providing a framework for our simultaneous report, which highlights the discovery of tubulysin-based ADCs, including use of site-specific conjugation to address in vivo stability of the C-11 acetate functionality.